modern transcriptome 
 profiling methods such as tiling arrays and whole transcriptome shotgun sequencing ( rna - seq ) have revealed that a larger number of rnas are 
 produced from the genome than previously thought .
furthermore , subsequent analysis 
 of these nonannotated transcripts has demonstrated the existence of 
 functional noncoding rnas , such as long intergenic noncoding rnas 
 ( lincs ) .
the identification of additional rnas also 
 raises the possibility that there may also exist additional nonannotated 
 protein - coding rnas .
the computational prediction of open reading 
 frames ( orfs ) ( i.e. , protein - coding regions ) relies on a number of 
 stringent criteria to avoid false discovery , such as a length cutoff , 
 aug start codon usage , and sequence conservation .
these 
 criteria are not perfect , and several types of orfs are often missed , 
 including orfs that use non - aug initiation codons as well as short 
 orfs ( sorfs ) that fall below the typical length cutoff of a 100 codons 
 ( i.e. , a 100 amino acid polypeptide ) .
frith and 
 colleagues , for example , utilized a new search algorithm to reanalyze 
 the mouse genome and predicted an additional 3000 protein - coding sorfs , which would correspond to an 10% increase 
 in the size of the mouse genome .
more recently , direct experimental evidence of the existence of 
 non - aug initiation sites and protein - coding sorfs has begun to emerge . 

ribosome profiling methods , which footprint the location of the ribosome 
 on rnas to identify protein - coding regions , revealed the existence 
 of a number of nonannotated protein - coding sorfs in the mouse genome . in these experiments , the addition of the drug 
 cycloheximide freezes the ribosome on start codons , and when cycloheximide 
 is used in combination with ribosome profiling , the start codons of 
 orfs can also be identified .
this analysis 
 led to the observation that while aug is the most common codon used 
 ( 45% of the time ) , cug and gug are also frequently used , which contradicts the dogma that translation 
 initiation is restricted to aug .
thus , ribosome profiling indicates 
 that cells often use non - aug start codons and reveals the existence 
 of nonannotated protein - coding sorfs , both of which would likely be 
 missed by classical algorithms for predicting protein - coding regions 
 in the genome .
in addition to ribosome profiling , mass spectrometry 
 ( ms ) peptidomics 
 and proteomics experiments have recently been implemented in the discovery 
 of sorf - encoded peptides ( seps ) .
these ms experiments 
 differ from ribosome profiling because they detect polypeptide generated 
 from a sorf and therefore validate the protein - coding potential of 
 the sorf by demonstrating the production of a stable protein product . 

because of transcript amplification and the number of reads per sequencing , 
 experiment ribosome profiling is more sensitive and will identify 
 the largest number of sorfs , but the bias of ms toward more abundant 
 proteins means that peptidomics and proteomics 
 will likely identify the most abundant cellular seps , which might 
 be the seps most likely to be functional .  
slavoff and colleagues 
 developed and utilized a peptidomics - based 
 strategy for the detection of novel human seps .
these studies were based on initial observations by yamamoto 
 and colleagues who identified four seps in k562 cells ( defined here 
 as fewer than 150 amino acids in length ) . to improve on these results , slavoff and coworkers utilized next - generation 
 rna sequencing ( rna - seq ) to identify all possible protein - coding mrna 
 transcripts , including nonannotated transcripts ( i.e. , transcripts 
 that exist but are not in the ncbi refseq database ) .
the rna - seq data 
 was translated into all possible reading frames to create a database 
 that should contain all of the polypeptide sequences that could theoretically 
 be produced in the cell . using this database , slavoff and colleagues 
 identified 90 human seps in these k562 cells , and 86 of these seps 
 were novel .
this work indicated that 
 seps represent a large class of nonannotated cellular polypeptides . 

recent work from others has also supported this conclusion , with vanderperre 
 and colleagues having characterized 1259 nonannotated polypeptides , the largest number reported to date using an 
 elegant combination of bioinformatics and mass spectrometry .
our goal here is to ( 1 ) determine whether we can identify a workflow 
 that provides the easiest route for sep detection , ( 2 ) determine whether 
 seps exist in other cell lines , and ( 3 ) determine whether we can find 
 seps in human tissues , specifically a human tumor sample .
our results 
 identify several workflows for sep discovery and demonstrate that 
 seps are ubiquitous and present in multiple cell lines and human tissues .
k562 cells were grown in rpmi1640 medium 
 supplemented with 10% fbs , penicillin and streptomycin at a density 
 of ( 1 to 10 )  10 cells / ml . mcf10a cells were grown 
 in megm complete medium ( life technologies ) , and mdamb231 cells were 
 grown in dmem medium supplemented with 10% fbs , penicillin , and streptomycin . 

tissue was obtained from the massachusetts 
 general hospital ( mgh ) department of pathology as a deidentified sample . 

