mass 
 spectrometry ( ms ) is a powerful tool to assess the relative abundance 
 of proteins among biological samples . numerous methodologies now support 
 relative quantification measurements , providing a routine means to 
 analyze protein expression patterns and post - translational modification 
 states as a function of biological perturbation . one of the most popular 
 methods for relative quantification through ms is stable isotope labeling 
 of proteins in samples prior to analysis . labeling can be achieved 
 by the application of combinatorial heavy isotopologues of c , h , n , 
 and o and can be introduced in proteins either by metabolic means 
 or through chemical derivatization processes . in vivo metabolic labeling 
 approaches include techniques such as stable isotope labeling in mammals 
 ( silam ) , stable isotope labeling by amino 
 acids in cell culture ( silac ) , and neucode 
 ( neutron encoding ) silac . the in vitro 
 chemical derivatization processes include techniques such as isotope - coded 
 affinity tags ( icat ) , dimethyl labeling , isobaric mass tags , and others . with the exception of isobaric mass tags , stable isotope derivatization 
 methods introduce a small mass difference to identical peptides from 
 two or more samples so that they can be distinguished in the ms1 spectrum . the relative - abundance ratio of peptides is experimentally measured 
 by comparing heavy / light peptide pairs , and then protein levels are 
 inferred from statistical evaluation of the peptide ratios . isobaric 
 tags , on the other hand , use a different concept for peptide quantification . 
 in isobaric labeling - based quantification , each sample is derivatized 
 with a different isotopic variant of an isobaric mass tag from a set , 
 and then the samples are pooled and analyzed simultaneously in ms . since the tags are isobaric , peptides labeled with isotopic variants 
 of the tag appear as a single composite peak at the same m / z value in an ms1 scan with identical liquid chromatography 
 ( lc ) retention time . the fragmentation of the modified precursor ion 
 during ms / ms event generates two types of product ions : ( a ) reporter 
 ion peaks and ( b ) peptide fragment ion peaks . the quantification is 
 accomplished by directly correlating the relative intensity of reporter 
 ions to that of the peptide selected for ms / ms fragmentation . the 
 fragment ion peaks observed at higher m / z are specific for peptide amino acid sequence and are used for peptide 
 identifications , which are eventually assigned to the proteins that 
 they represent . since every tryptic peptide can be labeled in an isobaric 
 labeling method , more than one peptide representing the same protein 
 may be identified , thereby increasing the confidence in both the identification 
 and quantification of the protein . this technology has proved to be 
 successful in numerous experimental contexts for comparative analysis 
 upon perturbation . the protocol 
 involves extraction of proteins from cells or tissues followed by 
 reduction , alkylation , and digestion . in the case of tmt 6-plex , up to six samples can be labeled with the six isobaric tags of the reagent . ( b ) in an ms1 scan , same - sequence peptides from the different samples 
 appear as a single unresolved additive precursor ion . following fragmentation 
 of the precursor ion during ms / ms , the six reporter ions appear as 
 distinct masses between m / z 126131 , 
 and the remainder of the sequence - informative b- and y - ions remains 
 as additive isobaric signals . the reporter ion intensity indicates 
 the relative amount of peptide in the mixture that was labeled with 
 the corresponding reagent . isobaric mass tags include 
 families of stable isotope chemicals 
 that are used for labeling of peptides . they generate relative quantitative 
 information in an isobaric labeling - based quantification strategy . isobaric mass tags have identical overall mass but vary in terms of 
 the distribution of heavy isotopes around their structure . the most 
 common isobaric tag is amine - reactive , but tags that react with cysteine 
 residues and carbonyl groups in proteins are also available . the amine 
 specificity of the amine - reactive isobaric mass tags makes most peptides 
 in a sample amenable to this labeling strategy . the tags employ n - hydroxysuccinimide ( nhs ) chemistry , and the structure 
 consists of three functional groups : an amine - reactive group and an 
 isotopic reporter group ( n - methylpiperazine ) linked 
 by an isotopic balancer group ( carbonyl ) for the normalization of 
 the total mass of the tags . the amine - reactive , nhs - ester - activated 
 group reacts with n - terminal amine groups and -amine groups 
 of lysine residues to attach the tags to the peptides . the labeling 
 is efficient for all peptides regardless of protein sequence or proteolytic 
 enzyme specificity . the labeling does not occur , however , if the primary 
 amino groups are modified , such as when n - terminal glutamine or glutamic 
 acid forms a ring ( pyro - glutamic acid ) or if the group is acetylated . 
 the nhs - based isobaric tags may lead to acylation of side chain hydroxyl 
 group of serine , threonine , and tyrosine residues under reaction conditions 
 normally employed for the acylation of primary amines . for successful quantification , labeling should be specific 
 to the targeted residues ( n - terminal amine and lysyl -amine 
 groups in a peptide ) and should proceed to completion . reversal of 
 peptide o - acylation reactions can be achieved by treatment with hydroxylamine 
 that has no disruptive effect on acyl modifications on primary amines . the mass normalization group balances the 
 mass difference among 
 the reporter ion groups so that different isotopic variants of the 
 tag have the same mass . the overall mass of reporter and balance components 
 of the molecule are kept constant using differential isotopic enrichment 
 with c , n , and o atoms . the 
 relative intensities of the reporter ion are used to derive quantitative 
 information on the labeled peptides between the samples . figure 2 shows chemical structure of commercially available 
 isobaric mass tags : tandem mass tag ( tmt ) and isobaric tags for relative 
 and absolute quantification ( itraq ) . the complete molecule consists 
 of a reporter group ( based 
 on n - methylpiperazine ) , a mass balance group ( carbonyl ) , 
 and a peptide - reactive group ( nhs ester ) . the overall mass of the 
 reporter and balance components of the molecule are kept constant 
 using differential isotopic enrichment with c , n , and o atoms . the reporter group ranges in mass from m / z 114117 , whereas the balance 
 group ranges in mass from 28 to 31 da , such that the combined mass 
 remains constant ( 145 da ) for each of the four reagents of the itraq 
 4-plex set . ( ii ) the tag reacts with peptide n - terminus or -amino 
 group of lysine to form an amide linkage that fragments in a similar 
 fashion to that of backbone peptide bonds when subjected to cid . following 
 fragmentation of the tag amide bond , the balance ( carbonyl ) moiety 
 is lost as neutral loss , whereas charge is retained by the reporter 
 group . the number in parentheses in the table indicates the number 
 of enriched centers in each section of the molecule . ( b ) chemical structure of a generic tmt reagent showing 
 the three functional groups : an amine - reactive group that labels the 
 n - terminus and -amine group of lysine in peptides , a mass normalization 
 ( balance ) group that balances mass differences from individual reporter 
 ions to ensure the same overall mass of the reagents , and a reporter 
 group that provides the abundance of a peptide upon ms / ms in individual 
 samples being mixed . the blue dashed lines indicate a cleavable linker 
 that enables the release of the reporter ion from the whole tag upon 
 ms / ms . the tmt reagent family consists of tmtzero , tmtduplex , tmt 
 6-plex , and tmt 10-plex sets , and each of them is based on the same 
 chemical structure . the application of isobaric tags 
 for simultaneous determination of 
 both the identity and relative abundance of peptide pairs was first 
 demonstrated by thompson et al . in 2003 . they synthesized peptides containing a tandem mass tag and showed 
 that this strategy could be used to obtain relative quantification 
 in ms / ms experiment . a year later , ross et al . published a similar 
 approach using the itraq approach . in 
 this study , they demonstrated for the first time the application of 
 isobaric mass tags with 4-fold multiplexing to identify global protein 
 expression trends in a set of isogenic yeast strains . an 8-plex series 
 of itraq reagent performs similarly and increases throughput of analyses 
 by a factor of 2 when compared to that of the 4-plex approach . a few year later , dayon et al . showed the increased multiplexing capability of tmt tags 
 and demonstrated its application by using 6-plex tmt reagents in relative 
 quantification of standard protein mixtures at various concentrations . 
