one major goal of proteomic profiling 
 is an accurate quantitation of proteins in samples with high complexity 
 and high dynamic range of protein concentrations , such as body fluids 
 ( serum , plasma , csf , etc ) . because high confidence peptide / protein 
 identification and at the same time high confidence quantitation is 
 a highly challenging task ,
multiple analytical approaches have been 
 developed based on separation of proteins in - gel ( 2de dige ) and/or 
 gel - free platform utilizing various methods of metabolic or chemical 
 labeling .
each quantitative platform , 
 including isobaric tags for relative and absolute quantitation ( itraq ) , 
 has strengths and limitations that have been experimentally compared .
itraq was developed in the early 2000s 
 to be applied for the multidimensional protein identification technology 
 ( mudpit ) approach in which proteins are fragmented with trypsin or 
 other proteolytic enzyme and subsequently chemically labeled with 
 isobaric tags .
this platform became a central technology in modern 
 proteomics research ; it is being widely used in all areas of research 
 with great utility .
as much as this approach seems to be straightforward , many aspects 
 of this proteomic platform add sources of variability , and these limit 
 the confidence in the output of protein identification and quantitation . 

the variability is introduced in multiple steps of sample preparation , 
 efficiency of chemical tagging , performance of instrumentation , and 
 method of acquisition used , as well as software ( algorithms ) and thresholds 
 defined for database searches .
importantly , 4-plex and 8-plex tags 
 provide overlapping mass of reporter ions ; however , their balance 
 groups are different , which has been postulated to have an impact 
 on yield of fragmentation in collision induced dissociation ( cid ) 
 leading to bias in quantitation .
nevertheless , itraq - based quantitation 
 is an attractive method in global proteomic quantitation .
first , it 
 can be used after processing of any sample , e.g. , cell lysates and 
 proteins obtained from organelles , and as such is not limited to only 
 those systems that can accommodate incorporation of stable isotopes 
 during cell culture . 

third , software for protein identification and 
 quantitation is fairly well developed and tested in numerous experimental 
 settings .
recently pichler and co - workers found that peptide 
 labeling with 4-plex tags yields higher identification rates compared 
 to 8-plex tags .
this conclusion is of 
 concern since experimental designs using 8-plex allow a much greater 
 level and ease of comparison .
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 ( running the control 
 and up to three experimental samples in each ) .
this increases the 
 amount of control sample needed , labor and supplies , and chromatography 
 and mass spectrometry time and likely introduces a source of variability .
the goal of this study was to compare experimental ratios of highly 
 complex samples tagged with 4-plex versus 8-plex reagents in controlled 
 ratios .
we used proteinpilot 4.0 software for data analysis , which 
 is associated with the absciex 4800 maldi - tof / tof mass spectrometer .
ammonium phosphate , -cyano-4-hydroxycinammic 
 acid ( chca ) , and trifluoroacetic acid ( tfa ) were from sigma aldrich 
 ( st . louis , mo , usa ) .
hplc grade water and acetonitrile ( mecn ; acn ) 
 were from fisher scientific ( pittsburgh , pa , usa ) .
human plasma samples were shipped 
 on dry ice from university of california  san diego ( ucsd ) to 
 university of nebraska medical center ( unmc ) and on arrival remained 
 frozen .
hiv was inactivated in all samples by addition of 10 l 
 of freshly prepared 10% triton x-100 and 50 l of a cocktail 
 of protease inhibitors ( sigma - aldrich st .
after 30 min samples were aliquoted , and those unused were stored 
 at 80 c . a 250 l portion from each sample was 
 filtered using a 0.2 m spin filter and immunodepleted using 
 an igy14 column ( sigma - aldrich ) to remove the following proteins : 
 albumin , 1-antitrypsin , igm , haptoglobin , fibrinogen , 
 1-acid glycoprotein , apolipoprotein a - i and a - iii , 
 apolipoprotein b , igg , iga , transferrin , 2-macroglobulin , 
 and complement c3 .
flow - through fractions containing unbound proteins 
 were concentrated using a vivaspin 15r ( sartorius , aubagne , france ) . 

