since the completion 
 of the human genome project in 2003 , a large 
 number of genetic variations have been uncovered in humans , including 
 single nucleotide variations , insertions and deletions . among these genetic variations , single nucleotide 
 variants ( snvs )
are recognized as the most common type of genetic 
 variations in the human genome , occurring on average on the order 
 of one per 860 base pairs where these variants are often associated 
 with particular diseases and differential drug response .
increasing evidence has shown that a small fraction of these variants 
 are responsible for the initiation or progression of cancer .
the genomic variations of cancer are expressed through aberrant 
 proteins and their altered functions .
therefore , the direct detection 
 of these variant peptides may provide unique opportunities for biomarker 
 development .
pancreatic cancer is a dismal disease , with a poor 
 long - term outcome 
 of less than 5% for the 5 year - survival rate .
however , the 
 possibility of patient s survival is considerably improved 
 when the patient is diagnosed at an early stage of the disease and 
 treated by surgical resection .
the current 
 pancreatic cancer biomarker used in clinical diagnosis , glycoprotein 
 ca 199 , can not satisfy this need because of its low sensitivity 
 and specificity . finding a novel biomarker or 

biomarker panel with higher sensitivity and specificity for pancreatic 
 cancer diagnosis is an urgent task .
a genome - wide 
 association study identified some susceptibility loci of pancreatic 
 cancer on chromosomes .
therefore , saav 
 peptide analysis in serum provides a new alternative for pancreatic 
 biomarker discovery .
although single nucleotide polymorphisms 
 ( snps ) in humans have 
 been extensively studied over the past decade , little attention has 
 been paid to single amino acid polymorphisms ( saps ) of proteins at 
 the proteome level , especially direct analysis in serum samples . with 
 the development of mass spectrometry techniques and bioinformatics , 

there has been a limited effort to identify single amino acid variant 
 ( saav ) peptides in tissues .
a mass spectrometry - friendly modified 
 database was created by adding variant sequences in the original international 
 protein sequence ( ipi ) database .
fifteen 
 variant peptides were identified using this database for variant peptides 
 analysis in lysosomes isolated from mcf7 breast cancer cells . to improve the efficiency of variant peptide 
 analysis
, a new variant database and an integrated data analysis workflow 
 were developed for variant peptide identification in three colorectal 
 cancer cell lines , where a total of 204 variant peptides were detected .
recently , a more comprehensive variant - associated database was constructed 
 by combining the unique variant sequences from the ms - canprovar database 
 ( http://bioinfo.vanderbilt.edu/canprovar ) and a humansavar 
 database ( http://www.uniprot.org/docs/humsavar ) .
this was 
 applied to variant peptide analysis in hepatocellular carcinoma ( hcc ) 
 and normal human liver tissues , where 282 unique saav sites were identified 
 and quantified .
recently , a novel 
 strategy developed by the zeng group may provide 
 a new direction for variant peptide analysis using a de novo sequencing 
 method .
the de novo sequencing strategy 
 is independent of variant databases and may identify some cancer specific 
 somatic mutations .
however , it is difficult to analyze thousands of 
 ms / ms spectra in a high - throughput mode .
moreover , the false positive 
 ratio control is another important challenge for large - scale variant 
 peptide analysis . to identify mutant proteins as cancer - specific biomarkers , 