 in this study , tmt 6-plex was also used to assess the differential 
 protein abundance in post - mortem cerebrospinal fluid samples after 
 brain injury vs antemortem samples . isobaric reagents are commercially available through vendors such 
 as ab sciex ( framingham , ma , usa ) and thermo scientific ( rockford , 
 il , usa ) . the itraq reagents available from ab sciex are set of 4-plex 
 and 8-plex mass tags that can be used to label and derive quantitative 
 information on up to four and eight different biological samples simultaneously . the 4-plex itraq reagents have reporter ion masses at m / z 114117 and a corresponding balancer group 
 added to accommodate the extra isotopes has masses of 2831 
 da such that they sum to about 145 da . the 8-plex reagents have reporter 
 ion masses at m / z 113119 
 and 121 with a balance group ranging from 2431 da . mass 120 
 is omitted in itraq 8-plex to avoid contamination from phenylalanine 
 immonium ion ( m / z 120.08 ) . thermo 
 scientific tmt reagents , available as tmtzero , tmt duplex , tmt 6-plex , 
 and tmt 10-plex , share an identical structure with each other but 
 contain different numbers and combinations of c and n isotopes in the mass reporter region . the identical structure 
 of tmt reagents facilitates efficient transition from method development 
 using tmtzero or tmt duplex to multiplex quantification using tmt 
 6-plex or tmt 10-plex . the chemical structure of the tmt tag enables 
 the introduction of five heavy isotopes ( c or n ) in the reporter group and five heavy isotopes ( c 
 or n ) in the balancer group to provide six isobaric tags 
 ( figure 3a ) . each of the six tags of tmt 6-plex 
 has a specific reporter ion that appears at m / z 126 , 127 , 128 , 129 , 130 , and 131 . tmt 10-plex is an expansion 
 of tmt 6-plex generated by combining current tmt 6-plex reagents with 
 four isotope variants of the tag with 6.32 mda mass differences between n and c isotopes . even though 
 the mass difference between these reporter ion isotopologues is incredibly 
 small , current generation high - resolution and high mass accuracy analyzers 
 can resolve these ions . the seemingly miniscule difference is sufficient 
 to achieve baseline resolution between the reporter ions when high 
 resolving power is employed ( 30 k at m / z 400 ) . figure 3b shows the substitution of n for c to 
 generate new reporter ions that are lighter than the original forms 
 used in tmt 6-plex . in cases where coalescence , fusion of the proximate 
 reporter ion signals into a single measurable entity , phenomenon is 
 observed , the artifact can be completely eliminated by lowering the 
 maximum ion target for ms / ms spectra . this modified setting does not result in any losses in identification 
 depth or quantification quality of proteins . the high - throughput tmt 10-plex reagent enables concurrent ms analysis 
 and relative quantification of up to 10 different samples derived 
 from cells , tissues , or biological fluids . the higher multiplexing 
 potential also facilitates incorporation of replicates , providing 
 additional statistical validation within any given isobaric labeling 
 experiments . ( a ) chemical structure 
 of tmt 6-plex reagents with c and n heavy 
 isotope positions ( blue asterisks ) . the 
 tags are isobaric , with a different distribution of isotopes between 
 the reporter and mass normalization ( balance ) groups . ( b ) the substitution 
 of n for c to generate new reporter ions 
 that are 6.32 mda lighter than the original forms used in tmt 6-plex . the tmt 6-plex reagents in combination with 
 four isotope variants of the tag with 6.32 mda mass differences were 
 used to generate tmt 10-plex reagent . the numbers of identified peptides and proteins in shotgun 
 proteomics 
 experiments have been compared for the three commercially available 
 isobaric mass tags : itraq 4-plex , tmt 6-plex , and itraq 8-plex . even though the number of identified proteins 
 and peptides was largest with itraq 4-plex , followed by tmt 6-plex , 
 and smallest with itraq 8-plex , the precision on the level of peptide the discrepancy 
 in peptide identification observed with different n - plex isobaric mass tags was suggested to be due to combination of 
 several factors , such as search algorithms and scoring functions , 
 fragment ions derived from cleavage of the label itself or within 
 the label from precursor ions , or disparate physiochemical properties 
 conferred to the peptides depending on the type of isobaric mass tags 
 used for their derivatization . however , 
 in a study by pottiez et al . on comparison of quantitative measurements 
 of proteins in human plasma samples by itraq 4-plex versus 8-plex 
 reagents , 8-plex tagging provided more consistent ratios than that 
 with 4-plex without compromising protein identification . the discrepancies in observations from pichler 
 et al . and pottiez et al . could be due to different instruments ( ltq 
 orbitrap versus mald - tof / tof 4800 platform ) and search algorithms 
 ( mascot and proteome discoverer software versus proteinpilot 4.0 with 
 paragon algorithm ) that were used for the data acquisition and analysis . nevertheless , the obvious advantage of 8-plex tagging is that it 
 allows investigation of eight experimental conditions in one analytical 
 experiment . for example , a study of one control and seven experimental 
 conditions can be performed in one 8-plex experiment but would require 
 at least three 4-plex experiments ( using the control and up to three 
 experimental samples in each ) . the three 4-plex experiments would 
 need more instrument time , likely introducing a source of variability , 
 and would be more laborious . n , n - dimethyl 
 leucine ( dileu ) is 
 an isobaric tandem mass tagging reagent that uses isotope - encoded 
 dimethylated leucine as reporters and serves as attractive alternative 
 for itraq and tmt . labeling with dileu , 
 however , requires activation of the reagents using 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium 
 chloride ( dmtmm)/n - methylmorpholine ( nmm ) in n , n - dimethylformamide ( dmf ) . the general structure of dileu 
 resembles that of other isobaric mass tags , with an amine - reactive 
 group ( triazine ester ) targeting the n - terminus and -amino 
 group of the lysine side chain of a peptide , a balance group , and 
 a reporter group . a mass shift of 145.1 
 da is observed for each incorporated label . by using dileu isobaric 
 tags , however , dileu - labeled peptides undergo better 
 fragmentation and hence generate higher reporter ion intensities than 
 itraq , thereby offering improved confidence for peptide identification 
 and more reliable quantification . intense 
 reporter ions ( dimethylated leucine a1 ion ) at m / z 115 , 116 , 117 , and 118 are observed for all pooled samples 
 upon ms / ms . even though deuterium affects the retention time of small- 
 to intermediate - sized peptides in reversed - phase chromatography , the increased polarity of the amine group offsets 
 the small deuterium number difference in 4-plex dileu tags . ( a ) ( i ) general structure of dimethyl leucine 
 isobaric ( dileu ) 
 mass tag . ( ii ) illustration of 
 formation of new peptide bond at n - terminus or -amino group 
 of the lysine side chain and isotope combination of isobaric tags 
 ( b ) chemical structure of diart isobaric reagents . positions containing heavy stable isotopes are illustrated 
 as numbers in the structure , and the table lists the elemental composition 
 of the corresponding numbers . during ms / ms , the diart - tagged peptides 
 yield reporter ions ranging from m / z 114 to 119 . deuterium isobaric amine 
 reactive tag ( diart ) is another alternative 
 to itraq and tmt for isobaric tagging in quantitative proteomics . like itraq or tmt , diart reagents have three functional groups , 
 an amine - reactive ( nhs ester ) group for coupling of peptides , a balancer , 
 and a reporter ( n , n-dimethylleucine ) 
 with a m / z range of 114119 
 ( figure 4b ) . up to six samples can be labeled 
 with diart reagents and analyzed by ms . diart reagents have high isotope purity ; hence , unlike that for 
 itraq , tmt , or dileu labeling , isotopic impurities correction is not 
 required during data analysis of diart - labeled samples . the performances of diart and itraq , including 
 their fragmentation mechanisms , the number of identified proteins , 
 and the accuracy of quantification , have been compared . regardless of the peptide sequence , diart tags 
 generate high - intensity reporter ions compared to those with itraq . since quantification accuracy is dependent on the intensity of reporter 
