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Danny Flamberg's Blog
Danny has been marketing for a while, and his articles and work reflect great understanding of data driven marketing.
Eric Peterson the Demystifier
Eric gets metrics, analytics, interactive, and the real world. His advice is worth taking...
Geeking with Greg
Greg Linden created Amazon's recommendation system, so imagine what can write about...
Ned Batchelder's Blog
Ned just finds and writes interesting things. I don't know how he does it.
R at LoyaltyMatrix
Jim Porzak tells of his real-life use of R for marketing analysis.
(For those who are saying "Dr. Who who?", instead of pointing you to a "Who's on First" writeup like I wanted to, instead I'll point you to the Wikipedia entry on Doctor Who.)
If you've known me for a while, you know I have a soft spot for the UK, esp. their TV and music. Yes, they make lots of garbage, but then they turn around and make magic. And both of those apply to the new Dr. Who.
(This is actually the 3rd go round; we won't talk about that depressing joke in the 90s.)
I actually disliked (ok, hated) the first season guy, the "Ninth Doctor", played by Christopher Eccleston. He really managed to muck it up pretty badly, and the few times I saw him, I realized that it could be a great show… if he weren't on it.
Eccleston was really annoying. Its ok for the Doctor to be quirky, and to be rude as part of that quirk is acceptable… but to be just a complete jerk is really not what the character is about.
But when he left and David Tennant joined up, I gave it another chance…
And it charmed me. For the first time in a while, I didn't wish I had Tivoed it so I could jump to the end and save some time. I really wanted to experience it as it played. Billie Piper as Rose is a great companion, Tennant has whimsy and grit, and the show makes lots of knowing nods to its past, including bringing back original characters and enemies from the early days. The plots unfold quite nicely, effects are far (really far) more clever than the original series and actually better than lots of the big-budget shows, and (dare I say it), you actually believe that there may be Time Lords and other such groups running around out there.
I recently saw The Girl in the Fireplace and though the ending was a stretch, it had lots of great parts with a fantastic performance by Sophia Myles. I was just flipping channels, and I couldn't get past this show. The Cybermen episode was also just as good.
The BBC is the "official" home, and as it runs on the SciFi network here in the states, http://www.scifi.com/doctorwho/ also has some info.
I don't have much time to see TV these days, so other than Lost, I don't watch much. And yes, Battlestar Galactica is impressive… but its also depressing. If I'm willing to take one of my few spare hours and watch a show, you know its gotta be good.
This is worth the time.
* * *


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Cervical Spine Pathologies and Treatments. Physician Name Physician Institution Date. Degenerative Disease of the Spine.
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Physician Name
Physician Institution
Normal Pathology
Degenerated Pathology
T2- and T1-weighted sagittals at midspine showing spinal canal stenosis from C4/C5/C6 level

subset: commoncrawl


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|Sangria by the glass
|Potato and Vegetable Soup
|Stir fried cabbage with Bacalhau
|Crispy Suckling Pig on Fried Rice
This isn't the best restaurant in Macau. But it was a good fix for a quick dinner the first night we got there. Especially since this was about 5th on my list - the first 4 including Litoral was fully booked. Shouldn't have been a surprise since it was the start of the Easter weekend.
Decent, competent and wholesome food that was reasonably priced. Not a combination you find too often these days.
G/F, 1/F, 393 Edf. Dynasty Plaza R/C
Alameda Dutor Carlos d'Assumpção
Tel: +853-2875 2599


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In the days after Pearl Harbor, it seemed possible that Honolulu could be invaded and conquered by the Imperial Japanese Army and Navy. Instead, it was victory and the passage of time that have erased much of the World War II-era city.
The attack that launched the United States into World War II led to a massive military buildup on the islands, then jet-age tourism and statehood. It was Florida without the bus, California without the winter, Mexico and the Caribbean without the need to know a word of the native tongue. The tourism boom made and remade the landscape.
A few businesses have survived the wrecking ball or retained the afterglow of the World War II era. Here are some favorites:


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Orangetheory Fitness Kicks Off 6-Week Weight Loss Challenge, With a Chance to Win Cash and Prizes
Colorado Springs, Colorado (companies21) January 6, 2017 - Orangetheory® Fitness Colorado Springs, the distinctive high-energy interval workout and first fitness franchise to use heart-rate-monitored training, will help thousands of men and women transform their bodies in the first two months of 2017 in its 6-Week National Weight Loss Challenge. The "New Year, New Challenge" kicks off on January 16 and offers cash and prizes to winners. And the prizes start from: 1st: $500; 2nd: $300 and 3rd: $150
The spokesperson stated, "We are excited to begin our next local Weight Loss Challenge and help people transform their bodies in six weeks with Orangetheory Fitness," said David Long, CEO and Partner at Orangetheory Fitness.
He also continued, "This Challenge is for anyone who is more interested in results than resolutions when it comes to losing weight, getting fit and making a healthy lifestyle a priority. We have the best one-hour workout in the country, with every detail designed to produce results based on science."
Participants in the Challenge must attend a minimum of three 60-minute Orangetheory Fitness sessions per week, with a goal of showing the greatest improvement over the course of the contest.
Orangetheory Fitness is a fast-growing fitness concept and the only heart-rate-monitored, high-intensity interval training based on science. Modern studios and experienced staff provide affordable personal training in a group setting, using the latest equipment including TRX, treadmills, rowing machines and free weights. Each unique 60-minute session is broken into intervals of cardiovascular and strength training, producing a tremendous metabolic increase and calorie burn for up to 36 hours after the workout.
The media person commented by adding, "Based on the percentage of body weight lost, achieve the body of your dreams starting today with the support system to make you successful. Nutritional Support: Free 1 on 1 coach consult and weekly ongoing support to help you achieve your goals. We also offer monthly member seminars. This year we are excited to get 2017 off to a great start. Registration starts from: December 30, 2016 to January 16, 2017 and the registration cost is $25."
Special Notes to Consider:
Pictures/Measurements/Weight-ins: January 12-16, 2017
Challenge: January 16, 2017 to February 27, 2017
Party Announcement: Saturday, March 11, 2017 - 12:30 pm
Nutrition Clinic: January 13, 2017 at 6:45 pm or January 15th at 12:30
About Orangetheory® Fitness
Orangetheory® Fitness is a one-of-a-kind, group personal training workout broken into intervals of cardiovascular and strength training. Backed by the science of excess post-exercise oxygen consumption (EPOC), Orangetheory's heart-rate-monitored training is designed to maintain a target zone that stimulates metabolism and increases energy. Led by skilled personal trainers, participants use a variety of equipment including treadmills, rowing machines, TRX, and free weights, burning up to an average of 900 calories per session. The result is the Orange Effect -- more energy, visible toning and extra calorie burn for up to 36 hours post-workout. For more details, visit http://fitnessincoloradosprings.com


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Patent application title: Detection of Disease Associated Proteolysis
William S. Hancock (Brookline, MA, US)
Haven Baker (Roslindale, MA, US)
Marina Hincapie (Framingham, MA, US)
Xiaoyang Zheng (Malden, MA, US)
IPC8 Class: AC12Q137FI
USPC Class:
435 23
Class name: Measuring or testing process involving enzymes or micro-organisms; composition or test strip therefore; processes of forming such composition or test strip involving hydrolase involving proteinase
Publication date: 2009-02-05
Patent application number: 20090035797
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Patent application title: Detection of Disease Associated Proteolysis
William S. Hancock
Haven Baker
Marina Hincapie
Xiaoyang Zheng
Origin: BOSTON, MA US
IPC8 Class: AC12Q137FI
USPC Class:
435 23
Described herein are methods and techniques to study the "degradome". The
degradome of a specific protease is the complete product of the natural
substrate repertoire of that enzyme in a cell, tissue or organism. The
complete set of proteases that are expressed at a particular moment or
circumstance by a cell, tissue or organism produces the collective
degradome. Included in the methods described herein are approaches that
allow the direct identification and characterization of degradome
peptides from approx. 400 to approx. 12,000 Da. The methods of the
invention avoid the inherent problems of studying the peptidome by
focusing on specific or unique proteolytic cleavages that occur as a
result of endogenous protease activity induced by specific diseases. Once
characterized, the presence of, or change in level of, specific peptides
of the degradome can be used, e.g., to identify specific peptides having
elevated levels compared to a reference normal/or to correlate identified
peptides with specific proteins and/or to identify protein fragmentation
patterns (e.g., peptide ladders) and the specific protease(s) that
brought them about and then correlate this information with the presence
or absense of a specific disease or condition. Thus, the methods of the
invention can be used, for example, to identify new diagnostic markers
and/or therapeutic targets, as specific clinical diagnostic methods for
individual patients and as methods of monitoring the progress of a
therapeutic regimen for the treatment of a patient.
1. A method for detecting disease associated proteolysis
comprising:providing a biological sample from a mammal;isolating from
said sample all low molecular weight peptides having a molecular weight
of not more than approximately 12,000 Daltons;directly detecting and
determining the amino acid sequences of said isolated peptides by
ionization mass spectrometry; andrelating the determined sequence
information to a reference standard.
2. The method of claim 1, wherein the sample is a bodily fluid selected from the group consisting of blood (e.g., plasma or serum), saliva, urine, nipple aspirate, ductal lavage, sweat or perspiration, tumor exudates, joint fluid (e.g. synovial fluid), inflammation fluid, tears, semen and vaginal secretions.
3. The method of claim 1, wherein the step of isolating comprises one or more methods selected from the group consisting of:reverse phase chromatography;ultrafiltration; andbulk electrophoresis.
4. The method of claim 1, wherein the step of directly detecting is carried out by electrospray ionization mass spectrometry.
5. The method of claim 1, wherein the step of directly detecting is carried out using an ion trap mass spectrometer.
6. The method of claim 1, wherein the step of directly detecting is carried out using a hybrid ion trap mass spectrometer coupled to a quadrapole, time-of-flight mass spectrometer.
7. The method of claim 1, wherein the step of directly detecting is carried out using a reversed phase-high performance liquid chromatography system connected to a nanospray ionization hybrid ion trap-fourier transform mass spectrometer.
8. The method of claim 1, wherein the step of relating comprises one or more methods selected from the group consisting of:identifying specific peptides having elevated levels compared to a reference normal;correlating peptides with specific proteins; andidentifying protein fragmentation cleavage sites and relating that cleavage site to the activity of a specific endogenous propeolytic enzyme.
9. The method of claim 1, wherein, prior to said step of directly detecting, further fractionating said isolated peptides by removing all peptides having a molecular weight of less than approximately 3,000-4,000 Daltons.