protein concentration was determined using a nanodrop spectrophotometer 
 ( thermo scientific , san jose , ca ) .
a total of 400 g of proteins 
 was pooled , and then aliquots of 50 g of proteins were used 
 in order to perform the itraq labeling .
a 50 g 
 sample of proteins was precipitated with ethanol , by adding 10 vol 
 of cold ethanol ( 200 proof ) to each sample , incubating for 3 h at 
 20 c , and centrifuging at 13,000 g for 
 15 min at 4 c .
proteins pellets were washed with 1 ml of 70% 
 ethanol and dried in a speedvac ( thermo scientific ) .
subsequent solutions 
 were provided by itraq reagent kits ( applied biosystem , carlsbad , 
 ca ) .
dried proteins were solubilized with dissolution solution , 
 and proteins were denaturated with 1 l of denaturant reagent . 

protein reduction with reducing reagent was performed for 1 h at 60 
 c . according to the manufacturer protocol
, samples used for 
 itraq 4-plex were alkylated with 84 mm iodoacetamide for 30 min at 
 room temperature , whereas for itraq 8-plex we used the cysteine blocking 
 solution from the itraq kit for 10 min at room temperature .
trypsin from 
 abi was reconstituted at 1 g/l with milli - q water , and 
 10 g of trypsin was added to each sample .
after digestion , peptides were labeled with 
 itraq label reagent ( abi ) ; 4-plex labeling was performed for 1 h at 
 room temperature , and after the incubation the reaction was quenched 
 with 100 l of mq water for 30 min at room temperature .
the 
 8-plex labeling was performed for 2 h at room temperature . labeled 
 peptides were combined in one tube ; we mixed a known quantity of peptides 
 from each tag ( see experimental design , figure 1 ) .
samples used in all three experiments ( 400 , 600 and 650 g ) 
 were taken from the same larger pool of immunodepleted plasma samples 
 ( see materials and methods for details of 
 immunodepletion ) . in all experiments regardless how much tagged peptides 
 were used for analyses , 50 g of peptide digest was always used 
 for itraq tagging to eliminate any effect of the tag to peptide ratio 
 between experiments .
samples were cleaned up using mixed cation exchange 
 ( mcx ) column ( water corp .
labeled peptides were solubilized 
 with 1 ml of 0.1% formic acid , passed through the column , and then 
 the column was washed with 5% methanol , 0.1% formic acid solution , 
 and then with hplc grade methanol .
then , 360 l of reconstituted 
 sample was supplemented with 1.44 ml of offgel solution .
next , samples 
 were fractionated on the basis of their isoelectric point ( pi ) using 3100 offgel fractionator ( agilent , inc .
offgel strips were rehydrated for 15 min at room temperature 
 with 40 l of offgel solution .
peptide samples were loaded onto 
 gel strips , splitting them equally between all 12 wells .
collected fractions were 
 cleaned with c-18 spin columns , according to the manufacturer s 
 protocol .
briefly , fractions were adjusted to 5% acetonitrile ( acn ) 
 and 0.5% trifluoracetic acid ( tfa ) and passed through activated columns . 

columns were washed twice with a 5% acn , 0.5% tfa solution , and peptides 
 were eluted with a 70% acn , 0.1% tfa solution .
subsequent fractionation 
 of offgel fractions was performed off - line using tempo lc system with 
 automatic high density spotting onto maldi target plates .
peptides 
 were solubilized in 12 l of 0.1% tfa , and 10 l of samples 
 were loaded onto a proteocol c18 trap cartridge ( michrom biosources , 
 auburn , ca ) and washed for 20 min at 9 l / min .
gradient of separation 
 was obtained using a ratio between two buffers : water / acn / tfa ( 98:2:0.1 ) 
 ( buffer a ) and water / acn / tfa ( 2:98:0.1 ) ( buffer b ) . to perform the 
 separation ,
the subsequent gradient was applied by altering buffer 
 b percentage : time 05 min , 5% to 15% ; 552 min , 15% 
 to 35% ; 5254 min , 35% to 80% ; 5464 min , 80% ; 6465 
 min , 80% to 5% ; and 6572 , min 5% .
peptide elution was monitored 
 with a uv cell at 214 nm absorbance . after the uv cell ,
eluted peptides 
 were mixed with a matrix solution ( 1.2 mg / ml in 75% acn and 0.1% tfa 
 solution ) at a flow rate 1 l / min using a harvard apparatus 
 syringe pump .
fractions were spotted every 30 s , and the voltage applied 
 to the plate during spotting was 2.8 kv .
spotted fractions were 
 submitted for data acquisition on a 4800 maldi - tof / tof mass spectrometer 
 ( abi ) .
ms spectra were acquired from 800 to 3000 m / z , for a total of 1000 laser shots by an nd : yag 
 laser operating at 355 nm and 200 hz .
ms / ms analyses were performed using 2 kv collision 
 energy with air as cid gas .
metastable ions were suppressed , for a 
 total of 1000 laser shots . protein identification and quantification 