 ions , as high - intensity reporter ions 
 are less susceptible to underestimation effect , diart labeling quantifies more peptides , including low - abundance 
 ones , and with reliable results . while 
 dileu uses a nontraditional activation chemistry ( dmtmm / nmm in dmf ) 
 to label peptides , diart uses the same labeling 
 protocol ( nhs - ester - based peptide coupling chemistry ) as that of tmt 
 and itraq , making it easy for users to switch between the techniques . however , unlike that for itraq or tmt , diart - labeled samples can not 
 be analyzed by the hcd - only instrument method due to easy fragmentation 
 of its reporter ions . nevertheless , diart 
 and dileu serve as a cost - effective alternatives to tmt and itraq 
 with comparable labeling efficiency . diart has been shown to be useful 
 in labeling large quantities of proteins from cell lysates prior to 
 tio2 enrichment in quantitative phosphoproteomics study . isobaric labeling - based quantification 
 can also be used for differential quantification of various protein 
 post - translational modifications . isobaric mass tags are available 
 that are especially designed to measure relative abundance of modified 
 cysteine residues or carbonylated residues in protein . carbonylation of proteins is caused 
 by the reactive oxygen and carbonyl species generated as byproducts 
 of lipid oxidation during oxidative stress . itraq hydrazide ( itraqh ) is a novel reagent for the selective labeling 
 and relative quantitative analysis of carbonyl groups in proteins . itraqh was synthesized from itraq and an excess 
 of hydrazine ( figure 5a ) . itraqh reacts with 
 a carbonylated peptide , resulting in the formation of a hydrazone 
 moiety . consistent with the isobaric labeling approach , peptides labeled 
 with different isotopic variants of itraqh reagents are indistinguishable 
 in ms scan . however , the itraqh reporter ions in the low m / z region of the ms / ms spectrum provide the relative 
 abundance information on the carbonylated proteins in the samples . the itraqh reporter ions have been used as targets for precursor selection 
 in precursor ion scan analysis , which allows selective acquisition 
 of ms / ms spectra of only the carbonylated peptides . this eliminates the need for the step involving enrichment 
 of modified peptides prior to lc ( a ) general 
 structure of itraq hydrazide ( itraqh ) for relative 
 quantitative analysis of carbonylation sites in proteins . ( b ) chemical structure of the carbonyl - reactive 
 glyco - tmt compounds . red asterisks indicate c , and blue asterisks , n. the table below the compound 
 structures shows isotope codes of the hydrazide- and aminoxy - functionalized 
 glyco - tmt compounds . the carbonyl - reactive tags can be used to quantify 
 a broad range of biologically important molecules including carbohydrates , 
 steroids , or oxidized proteins . ( c ) chemical structure of the cysteine - reactive 
 thermo scientific iodotmtzero isobaric mass tag . the iodotmt reagents 
 are iodoacetyl - activated isobaric mass tags for covalent , irreversible 
 labeling of sulfhydryl ( -sh ) groups . iodotmt 6-plex enable measurement 
 of protein and peptide cysteine modifications ( s - nitrosylation , oxidation , 
 and disulfide bridges ) by multiplex quantitative mass spectrometry . the workflow ( not shown in the image ) involves derivatization of modified 
 peptides or proteins with the reagent , enrichment of tmt tagged peptide 
 using anti - tmt antibody , and their subsequent elution . ms / ms to determine the sites of modification 
 and to measure their relative abundance across samples . on the basis of similar chemistry as that of itraqh , 
 the stable 
 isotope - labeled carbonyl - reactive tandem mass tags ( glyco - tmts ) have 
 been used for quantification of n - linked glycans . glyco - tmt reagents are derivatives of the original tmt 
 compounds but are functionalized with carbonyl - reactive groups involving 
 either hydrazide chemistry or aminoxy chemistry ( figure 5b ) . a study reported that aminooxy tmts outperformed their 
 hydrazide counterparts in labeling efficiency and quantification . the glyco - tmt compounds are coded with stable 
 isotopes and enable ( i ) isobaric quantification in ms / ms spectra and 
 ( ii ) quantification in ms1 spectra using heavy / light pairs . isobaric 
 quantification using glyco - tmt can be achieved by using the aminoxy 
 tmt-128 and tmt-131 as well as the hydrazide 
 tmt-126 and tmt-127 reagents ( figure 5b ) . the ms1 level quantification is accomplished 
 by the mass difference of 5.0105 da between the light tmt and the heavy tmt reagents ( figure 5b ) that is sufficient to separate the isotopic patterns of 
 all commonly existing n - glycans . glycan quantification using heavy 
 and light glycol - tmts provided more accurate quantification in ms1 
 spectra over a broad dynamic range compared with that from quantification 
 based on the reporter ions generated in ms / ms spectra . glyco - tmts with aminooxy - functionalized groups 
 are available commercially from thermo scientific ( rockford , il , usa ) 
 as aminoxytmtzero and aminoxytmt 6-plex reagents . labeling with aminoxytmt 
 reagents involves treating intact proteins or proteolytic digests 
 of proteins extracted from biological specimens with pngase f / a glycosidases 
 to release n - linked glycans . the free glycans are subsequently purified 
 from protein or peptide matrix and labeled at the reducing end with 
 the aminoxytmt reagents . the derivatized glycans from individual samples 
 are then combined and analyzed in ms to identify glycoforms in the 
 sample and to quantify reporter ion relative abundance at ms / ms level . cysteine sulfhydryls in proteins are potential sites of reversible 
 oxidative modification because of the unique redox chemistry of this 
 amino acid . s - nitrosylation is a redox - based 
 protein post - translational modification that occurs in response to 
 nitric oxide signaling and is involved in a wide range of biological 
 processes . it involves addition of a 
 nitric oxide ( no ) group to a specific cysteine residue of a protein 
 to form s - nitrosothiol . an analytical strategy to enrich and relatively 
 quantify cysteine - containing peptides in complex mixtures has been 
 reported . in this strategy , cysteine 
 residues in proteins are first derivatized with n-{2-((2-acryloyl)amino)ethyl-1,3 thiazolidine-4-carboxamide ) ( atc ) 
 followed by labeling with amine - reactive tmt tags for relative quantification 
 of the targeted peptides after the covalent capture . the workflow 
 involves reduction , derivatization of cysteine residue in protein 
 samples with atc tag , digestion with trypsin , and differential labeling 
 with tmt tags followed by pooling of the labeled samples . the atc - derivatized 
 cysteinyl peptides are subsequently isolated on an aldehyde resin 
 through the covalent capture technique and analyzed with lc the cysteine - reactive tmt reagents allow measurement of s - nitrosylation 
 occupancy and determination of individual protein thiol reactivity . however , the disulfide linkage between the ( reversible ) cysteine - reactive 
 tmt tag and protein thiol group can not survive the strong reducing 
 conditions normally used during enzymatic digestion for subsequent 
 shotgun proteomic analysis . an irreversible 
 cysteine - reactive tmt reagent containing a sulfhydryl - reactive iodoacetyl 
 reactive group called iodotmt has been developed . iodotmt reagents such as iodotmtzero and iodotmt 6-plex 
 are commercially available from thermo scientific ( rockford , il , usa ) . each isobaric iodotmt 6-plex reagent within a set has the same nominal 
 mass and consists of a thiol - reactive iodoacetyl functional group 
 for covalent and irreversible labeling of cysteine , a balancer , and 
 a reporter group . the quantification using iodotmt tags is achieved 
 by inspection of the reporter ion region in ms / ms spectra . an iodotmt switch assay uses an isobaric set of thiol - reactive 
 iodotmt 6-plex reagents to specifically detect and quantify protein 
 s - nitrosylation . the iodotmt switch assay workflow 
 includes irreversible labeling of s - nitrosylated cysteines followed 
 by enrichment of s - nitrosylated peptides using high - affinity anti - tmt 
 chromatography with competitive elution and finally multiplexed quantification 
 of protein s - nitrosylation via six unique tmt reporter ions . isobaric labeling - based quantification has many 
 advantages compared 
 to other stable isotope labeling techniques , one of which is the ability 
 to perform high - throughput quantification due to sample multiplexing . 