10. A method of identifying a potential biomarker for a disease or pathological condition, said method comprising the steps of:a) obtaining a plurality of bodily fluid samples from a plurality of donors, wherein said plurality of donors comprise healthy individuals and patients at high risk for and/or having said disease or pathological condition;b) isolating from said plurality of bodily fluid samples all low molecular weight peptides having a molecular weight of not more than approximately 12,000 Daltons;c) directly detecting and determining the amino acid sequences of said isolated peptides by mass spectrometry;d) determining a profile of low molecular weight peptide abundance in each of said samples;e) comparing said abundance profiles of said patients at high risk for and/or having said disease or pathological condition with said abundance profiles of said healthy individuals; andf) identifying any low molecular weight peptides that are present in the abundance profiles of said patients at high risk for and/or having said disease or pathological condition but not in, or at a reduced level in, the abundance profiles of said healthy individuals,wherein any low molecular weight peptide so identified is a potential biomarker for said disease or pathological condition.
11. The method of claim 10, further comprising repeating said method steps so as to identify a plurality of potential biomarkers for said disease or pathological condition.
12. The method of claim 11, wherein said plurality of potential biomarkers for said disease or pathological condition comprise a panel of potential biomarkers.
13. The method of claim 12, wherein said panel of potential biomarkers is a peptide ladder.
14. A method of separating small peptides from large peptides and proteins comprising:providing a column comprising polymeric, silica, zirconia reversed phase chromatographic particles equilibrated to retain hydrophobic compounds and not to retain salts and other hydrophilic compounds;applying a sample comprising a mixture of proteins and peptides to said column;washing hydrophilic compounds through said column;washing said column to remove mildly hydrophobic, small peptides from said column;washing said column to remove larger, more hydrophobic peptides away from proteins from said column; andwashing said column to separate proteins from mildly and very hydrophobic peptides.
15. The method of claim 14, further comprisingproviding a second column comprising a polymeric, silica, zirconia reversed phase chromatographic resin equilibrated to retain small, hydrophilic compounds eluted from said first column.
[0001]This application claims the priority of U.S. Provisional Application No. 60/619,162 filed Oct. 15, 2004 entitled, USING LIQUID CHROMOTOGRAPHY-MASS SPECTROMETRY TO ANALYZE NATIVE HUMAN SERUM PEPTIDES, the whole of which is hereby incorporated by reference herein.
[0003]Human serum is one of the most informative of bodily fluids as it provides the major link among many human organs, tissues and cells. It has been estimated that the serum proteome consists of tens of thousands of proteins/peptides, with a large concentration range from 35-50×109 pg/mL to 0-5 pg/mL.1 This proteome includes 22 abundant and well-characterized proteins, which represent 99% of the mass of the proteinaceous content of human serum.2
[0004]Advances in proteomics are continuing to expand the ability of investigators to analyze the serum proteome. In recent years it has been realized that in addition to the circulating proteins, human serum also contains a large number of peptides, frequently called the "peptidome." Many of these peptides are believed to be fragments of larger proteins that have been at least partially degraded by various enzymes such as metalloproteases. However, identifying these proteins of origin from a small amount of serum/plasma is difficult due to the complexity of the sample, the low levels of these peptides and the difficulties in getting a protein identification from a single peptide.
[0005]To date, researchers have used different approaches to characterize the low molecular weight (LMW) protein or peptide species in serum. In the past five years, Richter, Jurgens and colleagues have published a number of papers describing fractionation of both hemofiltrate and serum and use of MALDI-TOF and electrospray instruments for analysis.4,5 More recently, different approaches using ultrafiltration to extract the peptides from serum have been published. Tirumalai et al. adopted 2D-LC-MS/MS and identified 340 proteins.2 Forssmann et al. used MALDI-MS and obtained a differential peptide display.23 Zhou et al. used immunoaffinity or ligand affinity chromatography and 1D-LC-MS/MS technologies and identified 210 proteins.6 Harper et al. analyzed LMW proteins/peptides with isoelectric focusing and RPLC-MS/MS and identified 262 proteins.7 However, improved methods of analyzing the peptidome would be desirable.
[0006]The invention is directed to methods and techniques to study the "degradome." The degradome of a specific protease is the complete product of the natural substrate repetoire of that enzyme in a cell, tissue or organism. The complete set of proteases that are expressed at a particular moment or circumstance by a cell, tissue or organism produces the collective degradome. Included in the methods described herein are approaches that allow the direct identification and characterization of degradome peptides from approx. 400 to approx. 12,000 Da. The methods of the invention avoid the inherent problems of studying the peptidome by focusing on specific or unique proteolytic cleavages that occur as a result of endogenous protease activity induced by specific diseases. Once characterized, the presence of, or change in level of, specific peptides of the degradome can be used, e.g., to identify specific peptides having elevated levels compared to a reference normal and/or to correlate identified peptides with specific proteins and/or to identify protein fragmentation patterns (e.g., peptide ladders) and the specific protease(s) that brought them about and then correlate this information with the presence or absense of a specific disease or condition. Thus, the methods of the invention can be used, for example, to identify new diagnostic markers and/or therapeutic targets, as specific clinical diagnostic methods for individual patients and as methods of monitoring the progress of a therapeutic regimen for the treatment of a patient. The methods and techniques described herein enable the identification of protein substrates, which enable the discovery of the molecular basis of biological pathways or processes associated with proteolysis, including for example, cancer, inflammation, muscular, neurodegenerative and autoimmune diseases.
[0007]Cleavage of target protein substrates can directly or indirectly affect the activity or function of numerous proteins, enzymes and receptors, and other proteins within a biological pathway. This can lead to a cascade of events that may trigger intracellular signaling or may lead to changes in various cell activities, including, for example, cell spreading, migration, cell-cell adhesion, ectodomain shedding and cell death.
Thus, in general, the invention is directed to a method for detecting disease associated proteolysis comprising the steps of providing a sample of bodily fluid from a mammal; isolating from said bodily fluid sample all low molecular weight peptides having a molecular weight of not more than approximately 12,000 Daltons; directly detecting and determining the amino acid sequences of said isolated peptides by ionization mass spectrometry; and relating the determined sequence information to a reference standard. Preferably, the sample is a bodily fluid selected from the group consisting of blood (e.g., plasma or serum), saliva, urine, nipple aspirate, ductal lavage, sweat or perspiration, tumor exudates, joint fluid (e.g. synovial fluid), inflammation fluid, tears, semen and vaginal secretions. The step of isolating can include one or more methods selected from the group consisting of reverse phase chromatography, ultrafiltration and bulk electrophoresis, and the step of directly detecting can be carried out by electrospray ionization mass spectrometry, preferably using an ion trap mass spectrometer, more preferably, a hybrid ion trap mass spectrometer coupled to a quadrapole, time-of-flight mass spectrometer, and most preferably, a reversed phase-high performance liquid chromatography system connected to a nanospray ionization hybrid ion trap-fourier transform mass spectrometer. The relating step preferably includes one or more methods selected from the group consisting of identifying specific peptides having elevated levels compared to a reference normal, correlating peptides with specific proteins, and identifying protein fragmentation cleavage sites and relating that cleavage site to the activity of a specific endogenous propeolytic enzyme. The method can further comprise, prior to the step of directly detecting, further fractionating said isolated peptides by removing all peptides having a molecular weight of less than approximately 3,000-4,000 Daltons.
[0008]In another aspect, the invention is directed to a method of identifying a potential biomarker for a disease or pathological condition, the method including the steps of obtaining a plurality of biological samples from a plurality of donors, wherein said plurality of donors comprise healthy individuals and patients at high risk for and/or having said disease or pathological condition; isolating from the plurality of bodily fluid samples all low molecular weight peptides having a molecular weight of not more than approximately 12,000 Daltons; directly detecting and determining the amino acid sequences of the isolated peptides by mass spectrometry; determining a profile of low molecular weight peptide abundance in each of the samples; comparing the abundance profiles of the patients at high risk for and/or having the disease or pathological condition with the abundance profiles of the healthy individuals; and identifying any low molecular weight peptides that are present in the abundance profiles of the patients at high risk for and/or having the disease or pathological condition but not in, or at a reduced level in, the abundance profiles of the healthy individuals, wherein any low molecular weight peptide so identified is a potential biomarker for the disease or pathological condition. Preferably, this method further comprises repeating the method steps so as to identify a plurality of potential biomarkers for the disease or pathological condition and the plurality of potential biomarkers for the disease or pathological condition includes a panel of potential biomarkers.
[0009]In yet another aspect, the invention is directed to a method of separating small peptides from large peptides and proteins comprising providing a column comprising polymeric, silica, zirconia reversed phase chromatographic particles equilibrated to retain hydrophobic compounds and not to retain salts and other hydrophilic compounds; applying a sample comprising a mixture of proteins and peptides to said column; washing hydrophilic compounds through said column; washing said column to remove mildly hydrophobic, small peptides from said column; washing said column to remove larger, more hydrophobic peptides away from proteins from said column; and washing said column to separate proteins from mildly and very hydrophobic peptides. The method can comprise further providing a second column comprising a polymeric, silica, zirconia reversed phase chromatographic resin equilibrated to retain small, hydrophilic compounds eluted from said first column.