the search parameters were as follows : itraq 4-plex ( peptide labeled ) , 
 carbamidomethylation of cysteine , ncbi database ( created on december 
 2011 ) restricted to homo sapiens , itraq 8-plex ( peptide 
 labeled ) , methylthioalkylation of cysteine , ncbi database ( created 
 on december 2011 ) restricted to homo sapiens , for 
 itraq 4-plex and 8-plex , respectively .
our experimental design ( figure 1 ) used one large pool of human plasma immunodepleted of the 
 14 most abundant proteins .
regardless of how much of the resulting 
 peptides was used to create final ratios , we always used 50 g 
 during the reaction for itraq labeling .
this approach eliminated potential 
 variability that might be associated with efficiency of chemical labeling 
 when ratios to tag and peptides are not uniform .
we used 114 , 115 , 116 , 
 and 117 tags from 4-plex and from 8-plex kits and combined the following 
 amount of labeled peptides ( separately for the 4-plex and 8-plex ) 
 to achieve a 1:2:3:4 ratio from each kit : 10 g ( 114 ) , 20 g 
 ( 115 ) , 30 g ( 116 ) , and 40 g ( 117 ) . in experiment 2
we 
 repeated these conditions and added a third sample in which the 113 , 
 118 , 119 , and 121 tags from the 8-plex kit were used and peptides 
 again mixed in a 1:2:3:4 ratio . however , in experiment 3 we compared 
 labeling of 114 , 115 , 116 , and 117 tags from the 8-plex kit to labeling 
 with all eight tags from the same 8-plex kit .
relative to the first 
 two experiments , we scrambled tag assignment to the amount of peptides 
 used , which allowed us to limit another potential bias ( tag effect ) . 

in experiment 3 we also added 50 g of non - labeled peptides 
 to the sample labeled with four tags to make up for the difference 
 between amounts of peptides between those labeled with all eight tags . 
 in all three experiments we used the same conditions for fractionation 
 based on isoelectric point and subsequently rp - hplc in tempolc plate 
 spotter .
all data were processed by the same version of proteinpilot 
 with the same version of database . in figure 2
ideally we should observe ratios of 0.25 ( 114:117 ) , 0.5 ( 115:117 ) , 
 and 0.75 ( 116:117 ) .
first , as 
 confidence of protein identification decreases ( plotted on the x - axis ) , the ratios for individual proteins ( plotted on 
 the y - axis ) become more dispersed and the groups 
 start to overlap .
second , when we used tags from the 4-plex kit , ratios 
 of 114:117 showed lower than expected values , whereas when we used 
 tags from the 8-plex kit the ratio of 114:117 was as expected ( 0.25 ) . 

the two other ratios were very similar for both kits , and all were 
 slightly higher than expected .
proteins were plotted by decreasing value of confidence of identification 
 ( x axis ) and ratios that were calculated as relative 
 to 117 reporter ion ( y axis ) .
( a ) plot for 114 , 115 , 
 and 116 m / z reporter ions from itraq 
 4-plex kit .
( b ) ratios for the same m / z set of reporter ions from itraq 8-plex kit . in figure 3
we present a 
 comprehensive comparison of ratios derived from all three experiments 
 as a box - plot analysis .
as shown in panel a , comparison of the ratios 
 from experiment 2 shows a greater dispersion of data when tags from 
 the 4-plex kit were used .
comparison of the box ( containing the values 
 from 25% to 75% of the ratios ) reveals that the tags from the 8-plex 
 that have reporter masses similar to those of the 4-plex have a tighter 
 distribution than those from the 4-plex . in panel
b we present analysis 
 of the spread of ratios for tags 115 , 116 , and 117 from the 8-plex 
 kit relative to the 114 tag when labeled samples were mixed with an 
 equal amount of non - labeled peptides . in this experiment measured 

ratios indicated that the presence of non - labeled peptides skewed 
 results toward lower than expected values , which would have been 0.75 
 ( 115/114 ) , 0.50 ( 116/114 ) , and 0.25 ( 117/114 ) , respectively .
dispersion 
 of ratios was highest for 115/114 and lowest for 117/114 . in panel 