selected reaction monitoring ( srm ) approaches have been used to quantify 
 a few variant peptides from the mutant ras protein in cancer cell 
 lines and tissues . in one study ,
absolute 
 concentrations of three selected saav peptides among 290 serum samples 
 were investigated using a selected reaction monitoring ( srm ) approach , 
 and the results revealed that heterozygous and homozygous proteins 
 with various saps ( single amino acid polymorphisms ) have different 
 associations with particular traits in the population . although some progress has been achieved in saav peptide analysis , 
 the application of large - scale quantitative analysis of variant peptides 
 in serum is still limited .
the complexity and the wide dynamic 
 range of serum samples make 
 comprehensive analyses of the serum proteome a challenging task .
however , 
 screening serum glycoproteins as a powerful means to find novel markers 
 in serum biomarker discovery has been demonstrated in previous studies .
alterations in the glycosylation profile are now known to be associated 
 with many diseases .
the discovery and detailed characterization of 
 glycoprotein disease biomarkers is a primary interest of biomedical 
 research .
an isobaric labeling quantitative strategy was applied 
 to identify and quantify variant peptides in serum samples of pancreatic 
 cancer patients and other benign controls .
the largest number of variant 
 peptides to date in serum were identified and quantified , where 96 
 unique variant peptides were quantified in this quantitative analysis , 
 of which five variant peptides showed a statistically significant 
 difference between pancreatic cancer and other controls . the saav 
 peptide which showed a significant difference between pancreatic cancer 
 and controls
this study was 
 approved by the institutional review board of the university of michigan 
 medical school .
all serum samples used in this study were from age 
 and sex - matched patients with signed informed consent .
, serum samples 
 from 141 individual patients were investigated in this study , of which 
 cyst , chronic pancreatic , diabetes and obstructive jaundice served 
 as benign controls and 27 normal serum samples were used as healthy 
 controls .
twenty - six serum samples from patients of pancreatic cancer 
 were investigated to determine the significantly different proteins 
 against controls .
abbreviations : 
 pdac , pancreatic 
 ductal adenocarcinoma ; dm , diabetes mellitus ; cp , chronic pancreatitis ; 
 oj , obstructive jaundice ; n / a , not applicable ; m / f , male / female .
other races include black / african 
 amerian ; bi / multi racial / hispanic and unknown .
briefly , all 
 the serum samples were aliquoted 
 into different tubes and stored in a 70 c freezer .
the 
 serum samples were randomized into different sets before depleting 
 high - abundance proteins .
fourteen high - abundance proteins were depleted 
 in this study using a igy-14 lc10 column ( sigma , st .
pooled 
 samples were obtained by mixing 30 normal serum samples and used as 
 an internal standard .
amine - reactive , 6-plex tandem mass tag reagents 
 with a + 229.1629 
 mass shift were used to label the serum sample at the protein - level 
 as described previously .
briefly , an equal amount 
 of processed serum protein ( 100 g ) from the different disease 
 groups and internal standard ( pooled samples ) were labeled by tmt 
 reagent .
after the labeled samples from the same set were combined , 
 the buffer was exchanged into lectin affinity binding buffer for affinity 
 fractionation . to reduce the complexity of the serum sample , aleuria aurantia lectin ( aal ) affinity chromatography 
 was applied to enrich the glycoprotein fraction .
labeled samples were 
 diluted to 1 ml using binding buffer ( 20 mm tris , 0.15 m nacl , ph 
 = 7.5 , protease inhibitor 1:100 ) , and then were loaded onto an affinity 
 column packed with 600 l of agarose - bound aal lectin .
after 
 incubating for 15 min twice , binding proteins were eluted with four 
 volumes of elution buffer ( 200 mm fucose in binding buffer ) .
trypsin and asp - n 
 enzymes ( promega , madison , wi ) were used to digest the tmt labeled 
 samples in parallel . then , pngase f ( new england biolabs , ipswich , 
 ma ) was added and incubated at 37 c for 16 h. the digested samples 
 were desalted using c18 ziptips ( millipore , billerica , 
 ma ) and dried using a speedvac concentrator ( thermo savant , milford , 
 ma ) before lc  ms / ms analysis . the lc 
ms / ms 
 analysis was performed on an eksigent nano 2d lc system ( ab sciex ) 
 and orbitrap velos pro mass spectrometer ( thermo fisher scientific ) . 