 the ability to combine and analyze several samples within one experiment 
 eliminates the need to compare multiple lc ms / ms data sets , 
 thereby reducing overall analytical time and run - to - run variation . 
 moreover , the information replication within lc ms / ms experimental 
 regimes provides additional statistical validation within any given 
 experiment . this is desirable in an analysis 
 where conventional upregulation and downregulation measurements are 
 not nearly as meaningful as obtaining temporal expression patterns 
 of proteins throughout the experimental condition , such as in studies 
 involving different stages of cell differentiation , comparisons of 
 multiple drug treatments , identifications of protein  drug interactions , measurement of inhibitor dose response , or time 
 course comparisons . when each sample 
 is run separately or with limited multiplexing , as required in label - free , 
 metabolic - labeling and other ms1-based quantification methods , an 
 ion selected for fragmentation on one lc ms / ms run may not 
 be selected consistently in subsequent runs or spectra of suitable 
 quality may not be acquired . the isobaric labeling 
 strategy , however , is immune to the stochastic nature of data - dependent 
 mass spectrometry because a common precursor ion is fragmented that 
 corresponds to the same peptide species present in all of the labeled 
 samples , yielding quantitative data across samples within an isobaric 
 tagging experiment . isobaric labeling has been shown to surpass metabolic 
 labeling in quantification precision and reproducibility . isobaric labeling exhibits a wide dynamic 
 range in profiling both 
 high- and low - abundance proteins and proteins with wide array of physiological 
 properties . it can be used to identify 
 and quantify proteins across diverse molecular weight and pi ranges , 
 functional categories , and cellular locations . the isobaric mass tags do not interfere with peptide fragmentation , 
 and the peptide length distribution profile and amino acid content 
 of the isobarically derivatized peptides are similar to those obtained 
 using other ms - based approaches . in fact , 
 isobaric tags have been reported to improve the efficiency of ms / ms 
 fragmentation and result in increased signal intensities of native 
 peptides in samples of human parotid saliva that , in general , lack 
 the uniform architecture of tryptic cleavage products , e.g. , a basic 
 c - terminal amino acid residue . with an ms1-based quantification approach , the co - elution of light 
 and heavy peptides can compromise sensitivity as the ion current is 
 divided between multiple samples during ms analysis . occurrence of 
 multiple precursor ion species in the ms1 level can also create redundancy 
 in ms / ms scanning events of the same peptide bearing different labels . it is reported that 
 up to 50% of ms / ms scans acquired during data acquisition can be redundant . by contrast , labeling of samples by isobaric 
 mass tags does not increase the sample complexity during chromatographic 
 separation and ms analysis because they are isotope - coded molecules 
 with the same chemical structure and molecular weight , thus eluting 
 at the same chromatographic time and with the same peptide mass . in 
 fact , since differentially labeled but identical peptides from multiple 
 samples are efficaciously merged , an improvement in overall signal - to - noise 
 ratios occurs , allowing good - quality ms / ms data to be acquired from 
 low - copy - number proteins . moreover , the sequence 
 informative b- and y - ions in ms / ms spectra also show this summed intensity , 
 which aids sensitivity . the in 
 vitro labeling procedure used for isobaric labeling - based 
 quantification strategy is highly efficient and enables this method 
 to be applicable to wide variety of samples such as cultured cells , 
 human tissues and biofluids , and tissues from model animals . this 
 technique has been successfully applied to various biological studies , 
 demonstrating its validity and robustness for quantitative ms - based 
 proteomics . isobaric labeling , especially itraq has been used in identifying 
 and distinguishing protease - generated neo - n termini from n - termini 
 of mature proteins by performing terminal amine isotopic labeling 
 of substrates ( tails ) . after tryptic digestion of itraq - labeled 
 protein samples , n - terminal peptide separation is accomplished using 
 a high - molecular - weight dendritic polyglycerol aldehyde polymer that 
 binds internal tryptic and c - terminal peptides that now have n - terminal 
 alpha amines . the unbound itraq - labeled mature n - terminal and neo - n - terminal 
 peptides and naturally blocked ( acetylated , cyclized , and methylated ) the neo - n - terminal peptides specific to the protease of interest appear 
 only in the protease - treated sample and therefore show a high protease / untreated 
 itraq reporter ion intensity ratio , thus differentiating them from 
 trypsin cleavage products that are present in all samples in equal 
 amounts and therefore have expected itraq ratios of 1 . the applications of the isobaric labeling strategy 
 have also been extended to studies involving the characterization 
 of post - translational modifications such as phosphorylation and other modifications ( discussed in the section 
 above ) . initially , isobaric labeling experiments were carried 
 out on maldi - tof / tof and quadrupole time - of - flight 
 ( q - tof ) instruments . quadrupole and tof instruments are capable of detecting low m / z fragment ions in the region where reporter 
 ions are observed . however , the large ion selection window of the 
 tof / tof instrument can result in a relatively high background of chemical 
 noise for the reporter groups , compressing the dynamic range of the 
 ratios significantly . quadrupole ion 
 trap geometries generally produce suboptimal results because the reporter 
 ions often lie below the stability limit , as dictated by the precursor 
 peptide mass - to - charge ratio and pseudopotential well parameters used 
 for activation ( for example , activation q = 0.23 ) . the slow scanning q - tof instruments also have 
 less sensitivity for complex mixtures compared to that of linear ion 
 traps . isobaric quantification 
 using standard collision - induced dissociation 
 ( cid ) conditions is not feasible using ion traps . the  /3 rule  for ion - trap instruments restricts 
 the analysis of product ions with m / z values less than 2530% of the precursor ion . this low mass 
 cutoff limitation also applies to hybrid instruments containing an 
 ion - trap for fragmentation , such as the ltq - ft and the ltq - orbitrap . this limitation can , in principle , be overcome 
 by pulsed q dissociation ( pqd ) . pqd in 
 the ion trap facilitates detection of low m / z reporter ions , bridging the gap between the linear ion 
 trap with pqd and a quadrupole tof instrument . however , unlike conventional cid spectra , typical pqd spectra 
 are dominated by the unfragmented precursor ion , indicating poor fragmentation 
 efficiency and thus limiting its practical utility for quantification 
 of peptides by itraq or tmt approaches . have shown that by carefully optimizing instrument 
 parameters such as collision energy , activation q , delay time , ion isolation width , number of microscans , repeat count , 
 and number of trapped ions , low m / z fragment ion intensities can be generated that enable accurate peptide 
 quantification . a combined cid - pqd scan strategy 
 exploits cid for efficient peptide identification and pqd for quantification . the development of higher energy collision - induced dissociation 
 ( hcd ) in the ltq - orbitrap has also overcome the /3 rule limitation . in an ion trap cid is a resonance - based 
 process , whereas hcd is a beam - type cid event that results in a different 
 fragmentation pattern . during hcd , ions are accelerated as they leave 