c we show comparison of ratios from the second part of experiment 
 3 in which we used all tags from the 8-plex kit ; however , samples 
 were mixed in 1:1 , 1:2 , 1:3 , and 1:4 ratios and all were calculated 
 as relative to the 113 tag . in this data set experimental ratios matched 
 expected values for the following tags : 115/113 was 0.75 , 116/113 
 was 0.5 .
ratios for tags 117/113 and 121/113 were comparable to each 
 other ; however , both were below the 0.2 mark while we expected them 
 to be at the 0.25 mark . ratios of 118/113 , 119/113 , and 114/113 were 
 skewed to lower values quite substantially in some instances . besides 
 the fact that higher ratios had a larger spread of values in the top 
 and bottom quartiles , there was no obvious pattern of systematic skew 
 of data in either the top or bottom quartiles across all comparisons .
box - plot 
 of the ratios comparing effect of tags from itraq 4-plex and 8 plex 
 kits .
data presented are from experiment 2 in panel a and from experiment 
 3 in panels b and c. ( a ) .
ratios were calculated as relative to the 
 highest amount of peptides tagged with 117 for 4-plex and 8-plex and 
 121 for 8-plex only .
therefore , the first three box - plots represent 
 a 1:4 ratio ( expected value 0.25 ) , the second three box - plots represent 
 a 1:2 ratio ( expected value 0.5 ) , and the third set of box - plots represents 
 a 3:4 ratio ( expected value 0.75 ) .
box - plot analysis of ratios 
 from tagging peptides with 114 , 115 , 116 , and 117 tags from 8-plex 
 kit and mixed with an equal amount ( 50 g ) of non - labeled peptides . 

( c ) . box - plot analysis 
 of ratios of peptides tagged with all 8 tags from 8-plex kit .
the 
 amount of peptide digest was the same ( 100 g ) as in panel b. while manually analyzing ratios for individual 
 peptides labeled with the different tags , we have found three predominant 
 patterns of ratios regardless of whether the 4-plex or 8-plex assay 
 was used ( figure 4b ) . among them , linear dependence 
 ( pattern 1 , figure 4b ) is the most desirable 
 and expected and is representative of more than 80% of all patterns 
 found ( figure 4a ) .
more than 90% of peptides 
 ( figure 4a ) are included when these three patterns 
 are combined .
although more than 80% of peptides showed linear ratios , 
 we were interested in what impact the non - linear patterns have on 
 the overall quantitative ratio of a protein and whether these non - linear 
 patterns may skew quantitation . to investigate such possibility we 
 selected two proteins : serpin peptidase inhibitor , clade g , member 
 1 precursor and hemopexin precursor .
the peptides that contributed 
 to the overall ratios of these two proteins contained a mixture of 
 the three predominant peptide ratios as presented in figure 4 .
results of this analysis are presented in figure 5 and indicate that despite having a mixture of all 
 three patterns of peptide ratios , protein quantitation still shows 
 linearity . observed patterns of peptides ratios .
ideally , all peptides 
 labeled with itraq tags should show quantitative and linear ratios 
 representing controlled mixing of protein samples .
in reality , we 
 have found seven non - linear or no change patterns representing less 
 than 20% of the total number of peptides ( a ) .
expected linear pattern 
 is shown in panel b , and the two most predominant non - linear patterns 
 are shown in panels c and d , respectively . when combined , these three 
 patterns represent more than 90% of peptides .
two proteins , serpin peptidase inhibitor , clade g , member 
 1 precursor and hemopexin precursor , were selected for this comparison . 

selection was based on the fact that in both cases overall protein 
 ratios were calculated based on peptides representing a mixture of 
 patterns 1 , 2 , and 3 shown in figure 4 . in 
 both instances , serpin peptidase inhibitor , clade g ,
member 1 precursor 
 ( a ) and hemopexin precursor ( b ) proteins showed overall linearity 
 of ratios despite a mixture of linear and non - linear peptide ratios . one point of interest that pichler and co - workers 
 found was that for samples labeled with itraq 8-plex , the number of 
 peptide - spectrum matches and unique peptides was more than 70% lower 
 and the number of protein groups more than 60% lower , as compared 
 to itraq 4-plex .
we identified 72 proteins 
 with 99% and 98 with 66% confidence , respectively , using the 4-plex 
 itraq kit , and 64 and 90 proteins for the respective thresholds when 
 we used the 8-plex itraq kit .
when we used a 50% confidence cutoff 
 level , we found that samples labeled with 8-plex showed a decrease 
 in identifications of only 13% at the peptide level and 19% at the 
 protein level , and when compared to 4-plex these differences were 
 not statistically significant .
performed their quantitation and identification using a ltq 
 orbitrap and cid - hcd hybrid method and searches were performed using 
 mascot and proteome discoverer .
we used maldi 4800 with proteinpilot 
 4.0 with paragon algorithm , which has an impact on the number of proteins 
 identified .
itraq , as with any other analytical tool , 
 is under continuous scrutiny by scientists looking for ways of most 
 accurate measurements in quantitative proteomics .
because global 
 profiling with quantitation is a multistep experiment , the final output 
 may depend on wide range of factors ultimately contributing to skewed 
 or even false positive results .
one solution to prevent contribution 
 of errors originating from itraq data analyses is tightening thresholds ; 
 however , this approach must be used with caution because it may easily 
 lead to loss of important information . therefore , itraq is being constantly 
 evaluated and each study emphasizes different aspects of this approach . 