[0010]Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof and from the claims, taken in conjunction with the accompanying drawings, in which:
[0011]FIGS. 1A and 1B show SDS-PAGE analysis of human serum ultrafiltrate. FIG. 1A shows the staining of a 1D SDS-PAGE gel with the Coomassie® blue. FIG. 1B shows the same gel, destained and then stained with silver. Lane 1 is 12 μL of SeeBlue® pre-stained standards; lane 2 is 9 μg of protein from 1.5 μL of human serum diluted into 9 μL; lane 3 is 37 μg of sample, ultrafiltered from 57 μL of human serum, which has been precipitated in solvents and reconstituted; lane 4 is the same human serum filtrate, concentrated with a speed-vac and reconstituted. The molecular weights of the pre-stained standards are shown on the left of the gel. Bands in lanes 3 and 4 are defined from top to bottom as a, b, c, d, and e, respectively;
[0012]FIG. 2 shows RP-LC/MS base peak ion chromatograms from triplicate analyses of the 10,000 Da human serum filtrate. The sample (10 μL) was loaded for 60 minutes with a flow rate of 200 nL/min in 2% mobile phase B (0.1% formic acid, 100% acetonitrile). Peptides were eluted with a 60 minute gradient (2% B to 40% B in 35 min, 40% B to 60% B in 20 min) and washed in 80% B for 5 min;
[0013]FIGS. 3A and 3B show full MS scan and MS/MS spectrum, respectively, of peptide A.SEGGFTATGQR.Q from extracellular matrix protein 1. FIG. 3A: Full MS scan occurred when the solvent composition was at 15.5% B; the insert is a magnified view of the precursor ion showing a MW of 555.7634 Da for this peptide. FIG. 3B: MS/MS spectrum of this peptide. This is an example where the mass accuracy is less than 2 ppm;
[0014]FIGS. 4A and 4B show Venn diagrams from triplicate analyses of human serum ultrafiltrate. FIG. 4A illustrates the number of overlapping protein identifications from repeat analysis; 75 proteins were unique to run 1, 22 were present in both runs 1 and 2, 17 were present in both runs 1 and 3, and 55 proteins were present in all three runs. FIG. 4B illustrates the number of overlapping peptide identifications from the same analysis. Filter criteria: Xcorr(1+,2+,3+)=1.8, 2.5, 3.5;
[0015]FIG. 5 shows distribution of peptides by molecular weight. The 337 unique peptides listed in Table 2 were observed in at least two runs and have both an Xcorr(1+,2+,3+)≧1.8,2.5,3.5 and at least 2 ppm mass accuracy;
[0016]FIG. 6 shows the location of identified fibrinogen α/α-E chain peptides. The boxes refer to the sequence of the peptide ladders that were observed in the sample. The shaded box indicates the predominant cleavage sites;
[0017]FIG. 7 shows tissue specificity of the 61 proteins whose peptide(s) were identified in at least two analyses, with a mass accuracy of at least 2ppm. Tissue specificity was obtained from SwissProt (http://au.expasy.org/sprot/);
[0018]FIG. 8 shows cellular component information for the 61 proteins of FIG. 7. The cellular component was obtained from LocusLink (http://www.ncbi.nlm.nih.gov/projects/LocusLink/);
[0019]FIG. 9 shows the molecular function of the 61 proteins of FIG. 7. Molecular function information was obtained from LocusLink (http://www.ncbi.nlm.nih.gov/projects/LocusLink/);
[0020]FIG. 10 is a graph showing material eluted from a C18 reverse phase column over time versus percent hydrophobic phase; and
[0021]FIG. 11 is a schematic diagram of a two-column set-up for practicing the method of the invention.
[0022]Advances in proteomics are continuing to expand the ability of investigators and clinicians to analyze the serum proteome. In recent years, it has been realized that in addition to the circulating proteins, human serum also contains a large number of peptides. Many of these peptides are believed to be fragments from proteolytic processing by various enzymes, such as metalloproteases. Identifying these peptides from a small amount of serum or plasma is difficult due to the complexity of the sample, the low levels of these peptides, and the difficulties in getting a protein identification from a single peptide.
[0023]In the methods described herein, the protein/peptide population is depleted of high molecular weight material using, e.g., reverse phase chromatography, and/or centrifugal ultrafiltration and/or bulk electrophoresis. Unlike in previous protocols using ultrafiltration, the isolated low molecular weight peptides are not digested, e.g., with trypsin, prior to analysis. This means that what is identified is the peptides that are produced endogenously. The fraction depleted of high molecular weight material was then concentrated and analyzed by mass spectrometry.
[0024]To obtain additional data, the sample fraction depleted of high molecular weight material may be additionally depleted of the lowest molecular weight material (e.g., from 400-3-4,000 Da), and the "mid-range" low molecular weight material (3-4,000-12,000 Da) can be analyzed separately. Peptides in the range of 400-4000 Da can be sequenced by using conventional ion trap mass spectrometers (e.g., LTQ-MS from Thermo Electron (San Jose, Calif., USA). However, confident identifications of these peptides requires a high mass accuracy (<5 ppm) instrument such as the hybrid ion trap-fourier transform mass spectrometer (LTQ-FTMS). High mass accuracy combined with a peptide sequence allows for the identification of a protein substrate from a single peptide.
[0025]Sequencing of the mid-range peptides (4,000-12,000 Da) requires the LTQ-FT hybrid-ion trap, or another mass spectrometer system that combines sequencing capabilities with mass analyzer having a resolution of at least 25,000. Preferably, the mass spectrometer system includes a mass spectrometer having a mass resolution of at least 50,000 and may include separate mass spectrometer instruments having the indicated properties. Currently, the best solution for identifying individual mid-range peptides in the degradome is a hybrid instrument, the LTQ-FTMS, connected to a reversed phase-high performance liquid chromatography system with a nanospray ionization source. The mass accuracy of this instrument allows the user to identify multiply charged peptides and determine their charge state, thus confidently identifying the protein precursors with the use of single peptides. As described herein, peptides in the molecular weight range of 400-4000 Da and/or those having a molecular weight of 4,000-12,000 Da can be used for the purposes of identification.
[0026]The utility of the hybrid ion trap (LTQ-FTMS) was demonstrated by the identification and characterization of over 300 unique peptides per serum sample with 2 ppm or better mass accuracy. With confident identifications, the origin and function of native serum peptides can be determined. Interestingly, over 34 peptide ladders were observed from over 17 serum proteins. This indicates that a cascade of proteolytic processes affects the serum peptidome. To examine whether this result was an artifact of serum, matched plasma and serum samples were analyzed, with similar peptide ladders found in each.
[0027]The progenitor proteins identified herein are of clinical interest for disease monitoring (see, e.g., Table 2 and proteins listed therein, e.g., apolipoproteins (cardiovascular disease), complement factors and prothrombin (blood clotting disorders), thymosin (immune system), plectin (bullous pemphigoid), extracellular matrix protein (cancer), ephrin receptor (cancer), insulin-like growth factor binding protein (diabetes), and vitamin D-binding protein (cancer).
[0028]Many diseases, particularly cancer and inflammation-based disorders are associated with altered patterns of proteolysis, which can be associated with changes, e.g., in the regulation of metalloproteases and other enzymes.20 Table 6 shows the specificity of proteases responsible for the generation of the peptides listed in Table 3. The table shows the number of observed enzymatic cleavages between two specific amino acids for a given serum sample. From a table such as this, one can locate cleavage patterns that are indicative of specific proteases. It can be seen that enzymes with trypsin-like specificity represent only part of the observed cleavage sites. If one presumes that the observed peptides were produced by endoproteases, which cleave on the C-terminal side of amino acid residues, then the most common N-termini of the observed peptides were glycine (57 times), serine (39) and aspartic acid (36), while lysine (59) and arginine (76) predominate following the C-terminus. Exoprotease cleavages have also been observed.
TABLE-US-00001 TABLE 6 Examination of the N- and C-terminal amino acids of all unique peptides to indicate proteases involved in serum peptide generation. All peptides were observed in at least two runs and have both an Xcorr 1+, 2+, 3+) ≧ 1.8, 2.5, 3.5 and at least 2 ppm mass accuracy. N-terminus of Peptide C-terminal C-terminus of Peptide amino acid N-terminal C-terminal N terminal Grouped by of the amino acid of amino acid amino acid of Protease preceding identified of identified the following Characteristics Amino Acid peptide peptide peptide peptide Hydrophilic Lysine (K) 45 13 4 59 Basic Arginine (R) 68 4 26 76 Histidine (H) 5 14 26 3 Hydrophilic Aspartic Acid (D) 8 36 3 6 Acidic Glutamic Acid (E) 20 31 13 8 Hydrophilic Glycine (G) 31 57 37 28 Polar Serine (S) 25 39 25 29 Threonine (T) 6 36 28 13 Cysteine (C) 0 0 0 1 Asparagine (N) 3 9 15 11 Glutamine (Q) 9 9 19 3 Tyrosine (Y) 8 3 10 4 Hydrophobic Alanine (A) 29 18 20 23 Non-Polar Valine (V) 4 13 19 5 Leucine (L) 25 15 25 11 Isoleucine (I) 5 8 2 10 Proline (P) 20 12 13 5 Methionine (M) 7 1 0 2 Phenylalanine (F) 10 5 16 7 Tryptophan (W) 2 0 3 2
Nomenclature in this tables is as follows:
[0029]Analysis of changes in cleavage patterns such as these will be of value in linking altered proteolysis with disease. For example, in a clinical assay, any specific type of proteolytic cleavage, e.g., by an aspartic, cysteine, metallo, serine/threonine or other type of protease, can be determined as described herein by identifying the LMW peptides by mass spectrometry, and any changes in the observed cleavage patterns over time can be followed. As another example, a specific peptide determined to be a "biomarker" for the specific type of proteolysis can be detected as either in high or low abundance compared to a reference standard, and the determined value can be correlated with the extent of the related disease or condition. The protein substrate of the indicated proteolytic enzyme can also be useful as a biomarker. Biomarkers of this type can originate from any tissue or cellular compartment or can be generated by digestion of secreted and surface proteins.
[0030]For example, FIG. 7 lists the tissue of origin (as given in SwissProt) for 61 proteins based on peptide identifications herein with 2 ppm or better mass accuracy. As could be expected, the classification is dominated by those tissues responsible for the majority of secreted proteins in blood such as liver (23.3%), blood (16.6%), brain (13.3%), heart and muscle (both 10%). We also examined the same set of peptide identifications for cellular compartment of origin in expectation that the major fraction of the peptidome would be generated by digestion of secreted and surface proteins. The results are given in FIG. 8 and show that extracellular region is indeed the major compartment (36.7%), with smaller contributions from cytoplasmic (15%) and membrane (11.7%) sites.
[0031]Another way of assessing the origin of the degradome is to examine the gene ontology of the set of high confidence progenitor proteins using publicly available software (FuncAssociate http://llama.med.harvard.edu/cgi/func/funcassociate).21 Table 7 shows those attributes with an adjusted P-value of less than 0.001, and the most highly ranked categories include extracellular, organismal physiological process, endopeptidase and protease inhibitor, blood coagulation and clotting, acute-phase response and lipid transport and metabolism.