 the c - trap and then are fragmented in the nitrogen - filled collision 
 cell . the resulting fragments are returned to the c - trap and detected 
 in the orbitrap mass analyzer . this fragmentation technique allows 
 analysis of the low m / z region of 
 reporter ions in the orbitrap mass analyzer since there is no mass 
 cutoff for the multipole . hcd enables 
 efficient reporter ion generation with high mass accuracy detection , 
 but , in general , it suffers from poor peptide sequence - ion recovery 
 compared to that of the classical ion trap cid analysis . the combined 
 use of cid and hcd for efficient identification and relative quantification 
 of proteins with isobaric tags has been demonstrated . in this dual - fragmentation method , hcd is used to derive the accurate 
 quantitative information from the reporter ions , whereas cid provides 
 identification of the corresponding peptides . this method alternates 
 ms / ms spectra generated by cid fragmentation with ms / ms spectra obtained 
 from the same precursor ion by hcd fragmentation . since cid in the 
 ion trap occurs in parallel to acquisition of hcd ms / ms spectra in 
 the orbitrap , cid and hcd spectra 
 are subsequently combined by merging the peptide sequence - ion m / z range from cid spectra and the reporter 
 ion m / z range from hcd spectra . it should be noted that the extracted intensity values of the reporter 
 ions from each hcd spectrum should be normalized to low ion counts 
 when merging with the respective cid data , otherwise peptide scores 
 can be significantly reduced . the cid - hcd 
 method was shown to be superior to hcd alone in terms of sensitivity 
 and ability to identify proteins in complex mixtures . however , a recent study has shown that with fine - tuning 
 of the normalized collision energy values on orbitrap velos instruments , 
 an hcd - only method can perform better than a cid - hcd dual - fragmentation 
 method . this is due to the implementation 
 of the new hcd cell with an axial electric field to push the fragment 
 ions into the c - trap and mounted on orbitrap xl etd and orbitrap velos 
 instruments that allows an improvement in the analytical precision 
 of the acquired reporter ions . in addition , 
 the redundancy in precursor selection in the dual cid - hcd method compared 
 to that for the stand alone hcd method can result in a reduced number 
 of total peptide and protein identifications . the use of a stepped hcd scheme in q exactive instruments has been 
 shown to enhance the intensity of reporter ions without adversely 
 affecting peptide identifications . another method for analyzing isobaric labeled samples is to use 
 triple - stage mass spectrometry ( ms3 ) in a hybrid ion trap - orbitrap 
 platform . in this approach , a peptide 
 precursor ion is isolated and fragmented with cid - ms / ms to generate 
 a plurality of first - generation product ion species comprising different 
 respective m / z ratios . the most 
 intense product ion in ms / ms scan is then selected for hcd - ms3 , yielding 
 quantitative data . this method provides an experimental solution to 
 remove interference , thus eliminating the ratio distortion problem 
 ( discussed in the next section ) . a variant of this method referred 
 to as multinotch ms3 involves selecting and co - isolating 
 two or more of the first - generation product - ion species and fragmenting 
 them to generate a plurality of second - generation fragment ion species 
 including released isobaric tags ( figure 6 ) . the multinotch ms3 method significantly improves quantitative accuracy 
 and increases the sensitivity of the ms experiment up to n - fold , where n is the number of ms fragments selected 
 and simultaneously isolated . multinotch 
 ms3 involves selecting and co - isolating multiple ms / ms 
 product ion and fragmenting them to generate a plurality of second - generation 
 fragment ion species including released isobaric tags . the method increases the sensitivity and quantitative accuracy achieved 
 by isobaric labeling - based quantification approach . the ratios 
 of the intensity of the reporter ions reflect the relative 
 abundance of the peptides from which they are derived . the integration 
 of the relative quantification data for the peptides allows elucidation 
 of relative protein expression levels . this section discusses the 
 various aspects of data analysis in isobaric labeling - based quantification . isobaric labeling is usually very efficient ; 
 however , when primary amino groups are present elsewhere in the sample , 
 they may interfere with the labeling reaction since they can react 
 with the amine - reactive isobaric mass tags . hence , proper sample preparation 
 is imperative for the success of an isobaric labeling - based quantification 
 technique and includes either avoiding the use of primary amine - containing 
 buffers such as tris and ammonium bicarbonate or performing sample 
 cleanup prior to the isobaric labeling reaction . to improve detection limits and achieve a reliable estimate 
 of quantification , it is recommended that the labeling efficiency 
 be determined for each isobaric labeling experiment . the labeling 
 efficiency can be ascertained by searching the data separately against 
 protein database using tmt and itraq modifications as variable instead 
 of fixed modifications . using these parameters , both labeled and unlabeled 
 peptides can be identified and used to calculate labeling efficiency , 
 which is defined as the percent of labeled peptides among all identified 
 peptides . the labeling efficiency can be estimated aswhere nti and nki are the number of isobaric tag - labeled n - termini 
 and lysine residues , respectively , and , ntt and nkt are the total number of peptide 
 n - termini and lysine residues , respectively . additionally , due to isotopic contamination in isobaric mass tags , 
 the peaks for each reporter ion will have some contribution from adjacent 
 reporter ions . hence , prior to data analysis , each of the reporter 
 ion peaks must be corrected to account for isotopic overlap ( values 
 reported in the manufacturer s instruction sheet ) in order 
 to achieve accurate quantification . the uncorrected data will appear 
 distorted and confound the observed change in protein expression levels . a detailed procedure to calculate true peak 
 areas that account for overlapping isotopic contributions using the 
 reagent purity values provided by the manufacturer accurate 
 ratios can be determined 
 only when a single precursor ion is selected for fragmentation during 
 an ms / ms scan event . it has been observed that the presence of co - eluting 
 peptides within the isolation window used for the selection and subsequent 
 fragmentation of individual peptide ions typically results in an underestimation 
 or compression of actual protein abundance differences in the analyzed 
 samples . this effect 
 is ubiquitous and not dependent only on the instrument used to acquire 
 the data . the compression in relative 
 abundance is based on the assumption that the vast majority of proteins 
 in biological studies do not change significantly ; therefore , when 
 the peptides from these proteins co - fragment , the reporter ion intensity 
 ratios generated will be less pronounced in terms of fold changes . precursor ions of similar intensities can produce reporter ions that 
 span over 2 orders of magnitude in intensity . this means that very low intensity background ions can significantly 
 contribute to reporter ion signals when they get co - fragmented with 
 a selected precursor ion . additionally , if the coeluting peptides 
 display a nonequimolar distribution of reporter ions , then the net 
 effect of this co - selection is the unpredictable and context - specific 
 distortion of reporter ion intensities . in addition to the distortion in quantification accuracy due to coselection phenomena , the source of quantification error can also 
 be due to presence and interference from artifactual spectral peaks . 