gan and co - workers assessed the reliability of itraq from perspective 
 of different types of replicate analyses and took into account technical , 
 experimental , and biological variations .
mahoney and co - workers reported that measured variability was a 
 function of mean abundance , fold changes were biased toward the null , 
 and variance of a fold change was a function of protein mass and abundance .
ow and co - workers evaluated the quantitative dynamic range of itraq quantitation in 
 high- and low - complexity samples . although their study has similarities 
 in experimental design , there are also important differences , including 
 the use of non - mammalian samples to create a high complexity background , 
 spiking in strategy to measure ratios , strong cation exchange ( scx ) 
 separation in first dimension , and a qtof mass spectrometry platform . 
 in a subsequent paper
the authors used 
 a similar strategy of spiking in known proteins to evaluate accuracy 
 and precision of itraq based quantitation and proposed spiking as 
 a method to address accuracy and variance - stabilizing normalization 
 to address the issue of precision . in 
 another study evaluating accuracy of quantitation using itraq ratios 
 thingholm and co - workers used whole hela cell lysate as a model sample 
 and focused on phosphopeptides after enrichment on a tio2 column .
the authors used esi as ionization mode and 
 a ltq xl orbitrap as mass spectrometry platform .
they reported correlation 
 between reductions in identification efficiency with the size of the 
 isobaric tag .
taking all these studies together , we have gained knowledge 
 into understanding the itraq platform ; however , our study presented 
 here offers insight from a different perspective . here
we perform 
 a calculated experiment using immunodepleted human plasma , a body 
 fluid that is highly complex and has a high dynamic range of protein 
 concentration .
another way our study differs significantly is that 
 we use a maldi - tof / tof platform , followed by proteinpilot 4.0 data 
 analysis , both of which are offered from and supported by absciex , 
 the manufacturers of itraq .
other groups focused on software and mathematical 
 models for itraq data analyses and comparing algorithms across many 
 platforms . despite the collective effort ,
many outstanding questions related 
 to accuracy and sources of variability in itraq technique remain to be addressed , and more systematic 
 studies with direct comparisons across mass spectrometry platforms , 
 complexity of samples , and sample preparation methods are needed to 
 fully understand bias resulting from itraq quantitation .
we 
 were intrigued by the report of pichler and co - workers showing that 
 peptide labeling with 4-plex tags yields higher identification rates 
 compared to 8-plex tags .
the authors used 
 a ltq orbitrap mass spectrometer , cid - hcd hybrid method , and proteome 
 discoverer software ( thermo scientific ) .
the authors attributed the 
 differences in yields to differences in chemical structures of balance 
 groups and concluded that balance groups used in 8-plex tags are less 
 susceptible to fragmentation .
however on the basis of our previous 
 experience we found not only that were the differences in peptide 
 and protein identifications low but that the 8-plex tags resulted 
 in increased confidence of quantitation with limited impact on protein 
 identification . taking this together
we decided to test this effect 
 using a systematic experimental approach to examine this as well as 
 the accuracy of ratios obtained from intensities of the reporter ions 
 with simplified experimental design to remove as much bias as possible . 

we intentionally chose human plasma because of our past work on biomarker 
 discovery in body fluids ( csf , serum , and plasma analyses ) and because 
 it constitutes a highly complex mixture of proteins with high dynamic 
 range of relative concentrations .
plasma / serum and csf have been used in many biomarker discovery 
 studies , including using itraq platform ; however , in many instances 
 validation using larger population of clinical samples was disappointing , 
 leaving questions about the sources of such disconnect unanswered . 