TABLE-US-00002 TABLE 7 Sixty-one proteins identified according to the method of the invention grouped by function using FuncAssociate software (http://llama.med.harvard.edu/cgi/func/funcassociate). Number of Genes with this Gene Rank attribute Xa LODb P Value P-adjc Ontology Class of Proteins 1 30 1454 1.199 4.3e-21 <0.001 0005576 extracellular 2 27 2208 0.914 3.8e-13 <0.001 0050874 organismal physiological process 3 12 488 1.104 2.5e-09 <0.001 0009613 response to pest, pathogen or parasite/response to pest/pathogen/parasite 4 9 216 1.325 3.2e-09 <0.001 0004866 endopeptidase inhibitor activity/endoproteinase inhibitor/proteinase inhibitor 5 9 217 1.323 3.4e-09 <0.001 0030414 Protease inhibitor activity/peptidase inhibitor 6 7 93 1.588 3.4e-09 <0.001 0007596 Blood coagulation/blood clotting 7 12 503 1.090 3.5e-09 <0.001 0043207 response to external biotic stimulus 8 5 25 2.064 4.2e-09 <0.001 0006953 acute-phase response 9 7 100 1.554 5.6e-09 <0.001 0007599 hemostasis 10 7 109 1.514 1e-08 <0.001 0050878 regulation of body fluids 11 6 65 1.680 1.4e-08 <0.001 0006869 lipid transport 12 7 117 1.481 1.7e-08 <0.001 0050817 coagulation/clotting 13 6 69 1.652 2e-08 <0.001 0005319 lipid transporter activity/lipophorin 14 17 1367 0.828 2.3e-08 <0.001 0009605 response to external stimulus 15 5 39 1.837 4.4e-08 <0.001 0042157 lipoprotein metabolism 16 23 2692 0.699 4.6e-08 <0.001 0005515 protein binding 17 9 299 1.177 5.4e-08 <0.001 0005615 extracellular space 18 14 995 0.863 1.2e-07 <0.001 0006950 response to stress 19 9 330 1.132 1.2e-07 <0.001 0004857 enzyme inhibitor activity 20 6 106 1.452 2.6e-07 <0.001 0008015 circulation 21 8 287 1.137 5.6e-07 <0.001 0009611 response to wounding 22 4 27 1.909 5.9e-07 <0.001 0006958 complement activation, classical pathway 23 13 989 0.825 7.7e-07 <0.001 0006955 immune response 24 14 1236 0.763 1.6e-06 <0.001 0009607 response to biotic stimulus 25 13 1082 0.784 2.1e-06 0.001 0006952 defense response/defense response 26 4 37 1.755 2.2e-06 0.001 0006956 complement activation aTotal number of genes with this attribute. bThe natural log of the odds ratio, cadjusted P-value: fraction (as a %) of 1000 null-hypothesis simulations having attributes with this or a lower P value.21
[0032]FIG. 9 shows the types of biological activities associated with these 61 identified proteins, and a large proportion of the activities are associated with proteolysis, such as endopeptidase, peptidase activity, trypsin activity and other enzyme activity (total of 31%). Other interesting categories were receptor activity and receptor binding protein which together totaled 13%. These results again suggest that analysis of the peptidome will be valuable for the study of changes in disease associated proteolysis of surface receptors, secreted and shed proteins during processes such as apoptosis.
[0033]Thus, in general, the methods described herein can be practiced on any type of patent sample, including, without limitation, samples of bodily fluid taken from blood (e.g., plasma or serum), saliva, urine, nipple aspirate, ductal lavage, sweat or perspiration, tumor exudates, joint fluid (e.g., synovial fluid), inflammation fluid, tears, semen and vaginal secretions. Cells, tissues or organelles can also be sampled successfully if a protease cocktail is added immediately upon cell lysis.
[0034]The identified peptides can be used for the direct identification of precursor proteins and involved proteolytic enzymes, as described, or, e.g., pooled information from a number of patients can be used for clinical development of mass spectrometry-based diagnostics assays and for biomarker identification, particularly for the identification of multiple biomarkers that can be used simultaneously in panels. The identified peptides can also be used 1) to monitor the therapeutic response to specific protease inhibitors; 2) for selection and screening of drug candidates in preclinical studies; 3) for patient selection and response in clinical drug development; 4) for rational design of peptide inhibitors for specific proteases; 5) for the identification of novel proteases; and 6) to gain knowledge about the mechanism of disease etiology. The methods described herein can also be used to detect aberrant protein expression or function due to genetic mutations that may result in proteolytic degradation and subsequent peptide generation.
[0035]In an alternative use, the method of the invention can be used to explore the set of substrates for a selected protease, e.g., a metalloprotease, in a sample. A plasma or cell extract could be incubated with the protease and the cleavage points could be determined. This information would allow one to look for the same patterns in a specific disease and use the observation of an associated panel of peptides in the sample to show the protease was upregulated in that disease. Furthermore, information concerning the peptides of the degradome can be used to look at the pathology of a diseased sample by determining the cleavage patterns either in the tissue or in a fluid and then using that information to identify the protease. One can then image the tissue with an antibody to the peptide and localise the enzyme. Additionally, the method of the invention can be used to identify a set of proteases up or down-regulated in a specific disease or condition and then the proteases can be prepared by cloning or synthesis and attached to a chip as a protease array. A disease sample can then be added to the array along with fluorescent peptide substrates. If the peptide is in the sample it will compete with the fluorogenic peptide and a decrease in intensity will be observed.
[0036]The following examples are presented to illustrate the advantages of the present invention and to assist one of ordinary skill in making and using the same. These examples are not intended in any way otherwise to limit the scope of the disclosure.
Materials and Methods
[0037]Matched human serum and plasma sets were purchased from Bioreclamation, Inc. (Hicksville, N.Y.). Vivaspin® 4 centrifugal concentrators with a molecular weight cut off (MWCO) of 10,000 Da were obtained from ISC Bioexpress (Kaysville, Utah). Microcon YM-10 Centrifugal Filter Unit and Ziptip® with 0.6 μL C18 resin were purchased from Millipore (Bedford, Mass.).
[0038]Centrifugal concentrators were pre-rinsed with deionized water several times to minimize contamination of their membranes. For each sample, human serum (or plasma) was diluted in distilled and deionized water that contained 10-30% (v/v) acetonitrile in a ratio of 1:3 and then incubated at room temperature for 30 min. The diluted serum (or plasma) was transferred to a centrifugal filter with a MWCO of 10,000 Da and spun in a centrifuge to deplete the high molecular weight proteins (HMW). At the end of the process, approximately 80% of diluted filtrate was collected and peptides desalted using either Ziptip® with 0.6 μL C18 resin or reverse phase chromatography using a C18 column. (see below)
[0039]Alternatively, the filtrate can be subjected to a second round of ultrafiltration to separate the low mass range peptides (400-3-4,000) from the larger (3-4,000-12,000) peptides. The filtrate obtained from the 10,000 MWCO filter is apply to a centrifugal filter with a MWCO of 5,000 Da. And spin in a centrifuge. The small range peptides are collected in the filtrate, while the larger peptides (3-4,000-12,000 Da) are collected from the retained fraction. Both fractions are desalted using either Ziptip® with 0.6 μL C18 resin or reverse phase chromatography using a C18 column. (see below)
Protein Precipitation
[0040]The filtrate (200 μL) was first diluted with seven volumes (1400 μL) of cold acetonitrile (stored at -20° C. for at least 2 hours). After that, the mixture was vortexed vigorously for 5 seconds and spun at 1,100×g for 5 seconds. The supernatant was removed without disrupting the pellet at the bottom of tube. The pellet was washed with another 200 μL of cold acetonitrile. The supernatant was again removed, and the tube was left open at room temperature until the pellet was almost dry. Finally, the pellet was dissolved in 0.1% TFA in deionized water.
Sample Desalting and Peptide Isolation Prior to NanoLC-MS/MS Analysis
[0041](1) Using Ziptip® with 0.6 μL C18 Resin
[0042]Human serum (or plasma) filtrate contains a large amount of salt, which has to be removed before the sample is analyzed by LC-MS/MS. First, 200 μL human serum (or plasma) filtrate sample was concentrated with a speed-vac to 10 μL and then acidified with 0.5% TFA in deionized water. A Ziptip® with 0.6 μL C18 resin was used to carry out the necessary desalting according to the manufacturer's instructions. The analyte was then concentrated in a speed-vac and resuspended to a final volume of 20 μL of 0.1% FA in deionized water.
[0043](2) Using Reverse Phase Chromatography
[0044]The filtrate was desalted using a Supelco 4.6mm×20 mm C18 wide pore 3μ column. Eighty percent of the filtrate was loaded onto the column using a Shimadzu HPLC and a buffer system consisting of mobile phase A (0.08% trifluoro acetic acid (TFA) in water) and mobile phase B (0.07% TFA in 100% acetonitrile) and a flow rate of 1.5 ml/min. The column was first equilibrated with buffer A and salts were remove by washing the column with 100% buffer A for 3 min. This was followed by a 3 min step to 30% buffer B to elute the peptides and then a step to 90% for 3 min to remove very hydrophobic peptides and clean the column. FIG. 10 shows the peptides recovered in the 30% step.
[0045]Alternatively, a two-column reverse phase method can be used as a way of separating peptides into different populations. For example, human serum (or plasma) filtrate could be cleaned and at the same time separated into two different peptide populations using a combination of two reverse phase polymeric media of different hydrophobicities. Referring to FIG. 11, the sample, e.g., filtrate, is first loaded onto a weak (C4) hydrophobic phase column 10 to bind comparatively large and hydrophobic peptides with molecular weight range between 1,000 Da and 12,000 Da. Salts and small (400-1000 Da) and/or hydrophilic peptides flow through and are captured by a second more hydrophobic (C18) column 12. Alternatively, the hydrophobicities of the columns can be adjusted to a different cut-off value, e.g., 3-4,000 Da.
[0046]The two columns are operated in tandem using multidimensional High Pressure Liquid Chromatography (HPLC) instrumentation that has the capability of having both columns online for sample loading and valve switching to independently elute each column and elute the two peptide population into two different fractions. The two columns are placed on line, equilibrated with buffer A and a sample is then loaded on column 1. Salts and small/hydrophilic peptides flowing through column 1 are captured by column 2. The salts are removed by washing both columns with 4 ml of buffer A. Column 2 (C18) is taken off line and column 1 (C4) is eluted using 3 ml of 25% buffer B, followed by 3 ml of 90% buffer B. Column 1 is taken off-line, and column 2 is then eluted with a step to 90% buffer A.
[0047]Using the reverse phase approach, the low molecular weight (LMW) peptides (approx. 400-12,000 Da) can also be isolated directly from serum/plasma and separated from the high molecular weight (HMW) proteins using the principle that the hydrophobicity of a biological molecule increases with increasing molecular weight. The binding behavior of peptides and proteins depends on the surface area and the hydrophobicity of the polymeric reversed phase chromatographic resin. The cut-off is dictated by the percentage of mobile phase used to elute the peptides. It has been found that 35% organic solvent is the upper limit. Concentrations of mobile phase higher than 35% organic result in elution of higher MW proteins. This method uses reverse phase polymeric media with large pores to enhance mass transport and to enable extremely rapid separations of fragments/peptides from high molecular weight proteins thus minimizing the risk of endogenous proteolytic degradation. Separation of the fragments/peptides can be accomplished in less than 10 min. Plasma and or serum is diluted with 100 μl of 1×PBS, 7.2 M guanidine pH 7.4 and a final concentration of 5 mM reducing agent (either 5 mM DTT, or 5 mM TCEP) to break biomolecular interactions, and the sample is immediately injected onto the reversed phase column. HMW proteins and LMW peptides are bound to the first column and eluted with a step to between 25%-30% buffer B and 90% buffer B respectively, as described above. Salts and other non-bound species are washed off the resin by running between 5-10 column volumes of the acidic aqueous buffer through the column. The 25-30% acetonitrile eluted fraction is concentrated to 20 μl using a speed vac. Ten pl of the concentrated fraction is further separated by on-line LCMS 1-D reverse phase chromatography nano-LCMS on a linear ion trap, as described herein.