 the reporter ion region in orbitrap hcd ms / ms spectra contains many 
 signals that are nearly isobaric with reporter ions generated from 
 isobaric mass tags . these signals do not correspond to any plausible 
 chemical compositions and may , in part , be attributed to artifacts 
 related to amplifying and processing the transient signal of the orbitrap . depending on the mass tolerance used for picking 
 the reporter ion signals , the presence of these nonreporter ion signals 
 may distort the quantification results . peptide abundance ratios 
 are calculated by combining data from multiple fractions across ms 
 runs and then averaging across peptides to give an abundance ratio 
 for each parent protein . the measured relative abundance can be influenced 
 by the separation ( e.g. , scx ) stage in which the ms / ms was acquired , 
 a phenomenon termed as fraction effect . fraction effect for a given peptide is defined as a significant 
 dependence between the measured ratio and the fraction in which the 
 reading was taken . the error within a fraction group for a peptide 
 is smaller than the error between fraction groups and arises from 
 the additional variance from the repeated scx separation stage . the observation of fraction effect could be 
 due to differences in a peptide s concentration across fractions 
 that contribute to variability in precursor ion intensity measures 
 and subsequent reporter ion peak areas . in addition to fraction effect , the measured ratio is also dependent 
 on the precursor ion ( i.e. , peptide ) used to characterize a protein . the measurement error within a peptide group for a protein the difference 
 in quantitative value from one peptide to another , even though belonging 
 to the same protein , might result from factors such as post - translational 
 modifications and/or splice variants , tryptic digestion artifacts , peptide recovery , and stability . other factors of peptide effect include noise 
 peaks with high signal - to - noise in the reporter ion region , sequence of the peptide used for quantification 
 and the possibility of interference from the immonium ion signals 
 in the reporter ion region , various charge states 
 of the same peptide , and the number of isobaric tags per peptide . since interference due to coisolation 
 is dependent on sample complexity 
 and the number of co - eluting peptides , the ratio compression can be 
 partly alleviated by better fractionation of complex biological samples 
 at the protein or peptide level . ratio 
 compression was observed to be smaller for enriched phosphoproteome 
 samples compared to that for whole proteome samples due to their overall 
 lower sample complexity . another approach 
 involves using an optimized ( narrow ) ms / ms isolation width setting 
 so that fewer contaminant ions are present during precursor ion activation . the high mass resolving power ( m/m > 15  000 ) in the reporter ion 
 region 
 also minimizes interference from potential contaminant species that 
 may confound quantification data . delaying 
 peptide selection and fragmentation until the apex of the chromatographic 
 peak during lc ms / ms analysis has been shown to reduce co - fragmentation 
 by 2-fold . with the delayed fragmentation 
 approach , peptides were fragmented with 2.8-fold better signal - to - noise 
 ratios , significantly improving the quantification . a targeted mass spectrometric data acquisition methodology 
 with reporter ion - based quantification has been shown to be useful 
 in applications where it is essential to reidentify and requantify 
 a defined set of target proteins in a complex mixture . the gas - phase purification and ms3 methods also eliminate interfering 
 ions in complex mixtures . in q - tof instruments , ion mobility ( i m ) separations have the potential to mitigate quantitative inaccuracies 
 caused by isobaric interference since im - ms has the ability to separate 
 ions based on charge , m / z , and collision 
 cross section ( shape and size ) . the ratio correction can also be achieved by various computational 
 approaches post data acquisition . one of the strategies is to use 
 an algorithm that corrects experimental ratios on the basis of determined 
 peptide interference levels . in this 
 method , the measurement for spectrum purity in survey spectra ( signal - to - interference 
 measure ) was used to improve the accuracy of protein quantification . signal - to - interference at the time of an ms / ms event is calculated 
 by dividing precursor abundance by the sum of all ion signals observed 
 within the isolation window . consequently , values 
 close to one indicate little and values close to zero indicate a high 
 degree of interference caused by co - eluting components . other informatics 
 approaches include the intensity - based weighed average technique , variance - stabilizing normalization , and robust statistic - based metric called redescending 
 m - estimator . the interference from non - tmt 
 signals can be eliminated by mass difference processing in which tmt 
 reporter ions in hcd spectra are identified via accurate mass differences 
 between tmt reporter ions present within the same tandem mass spectrum 
 instead of applying fixed mass error tolerances for all tandem mass 
 spectra . this process leads to unambiguous 
 reporter ion identification and eliminates all non - tmt ions from the 
 spectra . zhang et al . developed an error model that relates the variance 
 of measured ratios to observed reporter ion intensity and provides 
 a p value , q value , and confidence 
 interval for every peptide identified . the identification and exclusion of outlier data , with grubb s 
 and rosner s tests , that alter or inappropriately skew the 
 average observed expression ratios has shown to result in a more statistically 
 robust estimation of relative protein abundance . the ability to consider outlier data , however , can occur 
 only for proteins in which there are more than three ms / ms measurements 
 of protein expression . in summary , even 
 though all of the suggested strategies have merit , some techniques 
 only partially remove the problem , and others come with decreased 
 throughput or utilize specialized mass spectrometric instrumentation . isobaric 
 labeling - based quantification accuracy is also influenced 
 by reporter ion signal intensity and may result in either an underestimation 
 or overestimation of quantification ratio if the signal intensity 
 is outside the detector s saturation point . the reporter ions intensities will range between two extremes : the 
 maximum intensity , which corresponds to saturation , and the minimum 
 intensity , which corresponds to the lowest intensity detected . however , not all reporter ion intensity peaks will lead to accurate 
 relative quantification . the peak intensities of high - abundance peptide 
 ions may be underestimated by a saturation effect of the detector , 
 which is instrument - dependent . nevertheless , 
 high - intensity peptides convey more reliable quantitative information 
 about the protein . larger variances of 
 peptide ratios have been observed for reporter ions of lower intensity because the noise associated with low - intensity 
 reporters constitutes a major handicap in determining the statistical 
 significance of the differential expression of a protein . therefore , peptides with higher reporter ion 
 intensities should be given higher weight when used to calculate a 
 protein s abundance . reporter 
 ion signal intensity can be increased by increasing the ms / ms acquisition 
 duration ; however , this comes at the expense of decreased sampling , 
 resulting in fewer protein identifications . it is therefore important to estimate the quantification limits 
 of the instrument and the method used in order to assess the reliability 
 of the obtained quantification measurement . this can be achieved by 
 spiking samples with known quantities of reference proteins prior 
 to analysis and confirming the expected protein ratio from the measured 
 reporter ion intensity ratios . in isobaric labeling significant quantification errors 
 arise if a quantified peptide is not unique to its corresponding protein . hence , relative quantification based on shared 
 peptides ( i.e. , peptides that match multiple proteins or protein isoforms ) 
 due to sequence homology should be interpreted with caution . for a distinct peptide , its relative abundance 
 ratio is a direct measure of the abundance ratio of its corresponding 
 protein . in contrast , the relative abundance ratio of a shared peptide 
 is a weighted average of the abundance ratios of all its corresponding 
 proteins , with the weighting factors being determined by the absolute 
 abundance of those proteins in the samples . even though isobaric quantification is not dependent on the 
 total number of spectra matching to each protein , a high number of 
 relative abundance ratios obtained from multiple peptide / spectra increase 
 the confidence in the observed protein ratios . both intact protein mass and abundance level influence 
 the reliability of the quantification results since highly abundant 
 proteins generate a larger number of peptides per protein . more data , whether from multiple observations 
 per protein or from increasing replication , increases the detection 
 of real signal and reduces false positives . the identification of 
 so - called one - hit wonders should be filtered intelligently based on 
 the goal of the study . these proteins 
 deserve special attention if isobaric labeling is used as a screening 
 tool since potentially important biological information or novel biomarkers 
 may be discarded before they are even considered . fold change has 
 been shown to be a function of protein mass and abundance , 
 with small , low - abundance proteins showing the largest variance . a protein is considered to be differentially 
 regulated if the measured fold change exceeds a certain threshold . 