we used one large sample of immunodepleted plasma securing identical 
 material for all experiments .
biological variability , although very 
 important , was not an objective of our study , and pooling multiple 
 samples averaged levels of proteins in the mix .
importantly , we used 
 a maldi - tof / tof 4800 mass spectrometer and proteinpilot software with 
 paragon algorithm , which are different than those used by pichler 
 and co - workers .
we have found that 
 8-plex tags performed with higher quantitation accuracy than the same 
 ( by m / z of reporter ions ) tags from 
 4-plex reagent , providing experimental ratios closer to theoretical 
 ratios without dramatically affecting peptide or protein identification . 
 also ,
when confidence of protein identification decreases , the spread 
 of ratios increases in both instances , however , to a lesser extent 
 when 8-plex tags are used ( figure 3a ) .
therefore 
 we conclude that more consistent ratios would be due to more complete 
 cid fragmentation of tags using maldi mass spectrometry .
box - plot 
 analysis of ratios from subsequent experiments showed that spread 
 of ratios is much tighter in two middle quartiles when 8-plex tags 
 are used .
additionally , labeling peptides with 8-plex tags yielded 
 more peptides with linear dependence of calculated itraq ratios , thus 
 better reflecting the ratio of controlled mixing . skewing of 
 the measured ratios in the 1:1 mixture of tagged and nontagged peptides 
 was an unexpected effect considering that the same amount of peptides 
 tagged with 8-plex yielded ratios close to their theoretical values . 
 in the mixture of tagged and nontagged peptides , for each peptide 

there were two different precursor ions that yielded identical or 
 very similar fragmentation spectra .
all spectra could contribute to 
 confidence of protein identification ; however , only half of the spectra 
 contributed to quantitation . if peptide fragmentation used for protein 
 identification and fragmentation of tags used for quantitation are 
 processed by algorithm as separate events and results
are merged at 
 the final step , such effect should not be observed . on the other hand , 

if quantitation and identification is considered by algorithm as one 
 event , 50% of spectra with null quantitation may induce a systematic 
 skew in the calculation of ratios .
therefore , completeness of tagging 
 may have a quite profound effect on quantitative output even if such 
 incomplete tagging is proportional to all of the peptides in the sample . 

also results from experiment 1 may also suggest that ratio can be 
 affected by either low level of precursor ion and thus more fragment 
 ions were under background level and/or poor fragmentation during 
 cid .
we observed in other itraq experiments examples in which the 
 intensity of reporter ions was clearly above background providing 
 good quantitation , but cid fragmentation of the tagged peptide was 
 so poor that identification was calculated with confidence below 1% 
 ( data not shown ) .
summarizing , we provide here experimental 
 evidence that under our experimental conditions , 8-plex tagging is 
 advantageous over 4-plex tagging in two aspects .
first , 8-plex tagging 
 provides more consistent ratios without compromising on protein identification . 

second , the 8-plex system of tagging allows investigation of eight 
 experimental conditions in one analytical experiment . a question that 
 remains to be addressed is whether , during itraq data acquisition , 
 the peptide and reporter ion fragmentation that leads to identification 
 and quantitation , respectively , should be considered as two separate 
 events or dependent on each other . this would need to be addressed 
 formally in subsequent experiments .
 methods for isobaric tagging of peptides , itraq or tmt , 
 are commonly used platforms in mass spectrometry based quantitative 
 proteomics . 
 these two methods are very often used to quantitate proteins 
 in complex samples , e.g. , serum / plasma or csf supporting biomarker 
 discovery studies . 
 the success of these studies depends on multiple 
 factors , including the accuracy of ratios of reporter ions reflecting 
 quantitative changes of proteins . 
 because reporter ions are generated 
 during peptide fragmentation , the differences of chemical structure 
 of itraq balance groups may have an effect on how efficiently these 
 groups are fragmented and thus how differences in protein expression 
 will be measured . because 4-plex and 8-plex itraq reagents do have 
 different structures of balanced groups , it has been postulated that 
 indeed differences in protein identification and quantitation exist 
 between these two reagents . in this study we controlled the ratios 
 of tagged samples and compared quantitation of proteins using 4-plex 
 versus 8-plex reagents in the context of a highly complex sample of 
 human plasma using absciex 4800 maldi - tof / tof mass spectrometer and 
 proteinpilot 4.0 software . 
 we observed that 8-plex tagging provides 
 more consistent ratios than 4-plex without compromising protein identification , 
 thus allowing investigation of eight experimental conditions in one 
 analytical experiment . 
Introduction
Materials and Methods
Results
Discussion