the digested peptides were loaded on a commercial new objective proteopepid 
 trap column ( 150 m  25 mm ) , and then separated on an 
 c18 analytical column ( 75 m  100 mm , 5 m , 300 
 a ) using a 100 min linear gradient from 2 to 32% solvent b ( 0.1% formic 
 acid in acetonitrile ) at a flow rate of 300 nl / min .
the 10 most intense 
 peaks from the ms spectrum were selected to run ms / ms analysis using 
 the hcd fragmentation mode .
the range of full scans is from m / z 400.0 to 1800.0 with a resolution of 
 30  000 .
the ms / ms scan was fixed at starting from m / z 100.00 with a resolution of 7,500 .
a  1.5 da isolation window was applied 
 to isolate precursor ions with dynamic exclusion of a 10 ppm exclusion 
 window .
every precursor ion was repeated twice within a duration time 
 of 20 s and was excluded for 20 s. the maximum injection time for 
 ftms full scan was set as 250 ms reaching an agc target value of 100  000 , 
 and the maximum injection time for ft msn scan was set as 200 ms reaching 
 an agc target value of 40  000 .
all the collected raw 
 data were analyzed by searching the swiss - cansaavs database using sequest in proteome discoverer 1.1 ( thermo ) . 

a total of 87  745 amino acid variant sequences and 73  910 
 uniprotkb / swiss - prot canonical protein entries are included in the 
 swiss - cansaavs database ( download from http://bioanalysis.dicp.ac.cn/proteomics/publications/saav/saav-database.htm on 11/21/2013 ) , of which 87,745 amino acid variant sequences were 
 constituted by combining data set  humsavar.txt  containing 
 human polymorphisms and disease mutations ( downloaded from www.uniprot.org/docs/humsavar on 11/25/2011 ) and ms - canprovar database ( downloaded from http://bioinfo.vanderbilt.edu/canprovar on 12/14/2011 ) .
the m / z tolerance of precursor ion and fragment ion were 
 set as 10 ppm and 0.05 da , respectively .
carbamidomethylation ( + 57.02146 da , c ) and tmt 
 6-plex ( + 219.163 da ) of lysines and protein n - termini were set as 
 fixed modifications .
oxidation ( + 15.99492 da , m ) and deamidation ( + 0.98402 
 da ,
the identified results were 
 filtered using a strict standard at 1% peptide - level false discovery 
 rate ( fdr ) .
the srm analysis was performed using a tsq 
 quantum ultra am ( thermo - finnigan , san jose , ca ) coupled with a surveyor 
 hplc system ( thermofisher , san jose , ca ) . for each serum sample ,
about 
 0.7 l of serum digest spiked with 4 pmol stable isotope labeled 
 standard peptides was injected for analysis .
the lc buffer system 
 was : buffer a , 0.1% formic acid / water and buffer b , 0.1% formic acid / acetonitrile . 

the peptides were separated and eluted at a flow rate of 250 l / min , 
 with a lc gradient from 5% to 35% b in 25 min on a 2.1  150 
 mm c18 analytical column ( 3 m , atlantis t3 , waters ) .
the samples 
 were analyzed using a spray voltage at 4000 v and a capillary temperature 
 of 300 c .
the srm analyses were performed with a scan time of 
 0.05 s and a scan width of 0.002 m / z , using a unit resolution of 0.7 da for both q1 and q3 .
the collision 
 energy for each transition and tube lens voltage for each peptide 
 were experimentally optimized by infusing each isotopically labeled 
 peptide solution .
the optimum transitions for each targeted peptide 
 were determined empirically using the synthetic reference peptides . 
 before running each batch of samples , a mixture containing 4 pmol 
 of each isotopically heavy peptide in 0.1% formic acid
was injected 
 to establish the retention time and chromatographic peak characteristics 
 of each reference peptide .
srm quantification analysis was performed 
 using xcalibur 2.1 ( thermoscientific ) software . for each targeted 
 peptide , the precursor peak and coelution of the associated transitions 