NanoLC-MS/MS Analysis
[0048]NanoLC-MS/MS analysis was performed on an UltiMate® Nano HPLC system (LC Packings-Dionex, Marlton, N.J.) and LTQ-FT® mass spectrometer (Thermo Electron, San Jose, Calif.). For the sample injection, FAMOS® well plate Microautosampler (LC Packings-Dionex, Marlton, N.J.) was used. The capillary column used for LC-MS/MS analysis (150×0.075 mm) was obtained from New Objective (Woburn, Mass.) and slurry packed in house with 5 μm, 200 Å pore size Magic C18 stationary phase (Michrom Bioresources, Auburn, Calif.). The mobile phase A for the LC separation was 0.1% formic acid in deionized water and the mobile phase B was 0.1% formic acid in acetonitrile. Half of the volume (10 μL) of filtrate obtained from the Ziptip desalting step was loaded on to the LC column. The electrospray ionization voltage was set at 1.8 kV and the normalized collision energy was set at 28% for MS/MS. The temperature of the ion transfer tube was 245° C. The flow rate was maintained at 200 nL/min after splitting. Data dependent ion selection was used to select the 8 most abundant ions from a MS scan for MS/MS analysis with LTQ FT system. A precursor ion was excluded from further LTQ MS/MS analysis for 1 min.
[0049]The chromatography gradient was set up to give a linear increase from 2% B to 40% B in 35 minutes and from 40% B to 60% B in 20 minutes and from 60% B to 80% B in 5 minutes. The dynamic exclusion list consisted of 200 precursor masses.
Data Processing and Analysis
[0050]Protein/peptide identifications were obtained using the SEQUEST algorithm from Thermo Electron (San Jose, Calif.). The human database used was extracted from the nr database on Sep. 9, 2004 (ftp://ftp.ncbi.nih.gov/blast.db/FASTA/). Search parameters used were as follows: no enzyme, no static modification, peptide mass tolerance set to 1.5 amu, fragment ion mass tolerance set to 0.0, number results scored set to 250. In this study, for a peptide to be considered a potentially positive identification the acceptance criteria required that the Xcorr be over 1.80 for singly-charged ions, 2.50 for doubly-charged ions, and 3.50 for triply-charged ions, additionally the mass error must be less than 2 ppm.
Analysis of the LMW Filtrate
[0051]Five hundred μL human serum was fractionated by ultrafiltration as described without being subjected first to enzymatic digestion, and the filtrate, depleted of high molecular weight (HMW) proteins, was collected. A commercial protein assay kit (BCA) was used to determine the protein concentrations of the starting material and the filtrate. The values were about 60,000 μg/mL and 190 μg/mL, respectively. A yield of ˜0.33 mg of the low molecular weight (LMW) fraction was obtained from ˜30 mg of proteinaceous material contained in a human serum sample (relative to a standard, bovine serum albumin). This amount of material represents approximately 1% of the initial protein mass and demonstrates the effectiveness of the ultrafiltration approach as a depletion method.
[0052]SDS PAGE analysis of the serum filtrate, concentrated either by cold acetonitrile precipitation or by use of a speed-vac, showed two weak bands in the low LMW region on the Coomassie® blue stained gel (see arrows, lane 3 and 4 in FIG. 1A). In order to improve detection sensitivity, the gel was destained and subjected to silver staining. The effectiveness of the ultrafiltration approach to successfully remove the HMW protein fraction was demonstrated by the fact that only bands having a molecular weight less than 10,000 Da were observed (see lanes 3 and 4 of the silver stained gel, FIG. 1B). Following speed-vac concentration, the salt was removed by a desalting step on a C18 Ziptip® prior to LC-MS/MS analysis.
[0053]A concern with the use of an ultrafiltration step to remove HMW proteins from serum relates to the potential loss of peptides that are strongly bound to carrier proteins, such as albumin. Although the addition of an organic solvent (10% acetonitrile) was used to disrupt hydrophobic interactions before the filtration step in an effort to minimize such losses, SDS PAGE analysis suggested that there may indeed be some peptide losses if one compares the intensity of bands "a" to "e" in lanes 2 to 4 of FIG. 1B. In this situation, however, the amount of peptide loss appears to be relatively consistent as yield measurements and SDS PAGE analysis of replicate isolations were found to be reproducible.
NanoLC-MS/MS Analysis
[0054]The LC-MS/MS analysis of a serum filtrate was performed at least three times for each sample. To assess reproducibility, the average coefficient of variation (CV) of the retention time was measured for five different peptides, and the value was found to be 0.15% for three runs. A typical comparison of the resulting total ion chromatograms is shown in FIG. 2. Peptides were identified by MS/MS fragmentation in a hybrid linear ion trap-Fourier transform mass spectrometer (LTQ-FTMS) with conservative values for sequence assignment (Xcorr(1+, 2+, 3+)=1.80, 2.50, 3.50).9 To get higher confidence peptide identifications, the hybrid FT-ICR instrument was used to acquire accurate mass measurements (within 2 ppm) during the survey MS scan (see, e.g., PCT Appl. No. PCT/US05/30713, which is hereby incorporated by reference herein) . Therefore, in addition to high Xcorr criteria for the assignment of peptide sequence, high mass accuracy obtained in the FTMS was used as another filter to assure accuracy of the assignment.
[0055]FIG. 3 shows an example of the type of data observed for the peptide SEGGFTAATGQR (presumed progenitor protein--extracellular matrix protein 1), which was identified as a doubly charged ion with an Xcorr of 3.34 and mass accuracy of 0.7 ppm. The extra filtering of data is particularly important in peptidome applications where many of the peptides are singly charged. Most of the proteins were identified from a single peptide (see FIG. 3A for typical results), and the decision not to use a trypsin digestion step precludes the use of trypsin specificity as an additional search criterion. In addition, the availability of an exact mass measurement greatly narrows down the number of possible candidates in a database search, which is particularly important for candidate sequences that contain several isobaric residues.
[0056]It has been noted previously that low level peptides may not be detected in a LC-MS/MS analysis due to the time constraints of the measurement, and, thus, repeated analyses are required.10 In addition, many correctly identified peptides, and particularly the lower abundance fragments, would also not be expected to be observed in replicate analyses because of the limitations of LC/MS. However, with the use of the LTQ-FT system, a total of 804 unique peptides, representing 359 unique proteins, were identified in human serum filtrate. Of these, 233 unique peptides and 55 proteins were identified in all of the triplicate runs (see FIG. 4). Table 2 lists 61 progenitor proteins and their 337 unique peptides, which were identified by MS/MS sequencing in at least two different runs and were measured with 2 ppm or better mass accuracy.
TABLE-US-00003 TABLE 2 Number of Unique ID Protein Name Peptides FIBA Fibrinogen alpha/alpha-E chain 118 ITH4 Inter-alpha-trypsin inhibitor heavy chain 38 CO3 Complement C3 18 CO4 Complement C4 22 APE Apolipoprotein E 16 KNG Kininogen 15 TYB4 Thymosin beta-4 14 APA4 Apolipoprotein A-IV 11 HEP2 Heparin cofactor II 7 F13A Coagulation factor XIII A chain 6 THRB Prothrombin 6 APC3 Apolipoprotein C-III 5 APA1 Apolipoprotein A-I 4 FIBB Fibrinogen beta Chain 4 APL Apolipoprotein L 3 A1AT Alpha-1-antitrypsin 3 A2HS Alpha-2-HS-glycoprotein 2 CO2 Complement C2 2 MYH4 Myosin heavy chain, skeletal muscle, fetal 2 CA14 Collagen alpha 1(IV) chain 2 ECM1 Extracellular matrix protein 1 2 ITAD Integrin alpha-D 2 LOX5 Arachidonate 5-lipoxygenase 2 NPL1 Nucleosome assembly protein 1-like 1 2 PLE1 Plectin 1 2 TTC3 Tetratricopeptide repeat protein 3 2 A2A1 Adapter-related protein complex 2 alpha 1 1 A2AP Alpha-2-antiplasmin precursor 1 APF Apolipoprotein F 1 ARVC Armadillo repeat protein deleted in velo 1 ARY1 Arylamine N-acetyltransferase 1 1 BASP Brain acid soluble protein 1 1 BAI3 Brain-specific angiogenesis inhibitor 3 1 CA12 Collagen alpha 1(II) 1 CA16 Collagen alpha 1(VI) Chain 1 CA1B Collagen alpha 1(XI) chain 1 C1QB Complement C1Q subcomponent, B chain 1 TP3B DNA Topoisonerase III beta-1 1 EPB2 Ephrin type-B receptor 2 1 GELS Gelsolin 1 HLP1 HAP1-like protein 1 1 HRG Histidine-rich glycoprotein 1 IBP5 Insulin-like growth factor binding protein 5 1 IRK4 Inward rectifier potassium channel 4 1 NEB1 Neurabin-I 1 OSF1 Osteoclast stimulating factor 1 P11A Phosphatidylinositol 3-kinase catalytic SU 1 PLX4 Plexin A3 1 BM1 Polycomb complex protein BMI-1 1 C215 Protein C21ORF5 1 MEFV Pyrin (marenostrin) 1 G3BP Ras-GTPASE-activating protein binding protein 1 RUN2 Runt-related transcription factor 2 1 ERR2 Steroid hormone receptor ERR2 1 T2AZ Stoned B-tflla-alpha and beta like factor 1 SYT1 Synaptotagmin I 1 TYB0 Thymosin beta-10 1 VIME Vimentin 1 VTDB Vitamin D-binding protein 1 VWF Von willebrand factor 1 Z151 Zinc finger protein 151 1 Proteins identified from human serum according to the method of the invention using an LTQ-FT analyzer and the NCBI database. All proteins were observed in at least two runs and have both an Xcorr(1+, 2+, 3+) ≧ 1.8, 2.5, 3.5 and at least 2 ppm mass accuracy.
[0057]As is consistent with the SDS-PAGE results, albumin or albumin related fragments were not observed in the LC/MS analysis. FIG. 5 shows that the major proportion of the peptides identified with a high probability assignment had a MW range of approximately 1000 to 2300, which is a range that is not easily detected in 2D gel electrophoresis.
Observance of Peptide Laddering
[0058]Many identified peptides were derived from the same protein sequence, a phenomenon referred to here as peptide laddering. These ladders are the result of progressive N- and C-terminal amino acid cleavages. In total, 34 peptide ladders were observed, derived from 17 different serum proteins with cleavage at diverse residues such as lysine, proline and glycine (Tables 3 and 4, and FIG. 6).