 the actual protein expression level is normally distorted by many 
 factors , with biological variation being the most significant and 
 which ultimately increases the cutoff point . the cutoff point that defines significant differential protein regulation 
 upon perturbation can be estimated by including sample replicates 
 in the experiment . the replicate samples can be 
 technical , experimental , or biological . according to gan et al . , the definition of replicates in terms of isobaric 
 labeling is as follows : a technical replicate will have two identical 
 samples from the same biological source in an isobaric experiment 
 set , whereas a biological replicate will have two distinct biological 
 samples from same condition in an experiment set . the experimental 
 replicate is the actual isobaric experiment replicate , the repetition 
 of the same samples in two or more experimental sets , and they must 
 have the same reference point or control . an illustration of the relationship 
 among technical , experimental , and biological replicates in isobaric 
 labeling experiments is depicted in figure 7 . an example defining the relation among technical , experimental , 
 and biological replicates in isobaric labeling ( itraq 4-plex in this 
 example ) experiments . a biological replicate 
 has two distinct biological samples ( x1 and x2 ) from the same condition 
 in an itraq set , whereas a technical replicate has two identical samples 
 ( x1 and x1 ) from the same biological source in an itraq set . an experimental 
 replicate is the repetitive analysis of the setup to assess the variation 
 of the identical sample in two different itraq sets ( y1 and x1 in 
 experiment 1 versus y1 and x1 in experiment 2 ) . r refers to a reference 
 sample that can be an individual sample or a pooled sample and allows 
 cross - set comparison . typically , a technical replicate assesses possible errors 
 contributed 
 from sample preparation , also commonly known as the sample variance . an experimental replicate 
 compares the variation of an identical sample in two different isobaric 
 experiment sets . during analysis of replicate samples , the theoretical 
 relative quantification ratio should be 1:1 ; however , due to associated variations , the observed relative protein 
 ratios might deviate from the theoretical value . the threshold should 
 be chosen such that it encompasses the majority of technical and biological 
 variation among the replicates . since in isobaric labeling multiple 
 samples hence , the ratio cutoff applied for significant protein 
 change via the isobaric labeling - based quantification approach is 
 lower than the cutoff applied for the label - free quantification approach ; however , the researcher will need to assess 
 whether such a change is biologically significant . for comparing biological replicates with isobaric labeling in multiple 
 experimental designs , this can be accomplished by first 
 comparing protein ratios of each sample against its reference within 
 individual experiments and then extending the information among multiple 
 experiments . the reference can be an individual sample or a pooled 
 sample prepared by mixing small aliquots of equal amounts of protein 
 from different individual samples . the 
 composition of reference samples does not contribute to missing quantitative 
 values , hence pooling to form a reference sample does not negatively 
 impact the ability to quantitate peptides from comparative individual 
 samples . the random biological variation in a pooled 
 sample is generally lower , as the biological variation can be normalized 
 by n samples before being introduced into the experiment . pooling provides a representative proteome of 
 all of the samples that are detected in comparative samples and is 
 needed for reliable quantification . , however , 
 have shown that using a masterpool is counterproductive since the 
 latter is also subjected to experimental noise and can result in highly 
 variable estimates when ratios are calculated . according to their study , more precise estimates of protein 
 relative abundance can be obtained by using the available biological 
 data . when a reference sample is used , 
 consistency of the reference is necessary throughout the entire experiment , 
 otherwise even small changes to the reference sample are sufficient 
 to alter the proteins that are reported as differentially expressed . regardless of using an individual sample 
 or a pooled sample as a reference , before employing isobaric quantification 
 results for follow - up studies , it is imperative to determine that 
 the data was normalized adequately and the shortlisted protein targets 
 hold merit . improper normalization might remove some of the biological 
 effects , resulting in attenuated estimates of the protein fold change . 
 like any other quantification technique , isobaric labeling - based quantification 
 is also biased toward identifying and quantifying a larger percentage 
 of the more abundant proteins , such as ribosomal proteins , heat shock 
 proteins , cytoskeletal proteins , transcription factors , and many others , 
 and often with multiple peptides . this is mainly 
 due to the fact that their precursor ions have higher signal intensity . the greater signal intensity increases the likelihood that a given 
 peptide will be selected for fragmentation during lc since , in most cases , the expression levels of these house - keeping 
 proteins remain unperturbed in related cell types or growth conditions , 
 they can be used as an effective means to determine the reliability 
 of data normalization . normalizing by 
 total intensity is not appropriate when the amount of protein is different 
 in the different quantitative sample such as samples that are enriched 
 for certain proteins by pull - down experiments . isobaric labeling experiments can 
 be used for phosphopeptide quantification , 
 and , in cases where the number of samples exceeds the number of isobaric 
 mass tags available for labeling , the throughput can be increased 
 by a hyperplexing method . amine - reactive isobaric mass tags have successfully 
 been used in the quantification of post - translational modification 
 such as phosphorylation . phosphopeptides exist in substoichiometric 
 quantities , and because of the high background of nonphosphorylated 
 peptides in a proteome digest , enrichment of phosphorylated peptides 
 is necessary prior to introduction into the ms . phosphopeptide enrichment 
 can be performed on isobaric - labeled peptides , or the phosphorylated peptide can be labeled 
 upon enrichment . labeling before enrichment 
 minimizes analytical variations caused by further sample manipulation 
 of individual samples during enrichment , whereas labeling after enrichment 
 might improve the yield of the labeling since the nonphosphorylated 
 peptides would otherwise compete for isobaric reagent and interfere 
 with the complete labeling of phosphopeptides . during the cid - hcd 
 dual method for the quantification and identification of isobarically 
 tagged phosphopeptides , cid with detection in the linear ion trap 
 provides better sensitivity and can be an advantage for low - abundance 
 precursors such as phosphopeptides . however , quantitative information 
 from the low mass region of subsequent hcd scans may not be available 
 for all such cid scans since hcd scans requires higher ion counts . wu et al . have examined the 
 optimal fragmentation conditions using the cid - hcd method for itraq - labeled 
 synthetic phosphopeptides in a complex phosphopeptide mix and phosphopeptides enriched from cells . during cid - ms / ms , the spectra derived from 
 phosphoserine- and phosphothreonine - containing peptides show facile 
 fragmentation of the phosphate group and dominance of neutral phosphate 
 losses from the precursor ions . the 
 neutral loss in hcd - ms / ms is much lower , and the sequence - specific 
 fragments are significantly more abundant . with the increasing charge 