the elisa kits of protein 
 aact , thbs1 , and ca199 were purchased from abcam , genway and 
 abnova , respectively . on the basis of the manufacturer s instructions , 
 the duplicate readings for each standard and sample
good 
 linear calibration curves were generated to determine the amount of 
 each protein .
isotopically labeled heavy 
 forms ( containing either a c - terminal [ c6n2 ] lys residue or [ c6n ] leu residue ) of 
 peptides were synthesized by anaspec , inc . as absolute quantification 
 peptides with known quantities .
all peptides were resuspended in 50% acetonitrile 
 ( acn ) , 0.1% formic acid , and then diluted into 5% acn containing 0.1% 
 formic acid during the preparation of dilution series .
the auc value was used to evaluate 
 the biomarker s performance by constructing receiver operating 
 characteristic ( roc ) curves .
multivariate analysis was done by logistic 
 regression to obtain the best - fitting multivariate model for each 
 biomarker panel .
the student s t test and 
 one - way analysis of variance ( anova ) were applied to calculate a p - value to determine statistical difference .
in this study , we investigate saav 
 peptides associated with pancreatic cancer in serum using a quantitative 
 proteomics strategy .
recently , several studies of saav proteomics 
 analyses conducted in cells or tissue samples have been reported .
however , serum is widely used in clinical diagnosis because of its 
 noninvasive advantage . therefore ,
identification of saav peptides 
 in serum may be a useful strategy to find potential markers .
as shown in figure 1 , the serum samples from 
 different disease groups and healthy controls were used to perform 
 saav peptide analysis , where cyst , chronic pancreatic , diabetes and 
 obstructive jaundice served as disease controls .
all serum samples were first depleted of 14 high abundance 
 proteins , then labeled at protein - level using tmt 6 plex isobaric 
 labeling reagents .
the digested peptides 
 were analyzed using an orbitrap velos pro mass spectrometer in duplicate . 

experimental workflow for quantitative analysis of single 
 amino 
 acid variant peptides associated with pancreatic cancer by isobaric 
 labeling strategy .
the saav peptides can not 
 be identified using standard databases 
 because the variant sequences are typically absent from generic proteomic 
 databases . as a result , a considerable portion of high - quality tandem 
 mass spectra collected is left unassigned . to identify saav peptides , 
 a database containing 87  745 amino acid variant sequences and 
 73  910 uniprotkb / swiss - prot canonical protein entries
was constructed 
 for database search , which enables one 
 to analyze saavs on a large - scale .
therefore , a database combining 
 amino acid variant sequences and uniprotkb / swiss - prot canonical protein 
 entries was applied to search the raw data . after filtering with a 
 1% fdr , 96 quantified saav peptides
were obtained in total from 141 
 serum samples , including 83 single amino acid variant sites ( supporting information table s1 ) .
after statistical 
 analysis , five saav peptides were determined to be significantly different 
 variant peptides ( p value < 0.05 , table 2 ) .
this is probably the largest scale quantification 
 data set of pancreatic cancer associated variant peptides in serum , 
 which is larger than that performed in a recent study of diabetic 
 serum samples .
bold and italic letter denote mutant 
 site ; n denotes deaminated modification . to increase the identification of saav peptides , trypsin 
 and asp - n 
 were used to digest the serum samples . in the total of 83 saav sites , 

47 saav sites were identified from trypsin digests and 36 saav sites 
 were detected from asp - n digests .
the result suggests different enzymes 
 increase the identification of saav peptides in serum . in this analysis , 

the matched results of their ms / ms spectra 
 clearly confirmed the variant sites . for example : ms / ms spectra of 
 the variant peptide gggagfisgltyleldnpagnkr and canonical peptide gggagfisglsyleldnpagnkr from complement 
 component c7 ( co7 ) are shown in figure 2 . a 
 + 14 da mass shift corresponding to the substitution of threonine ( thr ) 
 for serine ( ser ) at position 389
was indicated by a series of y13 , 
 y14 , y15 , y16 and y17 ions , which confirms the variant site .
the ms / ms 
 spectra of saav peptide degklqhlenelth and corresponding canonical peptide also clearly 
 indicate variant site ( e  v ) from alpha-1-antitrypsin ( supporting information figure s1 ) . to validate 
 the identification results of saav peptides , all the ms / ms spectra 

were manually checked based on the following criteria : ( 1 ) the fragment 
 ion peaks have a high signal - to - noise ratio , ( 2 ) and show sequential 
 members of the b- or y - ion series , and ( 3 ) the major peaks were matched . 