TABLE-US-00004 TABLE 3 No. of peptides in ID Peptide ladders identified in human serum. ladder FIBA a T.ADSGEGDFLAEGGGVR.G 13 R.GGSTSYGTGSETESPRNPSSAGSWNSGSSGPGSTGN 33 R NPGSSGTGGTATWKPGSSGPGPGSTGSWNSGSSGTGS TGNQNPGSPRPGSTGTWNPGSSE.R R.GSAGHWTSESSVSGSTGQWHSESGSFRPDSPGSGNA 5 .R R.REYHTEKLVTSKGDKEL.R 4 R.HPDEAAFFDTASTGKTFPGFFSP.M 4 L.GEFVSETESRGSESGIFTNTKESSSHHPGIAEFPSRG.K 8 K.SSSYSKQFTSSTSYNRGDSTFESKSYKMADEAGS 34 EADHEGTHSTKRGHA.K ITH4 a R.MNFRPGVLSSRQLGLPGPPDVPDHAAYHPF.R 30 CO3 a R.SSKITHRIHWESASLLRSEETKENEGFTVTAEGK.G 16 CO4 a R.TLEIPGNSDPNMIPDGDFNSYVR.V 4 R.NGFKSHALQLNNRQIRGLEEELQFSLGSKINVKVG 12 GNSKGTLKVL.R K.DDPDAPLQPVTPLQLFEG.R 4 KNG a R.GHGLGHGHEQQHGLGHGHKF.K 3 F.KLDDDLEHQGGHVLDHGH.K 7 K.RPPGFSPFR.S 2 K.DDPDAPLQPVTPLQLFEG.R 4 APE a A.KVEQAVETEPEPELRQQ.T 4 A.TVGSLAGQPLQERAQAWGERL.R 4 G.LVEKVQAAVGTSAAPVPSDNH.- 6 TYB4 a K.KTETQEKNPLPSKETIEQEKQAGES.- 14 HEP2 a G.GSKGPLDQLEKGGETAQSADPQWEQLNN.K 7 APA4 a K.GNTEGLQKSLAELGGHLDQQVEEFR.R 3 R.LAPLAEDVRGNL.K 5 F13A a R.AVPPNNSNAAEDDLPTVELQGVVPR.G 5 THRB a KSLEDKTERELLESYIDG.R 5 R.TATSEYQTFFNPR.T 4 FIBB a R.EEAPSLRPAPPPISGGGY.R 3 N.DNEEGFFS.A 2 APC3 a K.TAKDALSSVQESQVAQQA.R 2 A2HS a R.HTFMGVVSLGSPSGEVSHPRKT.R 4 APL a A.EEAGARVQQNVPSGTDTGDPQSKPLG.D 2 A1AT a A.EDPQGDAAQKTDTSH.H 2 CO2 a F.SHMLGATNPTQKTKESLG.R 2 a FIBA: Fibrinogen α/α-E CHAIN; ITH4: Inter-α-trypsin inhibitor; CO3: Complement C3; CO4: Complement C4; APE: Apolipoprotein E; TYB4: thymosin β-4; KNG: Kininogen; HEP2: Heparin cofactor II; F13A: Coagulation factor XIII A; APA4: Apolipoprotein A-IV; THRB: Prothrombin; FIBB: Fibrinogen β; A2HS: α-2-HS-Glycoprotein; APC3: Apolipoprotein C-III; APL: Apolipoprotein L; CO2: Complement C2; A1AT: α-1-antitrypsin.
TABLE-US-00005 TABLE 4 N-terminal ladders of fibrinopeptide A (FPA). No. MH+ Peptide Sequences 1 1536.69 T.ADSGEGDFLAEGGGVR.G 2 1465.66 A.DSGEGDFLAEGGGVR.G 3 1350.63 D.SGEGDFLAEGGGVR.G 4 1263.60 S.GEGDFLAEGGGVR.G 5 1077.53 G.EGDFLAEGGGVR.G 6 1380.59 T.ADSGEGDFLAEGGGV.R 7 1309.55 A.DSGEGDFLAEGGGV.R 8 1194.53 D.SGEGDFLAEGGGV.R 9 1107.50 S.GEGDFLAEGGGV.R 10 1050.47 G.EGDFLAEGGGV.R 11 921.43 E.GDFLAEGGGV.R 12 864.41 G.DFLAEGGGV.R 13 749.38 D.FLAEGGGV.R
[0059]These results suggest that proteases such as thrombin might be involved in the generation of the serum peptidome. Table 3 also shows that the number of peptides in a ladder is variable; the ladders observed ranged from 4 to 34 members. The most diverse ladder was derived from the fibrinogen a/a-E chain with numerous peptides that were identified from different parts of the protein. The location of the peptide fragments in the fibrinogen structure is shown by the shaded regions in FIG. 6. In addition, the shaded boxes in this figure mark the predominant cleavage sites, as measured by the frequent observation of certain high abundance peptides in the MS/MS analysis. As an example, Table 4 shows two ladders generated from residues 20 to 33 of fibrinogen (box 1 in Table 3) comprising 5 and 8 peptide members, respectively. The ladders were generated by a single processing of a C-terminal glycine residue as well as by multiple steps of N-terminal processing; the significance of this peptide will be discussed below.
[0060]It is to be expected that serum would contain a plethora of these ladder peptides, which could be generated from the enzymes activated during the clotting process. This expectation has resulted in the preference for plasma over serum in many proteomic studies. Certainly, the diversity of proteins with peptide ladders shown in Tables 3 and 4 supports this view, but it must be recognized that currently most clinical specimen banks consist of serum samples. However, the specificity of the laddering process may contain useful information when correlated with the presence of specific peptides according to the methods of the invention as many diseases are associated with coagulation defects.14,15 Interestingly, several of the cleaved proteins are not known to be involved in the clotting process, such as inter-α-trypsin inhibitor, apolipoprotein E and A-IV, thymosin-β-4 and kininogen. Furthermore, if non-specific proteolysis were a general phenomenon, it would be expected that all major serum proteins would exhibit the laddering process. In this study, however, cleavage products of the following abundant proteins were not observed: human serum albumin, immunoglobulins, transferrin, alpha-2-macroglobulin, haptoglobin, lipoprotein a, alpha-1-acid glycoprotein, factor H, ceruloplasmin, complement factor B, prealbumin, C9 complement, Clq complement and C8 complement.
[0061]One peptide of diagnostic significance, Fibrinopeptide A (FPA, T.ADSGEGDFLAEGGGVR.G; the residues on either side of the period refer to the first residue of the adjacent peptide), which was observed in this peptidome study (see FIGS. 4 and 6), is known to be released as part of the clotting process. An elevated level of this peptide may indicate an abnormal clotting process, such as disseminated intravascular coagulation, cellulites, ovarian cancer14 and systemic lupus erythematosus.15 Measurement of FPA is used in clinical chemistry to diagnose certain types of leukemia, which are associated with disseminated intravascular coagulation. The results described here suggest that the clinical measurement could be complicated, however, by multi-step proteolysis. Our data show that after the generation of the parent peptide, the C-terminal arginine of FPA was enzymatically cleaved by an unknown enzyme to generate the peptide (T.ADSGEGDFLAEGGGV.R) lacking the C-terminal glycine residue. The peptide ladder is generated by digestion of the FPA fragment by an N-terminal exopeptidase(s) to give the products shown in Table 3.
[0062]Another example of a peptide of potential diagnostic significance that exhibits laddering is shown in Table 2 for the sequence (R.SSKITHRIHWESASLLRSEETKENEGFTVTAEGK.G) and is derived from complement C3. One of the daughter peptides (R.SSKITHRIHWESASLLR.S) is known as the C3f fragment and is thought to be related to inflammation.16 It was reported that C3f fragments were produced less abundantly in chronic asthma mice than in control mice.17 The flanking arginine residue from both the N-terminal and the C-terminus of this peptide (R.SSKITHRIHWESASLLR.S) has been reported to be cleaved by factor I.18 Also Factor I may be responsible for the generation of peptide (R.EGVQKEDIPPADLSDQVPDTESET.R), part of C3g fragment by cleavage of the arginine residue flanking the C-terminus.19
[0063]To resolve the issues around the use of plasma or serum, the original serum study was repeated on a matched set of samples obtained from the same 36 year old healthy male. A total of 87 unique peptides from 34 proteins were identified from the plasma filtrate and 117 unique peptides from 50 proteins were identified from the serum filtrate. Table 5 shows that many of the abundant peptide ladders seen in serum were also present in plasma, for example fibrinogen was observed with a total of 37 peptides in both samples. While plasma collection processes can be variable, these studies do not suggest that there is a clear preference for plasma over serum in proteomic analyses. It should be noted that the number of unique peptides identified from the matched plasma and serum filtrate samples was lower than that of Sigma serum filtrate. This could be explained by the extra complexity of the commercial Sigma serum sample, which was prepared by the pooling of samples from multiple donors.
TABLE-US-00006 TABLE 5 Comparison of abundant proteins present in a set of plasma and serum samples from the same individual. Serum Protein Num. of Plasma ID Protein Name Rank peptides Rank Num. of peptides FIBA Fibrinogen alpha- 1 37 1 37 alpha/E chain CO3 Complement C3 2 12 2 8 CO4 Complement C4 3 11 3 6 TYB4 Thymosin beta-4 5 10 6 2 APOL1 Apolipoprotein-L 1 12 2 4 2 TTHY Transthyretin 18 1 5 2 FIBB Fibrinogen beta 23 1 9 1 chain KNG Kininogen 25 1 7 1
[0064]All identified peptides in the LC-MS/MS study had a molecular weight less than 4,000 Da if ZipTip C18 was used to clean the sample (see FIG. 5), which was different from the results of SDS PAGE analysis of unfractionated plasma (see FIG. 1B, lane 4). A possible reason for this result was that the use of a desalting and concentration step (ZipTip C18) before the MS analysis which could result in the loss of larger hydrophobic peptides and alternative sample preparation procedures are being evaluated to overcome such losses.