 state of the precursor ions , the neutral loss in hcd - ms / however , the study of the effect of normalized 
 collision energy on the hcd - ms / ms fragments and reporter ion abundances 
 shows that the hcd identified phosphopeptides and the hcd spectra 
 with reporter ion information are strongly dependent on precursor 
 charge state . the 2 + charged precursors 
 are more sensitive to the applied normalized collision energy values 
 than the 3 + charged precursor ions in hcd experiments . have shown that derivatization 
 with isobaric mass tags significantly increases the average ion charge 
 state of phosphopeptides compared to that of nonlabeled peptides , 
 resulting in a considerable reduction in the number of identified 
 phosphopeptides . interestingly , it was 
 demonstrated that adding a perpendicular flow of ammonia vapor between 
 the needle and the ms orifice in lc ms / ms analyses reduced 
 the average charge state of isobaric labeled peptides and resulted 
 in an increase in peptide identification . thus , the application of 
 isobaric labeling strategies for quantitative phosphopeptide analysis 
 requires simultaneous monitoring of peptide backbone dissociation , 
 loss of phosphoryl group , and the generation of reporter ions . with existing 
 isobaric mass tags , the maximum number of samples that 
 can be combined and analyzed in a single lc ms / ms experiment 
 is eight in the case of itraq and 10 with tmt . an effort to increase 
 the multiplexing capacity by the combined use of metabolic and isobaric 
 labeling has been demonstrated . in this 
 strategy , the mass separation of co - eluting intact peptides with the 
 same sequence in an ms1 scan achieved by duplex ( heavy and light ) 
 or triplex silac labeling was exploited to allow for the simultaneous 
 quantification of multiple sets of tmt 6-plex isobaric labels in a 
 single run . using a 3  6 hyperplexing experiment that enables 
 simultaneous quantification of 18 samples , yeast response to the immunosuppressant 
 drug rapamycin , which inhibits the kinase target of rapamycin ( tor ) , was monitored by measuring the changes in their protein abundance . in this study , three separate cultures of yeast 
 cells grown in light , medium , or heavy silac culture medium were treated 
 with 200 mm rapamycin , and samples were removed at 0 , 30 , 60 , 120 , 
 and 180 min . equal amounts of peptides from each sample were 
 labeled with 6-plex tmt reagents , mixed , and separated by scx before 
 lc  ms / ms analysis . the increased multiplexing capacity enabled 
 analyses of multiple biological replicates of a time - course study 
 in the same run , providing the statistical power required to identify 
 significant trends . the hyperplexing technique with combined metabolic 
 labeling and isobaric mass tags can also be extended to n - labeled samples . alternatively , the dimethyl chemical labeling 
 technique can be combined with isobaric mass tags to increase the 
 multiplexing capacity of quantitative proteomics . theoretically , the 
 combination of itraq 8-plex or tmt 10-plex reagents and triplex silac 
 would allow 24 or 30 channels to be monitored simultaneously . proteins 
 that are identified and quantified as differentially expressed 
 can be used for subsequent targeted studies using the isobaric labeling 
 technique to assess reproducibility of the entire procedure and to 
 validate the observed differences in protein expression levels between 
 samples . during biomarker discovery experiments , targeted investigations are necessary to verify proteins with higher variance in additional 
 patient samples or to obtain greater statistical power . for successful 
 targeted analysis , peptides that allow clear protein quantification 
 and isobaric mass tags are often used 
 for discovery studies to reveal proteins being differentially expressed 
 under any given conditions . however , stella et al . have shown that 
 isobaric mass tags can be used in combination with multiple reaction 
 monitoring ( mrm ) for targeted quantification . in this study , the instrument monitored the two reporter 
 ions and three transitions for each peptide selected from the target 
 membrane proteins . the relative quantification was achieved by comparing 
 the intensities of the reporter ions generated from the labeled precursor 
 peptide of two samples , wild - type ( reporter ion m / z 129 ) and prion protein(prp)-knockout ( reporter 
 ion m / z 131 ) cerebellar granule 
 neurons . byers et al . used isotopic 
 versions of tmt reagents for targeted quantification to verify protein 
 regulations observed in a discovery study . these isotopic sets of reagents are structurally identical to the 
 isobaric ones but have different numbers of heavy isotopes incorporated 
 and are referred to as light tmt and heavy tmt ( figure 8) . the labeling of peptides by these reagents results in an 
 increase in mass of 224 and 229 da , respectively , per introduced tag . chemical 
 structure of isotopic reagents , light tmt and heavy tmt , 
 used for targeted quantification . the 
 light reagent has no heavy isotope incorporated , whereas the heavy 
 reagent has five heavy isotopes incorporated ( 4  c and 1  n ) . labeling with these reagents introduces 
 mass differences into the peptides from different samples . in targeted 
 experiments , quantification is obtained by structural b and/or y ions 
 generated after collision - induced dissociation . the isobaric labeling - based 
 quantification technique has developed 
 as a powerful tool for obtaining the relative expression level of 
 proteins in quantitative proteomics studies . moreover , the ability 
 to multiplex with isobaric mass tags has expanded its applicability 
 to a wide range of sample types . isobaric mass tags are isotope - coded 
 molecules with the same chemical structure and molecular weight that 
 are used to differentially label peptides without introducing mass 
 difference and sample complexity . the isotopically derivatized peptides 
 display a single peak on an ms spectrum and yield a series of low - mass 
 reporter ions for quantification upon fragmentation in tandem mass 
 spectrometry . however , since peptide quantification ratios are measured 
 to determine protein relative abundance , the variance in peptide ratio 
 measurements will contribute into the protein - level variance , affecting 
 the accuracy of the quantification . herein , we have reviewed the studies 
 of different aspects of an isobaric labeling - based quantification 
 approach . this includes studies on different types of isobaric reagents 
 and their applications , sources of variation that affect quantification , 
 and the suggested combinations of experimental design and optimal 
 data acquisition methods to increase the precision and accuracy of 
 the measurements . we have also reviewed studies on challenges in data 
 analysis and the proposed solutions for data processing to increase 
 the confidence in the acquired data set .
mass spectrometry plays a key role 
 in relative quantitative comparisons 
 of proteins in order to understand their functional role in biological 
 systems upon perturbation . in this review , 
 we review studies that 
 examine different aspects of isobaric labeling - based relative quantification 
 for shotgun proteomic analysis . 
 in particular , we focus on different 
 types of isobaric reagents and their reaction chemistry ( e.g. , amine- , 
 carbonyl- , and sulfhydryl - reactive ) . 
 various factors , such as ratio 
 compression , reporter ion dynamic range , and others , cause an underestimation 
 of changes in relative abundance of proteins across samples , undermining 
 the ability of the isobaric labeling approach to be truly quantitative . 
 these factors that affect quantification and the suggested combinations 
 of experimental design and optimal data acquisition methods to increase 
 the precision and accuracy of the measurements will be discussed . 
 finally , the extended application of isobaric labeling - based approach 
 in hyperplexing strategy , targeted quantification , and phosphopeptide 
 analysis are also examined .