furthermore , two variant sites were further confirmed by synthetic 
 peptides containing variant sites ( supporting 
 information figure s2 ) . as a result ,
highly confident and large - scale 
 saav peptide data were identified in serum samples of pancreatic cancer .
ms / ms 
 spectra of the variant peptide gggagfisgltyleldnpagnkr ( a ) and canonical peptide 
 gggagfisglsyleldnpagnkr 
 ( b ) from complement component c7 ( co7 ) .
a + 14 da mass shift corresponding 
 to the substitution of threonine ( thr ) for serine ( ser ) at position 
 389 was indicated by the series of y13 , y14 , y15 , y16 and y17 ions , 
 which confirms the variant site .
these saav 
 peptides were further validated by srm analysis according to the following 
 rules : ( 1 ) these 
 peptides were quantified in more than half of the analyzed samples 
 in the experiments ; ( 2 ) no methionine ( m ) amino acid ( aa ) exists in 
 the peptide ; ( 3 ) the length of peptides is in the range of 725 
 aa . in the discovery phase , we used trypsin and asp - n enzyme to digest 
 serum samples in order to increase the identification of saav peptides . 
 in the srm analysis
, original serum samples were applied . on the basis 
 of the high cost of asp - n consideration ,
finally , the saav peptide dtpeagyfavavvkksas from serotransferrin 
 with a substitution of valine ( val ) for isoleucine ( ile ) at position 
 448 was selected for srm analysis .
the other four significant peptides 
 were not selected for validation because they did not meet the strict 
 filter criterion .
using trypsin digestion , the variant site was located 
 in the peptide of sdncedtpeagyfavavvk .
the optimum collision energies and transitions 
 of the saav peptide were determined empirically using a stable isotopic - labeled 
 synthetic peptide ( table 3 ) .
three optimum 
 transitions were selected for quantification in srm assay . denotes isotope labeling amino acid 
 residue ( 8 da shift ) .
retention time : 19.32 min . the same coelution time and a cv 
 of < 25% in the average relative 
 ratio of the srm transitions
figure 3a presents the same retention time 
 and similar average relative ratio from labeled peptide and targeted 
 peptide , which indicates no interference for these transitions .
simultaneously , 
 a series of different concentrations of diluted synthetic peptides 
 were analyzed in duplicate with serum matrix .
the curve showed excellent 
 linearity with a correlation of 0.99 ( r2 ) from 15 fmol to 15 pmol 
 ( figure 3b ) .
totally , 27 cancer , 20 cp 
 and 17 normal samples were analyzed in sequence .
the scatter 
 plot of the quantitative results of saav peptides is shown in figure 3c .
statistical analysis showed that a significant 
 difference existed between pancreatic cancer and cp , as well as between 
 cancer and normal samples ( p - value < 0.05 ) .
the results showed that there is no significant difference 
 between cancer serum samples and controls , which means the significant 
 difference of saav peptide ( dtpeagyfavavvkksas ) is not derived from 
 the difference of the parent protein ( supporting 
 information figure s3 ) .
unfortunately , the corresponding canonical 
 peptides were not quantified in our experiments . otherwise .
we could 
 quantify the fraction of saav peptides by comparing the abundance 
 of the saav peptide with the corresponding canonical peptides .
variant peptide 
 ( sdncedtpeagyfavavvk ) from serotransferrin was verified and quantified 
 by spiking heavy labeling peptide into serum samples .
the tic of the 
 variant peptide ( a ) , standard curve ( b ) and scatter plot of the amount 
 of variant peptide in different disease groups ( c ) . to evaluate 
 the performance of the saav peptide as a potential marker ,
auc values 
 were acquired by generating roc curves . as shown in figure 4 ,
an improved auc value ( 0.75 ) is obtained between pancreatic 
 cancer and cp . in previous work
further 
 elisa analysis of the two proteins found that the saav peptide is 
 complementary to these two candidate proteins .
a novel biomarker panel 
 was obtained by combining aact , thbs1 and saav peptide , where its 
 performance was investigated for diagnosing pancreatic cancer .
the 
 results revealed that the panel has a strong capability for differentiating 
 pancreatic cancer with healthy controls ( auc = 0.98 ) and cp controls 
 ( auc = 0.90 ) ( figure 5 ) .
performance of variant 
 peptide based on the results of srm assay . 
 the roc curve and auc value are presented .
performance of biomarker combination of saav peptide and thbs1 , 
 aact , the roc curve and auc value are shown .
currently , the performance of ca 199 marker can not 
 satisfy 
 the need to detect early pancreatic cancer . in this study , ca 199 elisa assay also was performed on 
 the same serum samples .
the ca199 performance between cancer 
 and cp or normal is an auc value of 0.79 and 0.89 , respectively , which 
 is consistent to other reports .
the biomarker panel 
 of aact , thbs1 and saav peptide has an improved performance compared 
 with ca199 .
if we combine ca 199 with the biomarker 
 panel , the performance of this combination in discriminating pancreatic 
 cancer with cp reaches to an auc value of 0.988 with 100% 
 sensitivity at 92% specificity , and reaches to an auc value of 0.998 
 in distinguishing cancer from normal controls with 100% sensitivity 
 at 96% specificity .
it should be noted that the same set of samples 
 was used in the different assays .
thus , a risk of overfitting may 
 exist . to reduce the effect of overfitting
first , the markers were simply linearly combined 
 with weights proportional to their marginal correlation coefficient 
 with the outcome instead of using optimized regression fits in the 
 multimarker analysis .
the 
 largest - scale quantification data set of saav peptides to date 
 was discovered in pancreatic cancer serum using quantitative strategies . 