[0065](1) Anderson, N. L.; Anderson, N. G. Mol. Cell. Proteomics. 2002, 1, 845-867. [0066](2) Tirumalai, R. S.; Chan K. C.; Prieto, D. A.; Issaq, H. J.;
[0067]Conrads T. P.; Veenstra T. D. Mol. Cell. Proteomics. 2003, 2, 1096-1103. [0068](3) Adkins, J. N.; Varnum, S. M.; Auberry, K. J.; Moore, R. J.; Angell, N. H., Smith, R. D.; Springer, D. L.; and Pounds, J. G. Mol. Cell. Proteomics 2002, 1, 947-955. [0069](4) Schrader, M.; Schulz-Knappe, P. Trends in biotechnol. 2001, 19, S55-S60. [0070](5) Richter, R.; Schulz-Knappe, P.; Schrader, M.; Standker, L.;
[0071]Jurgens, M.; Tammen, H.; Forssmann, W. G. J Chromatogr B Biomed Sci Appl. 1999, 726, 25-35. [0072](6) Zhou, M.; Lucas, D. A.; Chan, K. C.; Issaq, H. J.; Petricoin, E. F.; Liotta, L. A.; Veenstra, T. D.; Conrads, T. P. Electrophoresis 2004, 25, 1289-1298. [0073](7) Harper, R. G.; Workman, S. R.; Schuetzner, S.; Timperman, A. T.; Sutton, J. N Electrophoresis 2004, 1299-1306. [0074](8) Strader, M. B.; Verb erkmoes, N. C.; Tabb, D. L.; Connelly, H. M.; Barton, J. W.; Bruce, B. D.; Pelletier, D. A.; Davison, B. H.; Hettich, R. L.; Larimer, F. W.; Hurst, G. B. J. Proteome Res. 2004, 3, 965-978. [0075](9) Tabb, D. L.; McDonald, W. H.; Yates, J. R. III Journal of Proteome Research 2002, 1, 21-26. [0076](10) Shen, Y.; Jacobs, J. M.; Camp, D. G. II; Fang, R.; Moore, R. J.; Smith, R. D.; Xiao, W.; Davis, R. W.; Tompkins, R. G. Anal Chem. 2004, 76, 1134-1144. [0077](11) Cugno, M.; Scott, C. F.; Salerno, F.; Lorenzano, E.; Muller-Esterl, W.; Agostoni, A.; Colman, R. W. Thromb Haemost. 1999, 82, 1428-1432. [0078](12) Hannappel, E.; van Kampen, M. J Chromatogr. 1987, 397, 279-285. [0079](13) Smith, D. B.; Janmey, P. A.; Herbert, T. J.; Lind, S. E. J Lab Clin Med. 1987, 110, 189-195. [0080](14) Bergen, H. R. III; Vasmatzis, G.; Cliby, W. A.; Johnson, K. L.; Oberg, A. L.; Muddiman, D. C. Dis. Markers. 2003, 19, 239-249. [0081](15) Gando, S.; Nanzaki, S.; Sasaki, S.; Kemmotsu, O. Thromb. Haemost. 1998, 79, 1111-1115. [0082](16) De Bruijn, M. H. L.; Fey, G. H. Proc. Natl. Acad. Sci. U.S.A. 1985, 82, 708-712. [0083](17) Yeo, S.; Roh, G. S.; Kim, D. H.; Lee, J. M.; Seo, S. W.; Cho, J. W.; Kim, C. W.; Kwack, K. Proteomics 2004, 4, 3308-3317. [0084](18) Harrison, R. A.; Farries, T. C.; Northrop, F. D.; Lachmann, P. J.; Davis, A. E. Complement 1988, 5, 27-32. [0085](19) Chaplin, H.; Monroe, M. C.; Lachmann, P. J. Clin. Exp. Immunol. 1983, 51, 639-646. [0086](20) Boire, A.; Covic, L.; Agarwal, A.; Jacques, S.; Sherifi, S.; Kuliopulos, A. Cell. 2005, 120, 303-313. [0087](21) Berriz, G. F.; King, O. D.; Bryant, B.; Sander, C.; Roth, F. P. Bioinformatics 2003, 19, 2502-2504. [0088](22) Wu, S.-L., Jardine, I., Hancock, W. S., and Karger, B. L. Rapid Commun. Mass Spectrom 2004, 19, 2201-2207.
[0089]While the present invention has been described in conjunction with a preferred embodiment, one of ordinary skill, after reading the foregoing specification, will be able to effect various changes, substitutions of equivalents, and other alterations to the compositions and methods set forth herein. It is therefore intended that the protection granted by Letters Patent hereon be limited only by the definitions contained in the appended claims and equivalents thereof.
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Friday, June 23, 2017
Life Above All
6 comments:
- AnonymousJune 23, 2017 at 8:19 PM
I'm happy you find your motivation Sir!!!ReplyDelete
Glad to know you are alive :-P :-xReplyDelete
Aha! i figured it out... lolReplyDelete
its awesome to have you back ;-x
keep posting....
- AnonymousJune 26, 2017 at 5:45 AM
This piece is a very good guide for the young generation of mountaineers. As it is now a full fledged profession which often outweigh the feeling... the passion involved in it.ReplyDelete
Hi Satya, as one of the earlier commentators has pointed out, it is nice to know that you are still alive and kicking... both Margaret and I were wondering some time ago since you have gone AWOL from social media for quite some time now! Anyways, hope everything is tickety boo in your life (with your attitude it MUST be!). Hope to meet up for old time's sake when I visit India next (it has been more than 5 years now!)Cheers and Godbless from both of us.ReplyDelete
Dear Satya, Very happy to read you, would love to meet you when you travel to Belgium again :-) take care JaneReplyDelete


text:
These recipes with avocado demonstrate the immense popularity of the creamy green fruit. Avocados are reported to have numerous health benefits, including being rich in nutrients (especially Vitamin K and potassium), having lots of fiber and healthy fat content and protecting against eye disease. They are also in season this time of year.
Recipes with avocado can revamp traditional guacamole by adding unexpected ingredients, as seen with pineapple avocado appetizers, mango guacamole recipes and fennel apple guacamoles. Many Mexican mashup meals also exist, from Mexican taco nachos to DIY vegan burrito bowls.
Avocados can also be introduced to a variety of desserts, often adding a creamier texture. Chocolate avocado pies, gluten-free eggnog cakes and minty avocado fudge bars are all examples of this. Surprising avocado inclusions also extend to burger buns, blueberry muffins and breakfast pizzas.
100 Recipes With Avocado
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Vegan Sushi Burgers
Vegetarian Cobb Salads
Cheesy Seafood Sandwiches
Avocado-Shaped Caramel Chocolates
Summery Fire-Grilled Salads


text:
|Free Fall, monoprint, Asandra
Since I moved to California in February 2011, I lived in four different cities. Two located in Northern California and two in Southern. The impact of this can only be understood with the knowledge that all of this transpired during a 15-month period. In addition, every possession, including a car and a furry co-pilot named Sasha, came with me.
I grew up in New York, eventually relocated down the East Coast to Miami, then moved 3,000 miles across country to the East Bay area of California (close to San Francisco), and a month ago drove 377 miles to Southern California. One would certainly consider this a zigzag across the United States.
The response that I have often heard about my numerous relocations is that I am "wild." Hardly, I reply. Another common misconception: "You must not have a lot of possessions." Wrong again. However, the singular most common reaction is, "What do your Guides have to say about this?"
The expectation is, as an adept Channel, I should have known the exact, perfect location to land in not only immediately, but also effortlessly. Here is what many do not realize: I am no different from you! Yes, I am an experienced Medium, but do I have some extra-special privy to the Spirit world because I channel for a living? The answer is no. I am just like you: doing my best to hear, feel, sense, and respond, to the higher guidance.
Does that mean that I have been misguided, or not able to receive clear information? No. What it means is something that Spirit often says, "Life is a circuitous route." There is a tendency for us in our linear thinking, rational reality, to want to know why something is happening. Our expectation is that if we are receiving the guidance, the path will be void of road- blocks, traffic jams, or potholes. If we knew the cosmic reasoning for everything, however, there would not be any surprises, mysteries to explore, or lessons to learn. In other words, we would not grow.
For those of you still insisting on a more linear explanation for my zigzag journey, I can tell you that I used the resources I had available to me. This mostly required a lot of trusting the unfamiliar, and leaping into the unknown. Furthermore, whenever I second guessed myself and wondered if I had made incorrect choices, I realized that whatever I needed to face within me, as a result of leaping into the unknown, would still have surfaced. The great illusion is that we live life on the outside, but the truth is that we experience though our senses, react with our emotions, but live through our consciousness.
Have I settled yet? The answer again, is no; my new life is still a work in progress. I have learned something great though: that if I trust my inner guidance, the guidance of spirit is a given. They are right behind us, supporting us every step of the way. When a child is learning to walk, you cannot do it for them. At a certain point, you have to allow them to find their confidence and courage to walk forward. Sometimes we just have to learn to let go of getting it right and simply go in the direction of where we are inspired to go.
Asandra, copyright 2012
For more info: Asandra.com, TheGuidedPath.net, ContactYourSpiritGuides.com


text:
Small and Mighty: Zellous Hope Project
The Zellous Hope Project has received an $8,000 Small and Mighty grant from The Pittsburgh Foundation to continue providing resources to women transitioning from homelessness to permanent housing. Begun in 2011 as a program of Dress for Success Pittsburgh, this volunteer-run program packs essential household items such as dishes, cookware, towels, bedding and cleaning products into "Hope Chests". Additionally, the group provides "Hope Vouchers" to help pay for additional necessities such as a photo ID for employment or a tire replacement for a car used for work. This volunteer-run program also provides peer advocacy and support for addiction and recovery and referrals for various services. Last year, with a program budget of only $3,000, Zellous Hope Project served 19 families. With this grant, Zellous Hope Project will increase the women served through its Hope Chest and Hope Voucher Programs from 19 to 100 families, providing them with weekly wellness checks to assure their transitions go smoothly.
Small and Mighty grants are a direct result of the Foundation's 100 Percent Pittsburgh organizing principle, which focuses on engaging single women raising children and youth ages 12 to 24—two groups largely left behind in our region's economic resurgence. Small and Mighty offers a streamlined grant-seeking process to organizations with operating budgets less than $600,000. Zellous Hope Project was one of 18 local nonprofit organizations receiving support through this initiative, as their annual operating budget is just $21,500.


text:
With increased air routes and cruise ship promotions, travel firms have seen a strong start to the year, writes Kristy Dorsey
THIS week marks the start of daily flights between Edinburgh and Doha – capital of the world's richest country per capita – as the recovery in the travel industry works it way up to the premium end of the market.
Qatar Airways announced in November that the service, previously flying five times a week out of the Scottish capital, will become a daily operation from the start of this month. The announcement came just six months after the launch of Edinburgh's first direct flight to the Middle East.
Hailed at the time as a long-missing link in the capital's route network, the Edinburgh-Doha connection could have further to travel. Qatar Airways chief executive Akbar Al Baker said last year that two flights a day are possible if demand lives up to expectations. He's not the only one considering such possibilities. Turkish Airlines flies once a day between Edinburgh and Istanbul in winter and nine times a week in summer, but Ahmet Serhat Sari, the airline's general manager in Scotland, has said the carrier would consider increasing that frequency as well as flying to other destinations in Scotland if there is sufficient demand.
