acute myocardial infarction ( mi ) remains a leading cause of morbidity and mortality worldwide . according to the latest report of the american heart association , every 25 seconds , an american will have a coronary event , and approximately every minute , someone will die of a coronary event . in 2010 , 785,000 americans experienced an mi , and approximately 470,000 had a recurring mi . heart failure can result from adverse remodeling of the collagenous scar that replaces the damaged myocardium in the left ventricle ( lv ) after mi . lv remodeling is mediated by cell survival , inflammation , angiogenesis , and turnover of the extracellular matrix ( ecm ) . markers of lv remodeling can be either determined in the circulation ( e.g. , serum or plasma ) or detected in the heart by imaging technologies or biopsy . post - mi , levels of specific matrix metalloproteinases ( mmps ) increase and mediate left ventricular remodeling . mmp-9 has been reported as a prognostic indicator of cardiac dysfunction in mi patients [ 2 , 3 ] . mmp-9 deletion has also been shown to improve remodeling of the lv in mice [ 4 , 5 ] . we hypothesized that the analysis of plasma proteins post - mi in wild - type ( wt ) and mmp-9 null mice will identify prospective markers of early mi that are mmp-9 dependent . termed as the most complex proteome , plasma is an intricate body fluid , containing a wide diversity of proteins . plasma has been investigated using targeted evaluations , to measure markers that detect mi or predict outcomes following mi . for examples , the muscle form of creatine kinase ( ck - mb ) , troponins , and c - reactive protein are used clinically to determine both presence of mi and extent of myocardial damage [ 7 , 8 ] . mmp-9 , galectin-3 , and brain natriuretic peptide have been used to evaluate lv responses to mi [ 911 ] . plasma has also been investigated using proteomic approaches , but this has been fraught with technical issues , primarily because the range of protein levels in the plasma is 10 , and the ten most abundant proteins account for 90% of the total protein concentration [ 12 , 13 ] . serum albumin is a high abundant protein in plasma , and it is the leading candidate for selective removal prior to proteomics analysis of less abundant proteins in plasma . albumin can also be removed by ligand chromatography [ 14 , 15 ] , and isoelectric trapping . nonetheless , the use of depletion methods may also result in specific removal of low abundant cytokines , lipoproteins , and peptide hormones of interest . accordingly , we hypothesized that using a fractionation protocol for the analysis of plasma proteins post - mi in wild - type ( wt ) and mmp-9 null mice would identify prospective markers of early mi that are mmp-9 dependent . in our study , we performed protein fractionation prior to protein separation by 1d - page and ms analysis . by doing so , we avoided using depletion methods and concomitantly reduced the presence of albumin and enriched for lower abundance proteins . all animal procedures were conducted according to the   guide for the care and use of laboratory animals  ( nih notice number : not - od-12 - 020 ) and were approved by the institutional animal care and use committee at the university of texas at san antonio . male 48 months old c57bl6/j wild - type ( wt ) ( n = 3 ) and mmp-9 null mice ( n = 3 ) were used in this study . animals were housed at constant temperature ( 22  2c ) on a 12  h light / dark cycle . they were fed ad libitum on standard laboratory mice chow and had free access to tap water . mi was made by permanent ligation of the left anterior descending coronary artery as described previously . animals without mi ( day 0 ) were used as controls ( n = 3/genotype ) . at one day post - mi , mice were anesthetized with 5% isoflurane , plasma was collected , the coronary vasculature was flushed with 0.9  m saline , and the hearts were excised . the hearts were separated between right and left ventricles and were stained with 1% 2,3,5-triphenyltetrazolium chloride ( sigma ) and photographed for measurement of infarct area . plasma was collected at days 0 and 1 post - mi , snap frozen and stored at 80c . at sacrifice , heparin ( 100  l of 1000  usp  units / ml ) was injected intraperitoneally , and 5  min after heparin injection , blood was collected from the carotid artery of the mouse . plasma was fractionated using the gellfree 8100 fractionation system ( protein discovery , inc . ) . five hundred micrograms of total protein were reduced for 10  min at 50c with 1x acetate sample buffer ( protein discovery , inc . ) and 0.053  m dithiothreitol ( dtt ) . after samples being cooled down to room temperature , 15  mm iodoacetamide was added , and samples were alkylated in the dark for 10  min .   for protocol optimization , we used six different protocols where samples were either run in an 8% , 10% , or 12% tris - acetate cartridge combined with one of three fractionation programs . we tested three different fractionation programs shown in figures 2 , 3 , and 4 . for all of the programs , mes was used as the running buffer ( 0.05  m  mes , 0.05  m  tris , 0.1%  sds  ph 7.9 ) . for each of the six protocols tested , twelve fractions ( 150  l / fraction ) were collected per sample and proteins were visualized on a 12% bis - tris gel by sds - page . the proteins in fraction 1 were separated in a 1020%  tricine / peptide gel . the gel region containing visually detectable proteins from the lane for fraction 12 ( the highest molecular weight fraction ) on the bis - tris gel was excised into three slices . each slice was separately destained and dehydrated and the proteins digested in situ with trypsin ( promega ) . the digests were analyzed by capillary hplc - electrospray ionization tandem mass spectrometry ( hplc - esi - ms / ms ) on a thermo fisher ltq orbitrap velos mass spectrometer fitted with a new objective digital picoview 550 nanoesi source . online hplc separation of the digests was accomplished with an eksigent / ab sciex nanolc - ultra 2-d hplc system : column , picofrit ( new objective ; 75  m i.d . ) packed to 15  cm with c18 adsorbent ( vydac ; 218ms 5  m , 300   ) . precursor ions were acquired in the orbitrap in profile mode at 60,000 resolution ( m / z  400 ) ; data - dependent collision - induced dissociation ( cid ) spectra of the six most intense ions in the precursor scan above a set threshold were acquired at the same time in the linear trap . mascot ( versions 2.3.02 ; matrix science ) was used to search the uninterpreted cid spectra against a combination of the mouse subset of the ncbinr database ( mus . ( 145,083 sequences ) ) and a database of common contaminants ( 179 sequences ) . methionine oxidation was considered as a variable modification ; trypsin was specified as the proteolytic enzyme , with one missed cleavage allowed . a secondary search of the cid spectra using x ! tandem and mascot results , and determination of protein and peptide identity probabilities were accomplished by scaffold ( version 3 ; proteome software ) . the thresholds for acceptance of peptide and protein assignments in scaffold were 95% and 99.9% , respectively . total proteins ( 10  g ) were loaded onto either 412% bis - tris gels ( proteins > 50  kda ) or 1020%  tricine gels ( proteins < 50  kda ) and run by sds - page . proteins were transferred to a nitrocellulose membrane which was hybridized overnight at 4c with primary antibody . primary antibodies used were antiserum amyloid a1 ( number af2948 , r&d ) , anti - alpha-2 macroglobulin ( number ab52651 , abcam ) and antineutrophil - associated gelatinase lipocalin ( ngal , aka lipocalin 2 ; number ab63929 , abcam ) ( number ab63929 , abcam ) . immunoblot intensities ( arbitrary units ) were assessed using a one - way anova with newman - keuls multiple comparison test . infarct areas were similar between wt and mmp-9 null mice ( p = 0.85 ; figure 1 ) , indicating that both groups received a similar injury stimulus . the different fractionation programs are shown in the figures . when using fractionation program number 1 and an 8% acetate cartridge , proteins were only observed in fractions 10 to 12 ( figure 2 ) . by changing voltage intensities and step duration the protein ' profiles differed depending of the type of cartridge used ( figure 3 ) . since serum albumin is approximately 66  kda , we focused on protocols that provided fractions with reduced albumin content . the combination of program number 3 with the 8% acetate cartridge yielded fractions with these characteristics , where most of the albumin was seen in fractions 2 to 11 ( figure 4 ) . these conditions were considered optimal for our examination , and fractions 1 and 12 , which showed reduced levels of albumin , were further analyzed by hplc - esi - ms / ms on an orbitrap velos . supplemental tables  1 and  2 list the proteins identified in both fractions ( see supplementary material available online at doi:10.1155/2012/397103 ) , per genotype and time point . of the 145 proteins identified in the wt mice , 12 proteins were present only at day 0 , and 45 proteins were just observed 1 day post - mi ( figure 5(a ) ) . in the mmp-9 null mice , 195 proteins were identified ; of which 19 were unique to day 0 and 61 proteins were observed only post - mi ( figure 5(b ) ) . the molecular weight of proteins observed in fraction 1 ranged from 7  kda to 69  kda , although fragments of higher molecular weight proteins ( e.g. , c - terminus of alpha-2 macroglobulin ) were also present . the majority of proteins observed in fraction 12 had molecular weights ranging from 45  kda to 263  kda ; nevertheless , lower molecular weight proteins such as transthyretin ( 16  kda ) were also observed . the unweighted spectrum counts were used to provide measure of relative abundance ( figure 6 ) . two percent of the proteins identified in wt animals were ecm proteins , while ecm proteins accounted for 3% of the total identified proteins in mmp-9 null mice . serum amyloid a ( saa ) , a marker of inflammation , was observed at both time points . saa was identified in fraction 1 of both genotypes and levels at day 0 were significantly different ( p < 0.001 ) from levels at day 1 post - mi ( figure 7(a ) ) . ngal levels post - mi were significantly higher than at day 0 ( p < 0.05 ) but no differences were observed between genotypes ( figure 7(b ) ) . alpha-2 macroglobulin , a generic mmp inhibitor and an mmp-9 substrate , was observed only post - mi in the wt group . nevertheless , alpha-2 macroglobulin precursor was observed in both groups at days 0 and 1 post - mi . quantification of alpha-2 macroglobulin showed a significant difference between genotypes 1 day post - mi ( figure 7(c ) ) . the discovery of plasma markers remains challenging due to the complexity of the samples and the wide range of protein concentrations . in addition , the analysis of proteomics data is a complex multistep process . therefore , to overcome these problems , effective sample preparation is of outmost importance . efficient sample preparation will reduce component complexity and enrich for lower abundance proteins while depleting or reducing the most abundant ones . we used a novel fractionation technique to interrogate plasma from wt and mmp-9 null mice at day 1 post - mi . this novel technique provides all of the advantages of 1-d gel electrophoresis , with the additional benefits of increased loading capacity and high yield liquid phase recovery . the most significant findings of this study were that ( 1 ) multiple proteins were identified in post - mi plasma , compared with day 0 control plasma ; ( 2 ) serum amyloid a is good marker of early mi but it is not mmp-9 dependent ; ( 3 ) alpha-2 macroglobulin may be an mmp-9 dependent marker . these results combined indicate that a fractionation followed by 1-d  gel / lc / ms analysis strategy is effective to isolate and identify plasma proteins changes in response to mi . we identified a total of 145 unique proteins in the wt samples and 195 unique proteins in the mmp-9 null samples . known markers of inflammation , such as haptoglobin and saa , were among the proteins identified . studies from the malm preventive study , sweden , have shown that elevated plasma levels of haptoglobin are a risk factor for mi . recently , devaux 's group identified haptoglobin as a potential biomarker of prognosis of heart failure in patients with acute mi . interestingly , they state that low levels of haptoglobin early post - mi favor heart failure . we identified haptoglobin as a potentially increased post - mi marker ; nevertheless , studies with longer time points post - mi will have to be developed to confirm the role of haptoglobin in progression to heart failure . saa is a known marker of inflammation , and saa levels inversely correlate with cardiac function . one - day post - mi the levels of saa were significantly higher , in both genotypes , confirming an association with mi . we plan to investigate if changes in plasma saa are correlated with progression to heart failure post - mi . from the proteins present only post - mi ngal is a marker of kidney injury , as well as matrix degradation and inflammation . this protein has previously been reported to be associated with mi and heart failure [ 24 , 25 ] . a recent paper by akcay and colleagues shows that 1-year mortality rates were significantly higher in patients with high levels of ngal . our results were in accordance with the previous reports , showing a robust increase in ngal levels post - mi . although the association between mmp-9 and alpha-2 macroglobulin has been previously reported , this is the first time that alpha-2 macroglobulin is associated with mmp-9 in the myocardial infarction setting . the higher levels of alpha-2 macroglobulin observed in the mmp-9 mice post - mi suggest that this protein may be an mi biomarker that is mmp-9 dependent . the protocols developed in this study can be used for other biological samples besides plasma . we are currently developing a fractionation method to investigate secreted proteins in cell culture media . like in plasma , albumin is highly abundant in the commercially available serums used to supplement culture media . in vitro , the levels of secreted proteins are very low compared to the values observed in culture serum , making it very difficult to identify and quantify the proteins produced by the cells . fractionation of samples is an easy and reproducible technique that can be used in a variety of models and biological samples . mouse models of mi are very useful and important given the unique ability to genetically manipulate these animals . however , it is important to remember that the mi mouse model does not fully mimic the human disease . thus , postinfarct remodeling of the lv likely has differences between the mouse and human that will need to be taken into account before full translation can occur . thus , the discovery and development of biomarkers has high potential for providing a real benefit for screening , diagnosis , prognosis , prediction of recurrence , and therapeutic monitoring of mi patients . in conclusion , by performing plasma fractionation prior to proteomics analysis , we were able to reduce the presence of high abundant proteins , such as albumin , and enrich samples for the detection of lower abundance proteins . we compared plasma samples from wild - type and mmp-9 null mice post - mi , and identified alpha-2 macroglobulin as a prospective mi marker which may be mmp-9 dependent . this technical report revealed that a fractionation approach is a useful technique to evaluate plasma samples .
following myocardial infarction ( mi ) , matrix metalloproteinase-9 ( mmp-9 ) levels increase , and mmp-9 deletion improves post - mi remodeling of the left ventricle ( lv ) . 
 we provide here a technical report on plasma - analysis from wild type ( wt ) and mmp-9 null mice using fractionation and mass - spectrometry - based proteomics . 
 mi was induced by coronary artery ligation in male wt and mmp-9 null mice ( 48 months old ; n = 3/genotype ) . 
 plasma was collected on days 0 ( pre- ) and 1 post - mi . 
 plasma proteins were fractionated and proteins in the lowest ( fraction 1 ) and highest ( fraction 12 ) molecular weight fractions were separated by 1-d sds - page , digested in - gel with trypsin and analyzed by hplc - esi - ms / ms on an orbitrap velos . 
 we tried five different fractionation protocols , before reaching an optimized protocol that allowed us to identify over 100 proteins . 
 serum amyloid a substantially increased post - mi in both genotypes , while alpha-2 macroglobulin increased only in the null samples . in fraction 12 
 , extracellular matrix proteins were observed only post - mi . 
 interestingly , fibronectin-1 , a substrate of mmp-9 , was identified at both day 0 and day 1 post - mi in the mmp-9 null mice but was only identified post - mi in the wt mice . in conclusion , plasma fractionation offers an improved depletion - free method to evaluate plasma changes following mi .