a total of 96 unique saav peptides were quantified in this quantitative 
 analysis , of which five variant peptides showed a significant difference 
 between pancreatic cancer and other controls .
the saav peptide from 
 serotransferrin protein showed its potential as a potential biomarker 
 by further srm validation analysis .
a promising combination of aact , 
 thbs1 proteins and the saav peptide was achieved , which has an excellent 
 performance in discriminating pancreatic cancer with other controls . 

these results show that large - scale analysis of saav peptides in serum 
 may hold a promising future for biomarker discovery research .
 single 
 amino acid variations are highly associated with many human 
 diseases . 
 the direct detection of peptides containing single amino 
 acid variants ( saavs ) derived from nonsynonymous single nucleotide 
 polymorphisms ( snps ) in serum can provide unique opportunities for 
 saav associated biomarker discovery . in the present study , 
 an isobaric 
 labeling quantitative strategy was applied to identify and quantify 
 variant peptides in serum samples of pancreatic cancer patients and 
 other benign controls . 
 the largest number of saav peptides to date 
 in serum including 96 unique variant peptides were quantified in this 
 quantitative analysis , of which five variant peptides showed a statistically 
 significant difference between pancreatic cancer and other controls 
 ( p - value < 0.05 ) . 
 significant differences in the 
 variant peptide sdncedtpeagyfavavvk from serotransferrin were detected between pancreatic cancer 
 and controls , which was further validated by selected reaction monitoring 
 ( srm ) analysis . 
 the novel biomarker panel obtained by combining -1-antichymotrypsin 
 ( aact ) , thrombospondin-1 ( thbs1 ) and this variant peptide showed an 
 excellent diagnostic performance in discriminating pancreatic cancer 
 from healthy controls ( auc = 0.98 ) and chronic pancreatitis ( auc = 
 0.90 ) . 
 these results suggest that large - scale analysis of saav peptides 
 in serum may provide a new direction for biomarker discovery research . 
Introduction
Materials and Methods
Results
and Discussion
Conclusions