After the devastating fall-out from the last recession, the travel market has been in slow recovery mode for a year or so as consumer confidence has gradually crept back in. ABTA's Travel Trends Report for 2015 found 20 per cent of holidaymakers will spend more on their trips this year, while only 15 per cent plan to spend less. Meanwhile, recent figures from the Office for National Statistics show UK holiday departures in 2014 hit 39 million for the first time since 2008. The positive trend has continued into the current year, with industry analyst GfK reporting a 7 per cent increase in summer bookings in the latest week up to 14 February.
Stuart Leven, UK chief executive of Royal Caribbean, said the cruise operator has seen a similar increase in bookings in the first part of 2015. Customers are also opting for longer cruises.
"From Scotland, we have seen a very strong start to the year," said Leven, who was in Glasgow last week speaking at the annual dinner of the Scottish Passenger Agents Association. "We are ahead of where we were at this time in 2014."
Of those Scottish travellers who book a cruise, 56 per cent will book again in the following two years. The UK average, by comparison, is 50 per cent. In addition, Scots generally cruise for longer than people from other parts of the UK. For these reasons, Royal Caribbean is investing north of the Border in promoting its new ship, Anthem Of The Seas. It will begin sailing in April from Southampton with a range of restaurants, bars and activities including virtual skydiving and surf simulators to a Broadway-style theatre and a "Bionic Bar" staffed by robots.
Despite the host of amenities, Leven said cruising is no longer the elitist pursuit it once was. He argues it should be compared to the cost of similar holidays in an all-inclusive resort.
Cruising does, however, suffer from misconceptions based on gentrified images from the past.
"The conceptions that people have of cruises is that they are for older people, not families going on holidays," he said. "There is also this idea that cruising is not an active holiday – you lay on the deck in the sun, you get off in the afternoon for a walk through some town, then you come back on board and eat your dinner, and that's pretty much it. That certainly is not what we believe a cruise to be."
Break that barrier, Leven said, and Royal Caribbean sees "great potential for growth in the Scottish market".
Executives at Emirates are likewise upbeat. The carrier has flown 2.8 million people between Glasgow and Dubai since that service began in 2004, with growth quickening after the introduction of a second daily flight and first class seating, both in 2012.
Part of the route's success lies in the fact that leisure travellers can easily connect onwards to the likes of Australia, Thailand, Mauritius or the Seychelles, opening up alternatives to the UK's usual foreign holiday destinations. Laurie Berryman, UK vice-president of Emirates, said about three-quarters of Scots leisure travellers using the route are bound for further destinations.
But Emirates has also seen a big increase in demand for its premium services – business and first class – which has been driven in large part by the return of corporate spending. Premium customers have use of the Emirates executive lounge in Glasgow, as well as a complimentary chauffeur service to and from the airport.
As a major gateway for passengers and cargo, Berryman said Glasgow remains of "huge importance" to the carrier.
"Its robust economy and tremendous appetite for both global commerce and travel has resulted in significant growth for our operation in a relatively short amount of time," Berryman said last week on a visit to the city.
"Testament to our belief in Glasgow as the cultural, sporting and academic heart of Scotland, we have invested heavily in the area beyond simply the airport, including sponsorship of the Emirates Arena and the upcoming Rugby Sevens. Glasgow remains a key location for us, and we look forward to growing our operation over the coming years."
That could include introducing the Airbus A380 on the route, though Berryman said that would be a "big step up". Compared to the Boeing 777-330 currently in use, the A380 has 34 additional first class seats, 62 extra business class seats and an additional 117 seats in economy. An alternative would be to add a third daily flight.
"Looking ahead, Scotland has got a very exciting future aviation-wise," Berryman said. "It is good to see confidence again in the aviation business, and in travel overall."
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text:
Wednesday, February 18, 2015
Analog Science Fiction and Fact, April 2015
A pretty decent issue, mostly.
SHORT STORY: TRANSFER POINT, Barry N. Malzberg & Bill Pronzini
A customs inspector at a lunar immigration station is told that the station will be closed, and the new one will be established on Mars. He starts his day a bit baffled about the news. He is also feeling somehow strange, strangely light headed or something. A short, light story with a fifties feel in it. ***+
NOVELETTE: THE EIGHTH ITERATION, Bond Elam
A colony is facing internal struggle where the colonists have divided into factions. A leader of "moderates" is facing a capture and possibly trial. He and his female friend escape and find the ruins of a similar compound they live. Apparently due to problems in cold sleep they woke up without any memories. The old compound shows signs of a struggle. The story start without any background straight away in middle of action. At first that felt distracting, but it suited to the story- the protagonists themselves had woken in the middle of action without any memories. I am not sure if the factions would appear as fast and as rigid as in the story, but pretty good anyway. ***½
SHORT STORY: DAILY TEDS, Ron Collins
A guy discovers a way to send a copy of himself to the future. Soon there are a lot of copies with increasing deviating behavior compared to the original. And all copies are making more copies. That can only lead to serious problems. Light story about a possible end of the world. ***+
SHORT STORY: DOWN PLEASE: THE ONLY RECORDED ADVENTURE OF LARS FOUTON, CAPTAIN'S LIFT OPERATOR ON THE STARSHIP MAGNIFICENT, Adam-Troy Castro
A story about the elevator operator of the most advanced space ship of the alliance. He drives the personal elevator of the ship's commander and his guests. The elevator goes from the bridge to the personal and guest's quarters, nowhere else. Why would a super advanced and powerful spaceship have a lift which needs a person to operate it? Well, that IS the question. A pretty nice and fun story. Pretty bad job, though. ****-
SHORT STORY: PARTIBLE, K.J. Zimring
An anthropologist gets a notification that his son is on the custody of the immigration officials. That's kind of strange as he doesn't have a son. As matter of fact, he hasn't even had sex with the woman who is the mother of the child. He did know her and was in love with her, but sex - no. Especially surprising is when the DNA test turns out to be positive. A good and moving story. ***½
SHORT STORY: THE LAST DAYS OF DOGGER CITY, Mjke Wood
A city or large structure built on the coast of Norway (?) starts to fall into the ocean. The future is bleak, Yellowstone has apparently blown up and practically no communication is available from anywhere. A woman loses her son and must look for him in a falling building. Just an action scene. The more interesting parts of the story are missing. ***
NOVELETTE: DANCING IN THE DARK, Ramona Louise Wheeler
Comets are being chased for their volatiles in a solar system which has been colonized by humans a few hundred years ago. A group which is hunting for them discovers the largest ever. Its' trajectory seems strange; it is like something changed the orbit recently. The found that something seems to be eating comets. Is it a strange lifeform? Another story which feels like a third or fourth chapter ripped from a novel. ***
PROBABILITY ZERO: WHEY STATION, Guy Stewart
Usually I haven't reviewed the Probability Zero stories, as they are very short fillers. Usually they are fun but forgettable stories. This time – I haven't the slightest idea what happens in this story. I asked about it on Analog's Facebook page, but didn't get any answers. Could someone please explain to me WTF happens in this story?
3 comments:
- GuyStewart said...
Re: "Whey Station"
I should give you some background:
"Whey Station" is a play on words. Clifford D. Simak (who is a fellow Minnesotan gone these last 27 years. He was awarded 3 Hugos, a Nebula, and a SFWA Grandmaster as well as a Bram Stoker Award for Lifetime Achievement.
He's something of a personal hero of mine and after re-reading Way Station (1963) (In which a Civil War veteran is a caretaker of a secret Way Station, a transfer point for aliens. But the outside world is snooping around, and their blundering may endanger all of humanity. It takes place on a farm in Wisconsin and we spent a summer on a tiny farm in the same state), I had an idea for a really, really short story playing with a "what if?" In this case, I wondered what would have happened to such a farm if the caretaker died suddenly and his family quickly sold it -- without having any idea what it was? Ni and Rey bought this farm and in the process of renovating activate the TRANSFER POINT again.
I upgraded the ambient technology (chest freezers, flat screen displays, and plastic tables) and then had the screen activate with an incoming transfer.
At the beginning of Chapter 5 of Simak's book, WAY STATION, you'll find the message:
It's letting the caretaker of the station, Enoch Wallace, know exactly what to expect of the alien transferring through and what kind of preparations he should have.
My message:
wstat120254 2 whey-stat 18328 NatOnyxyfmII. N2checkon. 2LIQ-Tank. SOLN 27. DEP4 wstat31591 @615.5.05.00084.0000141. Rog?
wstat120254 2 = the station Rey and Ni purchased (secondary to the one presumed to have been in Wisconsin)
whey-stat 18328 = the end station (note: one number different from Simak's destination station)
NatOnyxyfmII = Native of Onyxyfm II (an alien people from another story I wrote)
N2checkon = two pieces of check on luggage
2LIQ-Tank = second-type of liquid tank (liquid nitrogen, if you wanted to know) necessary for the traveler
SOLN 27 = lifted it from Simak
DEP4 wstat31591 = Departing for whey (way) station 31591
@615.5.05.00084.0000141 = a time expressed in some sort of universal, intergalactic time; I thought it should be more complex than Simak did...
Rog? = 21st century US is a bit less formal than 1960s US, so "roger" instead of CONFIRM...
That's it. Hope it doesn't disappoint...I didn't mean for it to be opaque...
Maybe you can read something else I wrote -- not everything I do is so dependent on having read something else.
But thanks for asking! I'll keep this letter handy for the next person who asks...
Guy Stewart
- August 24, 2015 at 5:16 AM
- tpi said...
- This comment has been removed by the author.
- August 25, 2015 at 11:31 PM
- tpi said...
Thank you for you comment. I am familiar with Way Station. It is one of my favorite books, but it has been probably more than ten years I last read it and didn't remember those "codes". Also as non-English speaker I tend to be very bad at puns - especially in non-expected puns. When I read English I don't "sound out" the words in my mind. So "Whey" and "Way" are totally different words for me, even if they are pronounced in the same way. If I know that there is a pun, I must consciously sound out the words, and then I usually notice it. That sometime makes jokes hard to notice, but on the other hand I never, ever have to worry about how to pronounce words when I read, or find unpronounceable words (like for example "wrtxxd") hard to read. This probably comes from my native language, Finnish, which is written 99,9% phonetically. If you see a word, you always can pronounce it correctly (when I was small and really hadn't started on English yet, I read about "spelling bees" in American schools from a magazine. I remember I was very, very baffled. If you hear a word, why should you not be able to spell it?)
- August 25, 2015 at 11:34 PM


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Don't have enough monsters? Or just want to show off your painting skills. Why not try entering the Monster Mash! - Click here for more info.Dismiss Notice
Contest October-November 2017 Short Story Contest Entry thread (new entries closed)
Discussion in 'Fluff and Stories' started by Scalenex, Sep 22, 2017.
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