this was done in accordance with all of the rules and regulations 
 of the harvard irb .
aliquots of k562 , 
 mcf10a , and mdamb231 cells ( 2  10 cells per experiment ) 
 were placed in falcon tubes , washed three times with cold pbs , pelleted , 
 and transferred into 1.5 ml protein lobind tubes ( eppendorf ) . boiling 
 water ( 300 l ) was directly added to the cell pellet , and the 
 cells were boiled for an additional 20 min .
this step eliminates protease 
 activity to maintain the integrity of the peptidome for subsequent 
 lc  ms analysis .
after the samples were cooled on ice , the cells 
 were sonicated on ice for 20 bursts at output level 2 with a 30% duty 
 cycle ( branson sonifier 250 ; ultrasonic convertor ) .
acetic acid was 
 added to the cell lysate until the final concentration of acetic acid 
 was 0.25% by volume .
the sample was then centrifuged at 14  000 g for 20 min at 4 c to precipitate large proteins 
 and reduce the complexity of the sample .
the supernatant was passed 
 through a 30 kda molecular weight cutoff ( mwco ) filter , and the small 
 proteins and polypeptides were isolated in the flow - through . an aliquot 
 of the flow - through was taken for a bca assay to measure the protein 
 concentration . the remaining sample
was then evaporated to dryness 
 at low temperature in a speedvac and used for lc 
this supernatant was loaded 
 onto a 16% tricine gel ( novex , 1.0 mm ) and run at 120 v for 80 min 
 instead of being passed through an mwco filter .
dual 
 xtra standards ( bio - rad ) was used as the molecular - weight marker , 
 and the gel was sectioned below the 15 kda marker to afford three 
 sections : 25 , 510 , and 1015 kda .
each gel 
 slice was placed in 1.5 ml protein lobind tubes ( eppendorf ) and washed 
 with 1 ml of 50% hplc grade acetonitrile in water three times .
frozen human breast 
 tumor sample ( 200 mg ) was immersed in boiling water ( 200 l ) 
 for 10 min .
the aqueous fraction was collected and saved in a clean 
 tube , and the tissue was dounce - homogenized in 500 l of ice - cold 
 acetic acid ( 0.5% v / v ) . the aqueous fraction and
the homogenate were 
 combined and centrifuged at 20  000 g for 20 
 min at 4 c .
the supernatant was transferred to a new lo - bind 
 tube and evaporated to dryness at low temperature in a speedvac .
the 
 dried sample was suspended in pbs and loading dye , followed by separation 
 in a 16% tricine gel ( novex , 1.0 mm ) . the excised gel bands ( < 15 
 kda ) were analyzed by lc 
after trypsin 
 digest the samples were dried in a speed vac and suspended in erlic 
 buffer a ( 90% acetonitrile 0.1% acetic acid ) .
samples were then fractionated 
 using an hplc ( agilent 1200 hplc ) equipped with an erlic column ( polywax 
 lp column , 200  2.1 mm , 5 m , 300  ( polylc ) ) .
samples 
 were separated using a stepwise gradient with the following steps : 
 05 min , 0% b ; 515 min , 08% b ; 1545 
 min , 835% b ; 4555 min , 3575% b ; 5560 
 min , 75100% b ; 6070 min , 100% b ( a : 90% acetonitrile , 
 0.1% formic acid ; b : 30% acetonitrile , 0.1% formic acid ) .
an automated 
 fraction collector was used to collect 25 equivalent fractions that 
 were concentrated then analyzed by lc 
erlic samples were digested 
 prior to erlic and did not require any additional sample prepl prior 
 to lc  ms .
gel slices from page separation were extracted and 
 then digested with trypsin overnight . the resulting peptide mixture 
 was separated from any residual gel slices and analyzed on an orbitrap 
 velos hybrid ion trap mass spectrometer ( thermo fisher scientific ) . 
 regions between 395 and 1600 m / z ions were collected at 60k resolving power for the ms1 , and these 
 data were used to trigger ms / ms in the ion trap for the top 20 ions 
 in the ms1 ( i.e. , top 20 experiment ) . active dynamic exclusion of 
 500 ions for 90 s was used throughout the lc  ms / ms method . 

samples were trapped for 15 min with flow rate of 2 l / min on 
 a trapping column 100 m i d packed for 5 cm in - house with 5 
 m magic c18 aq beads ( waters ) and eluted onto 20 cm  
 75 m i d analytical column ( new objective ) packed in - house with 
 3 m magic c18 aq beads ( waters ) .
peptides were eluted with 
 300 nl flow rate using a nanoacquity pump ( waters ) using a binary 
 gradient of 232% b over 90 min ( a : 0.1% formic acid in water ; 
 b : 0.1% formic acid in acetonitrile ) .
the sequest algorithm was used to analyze the acquired 
 ms / ms spectra using a database 
 derived from three - frame translation from the rna - seq data for that 
 cell line .
rna - seq data from k562 , mcf10a , or mdamb231 cell lines 
 were assembled into a transcriptome using cufflinks and then translated in three ( forward ) frames in silico . 

the search against this database was performed using the following 
 parameters : variable modifications , oxidation ( met ) , n - acetylation , 
 semitryptic requirement , two maximum missed cleavages , precursor mass 
 tolerance of 20 ppm , and fragment mass tolerance of 0.7 da .
search 
 results were filtered such that the estimated false discovery rate 
 of the remaining results was at 1% . for this purpose
the sf score 
 of greater than 0.7 was the required with a mass accuracy of < 3.5 
 ppm . after analysis ,
the data were filtered based on a combination 
 of the preliminary score , the cross - correlation , and the differential 
 between the scores for the highest scoring protein and the second 
 highest scoring protein . a list of peptides that passed the search 
 criteria
was then searched against the uniprot human ( swissprot ) protein 
 database using a string - searching algorithm .
peptides found to be 
 identical and to overlap with part of annotated proteins were eliminated 
 from the list .
the remaining peptides were then searched one more 
 time against nonredundant human protein sequences using the basic 
 local alignment search tool ( blast ) .
peptides that 
 were identical or different by one amino acid from the nearest protein 
 match were discarded .
the final list of peptides , candidate 
 seps , were searched against human reference ( refseq ) rna sequences 
 using blast to assess their location relative to the annotated transcripts , 
 which can be categorized into 5utr , 3utr , cds , and 
 noncoding .
if the peptides had no match in the refseq rna sequences , 
 then they were derived from rnas that were present in the rna - seq 
 data that had not been annotated in refseq ( i.e. , nonannotated rnas ) .
total 
 rna ( 3000 ng ) was purified from mcf10a and mdamb231 cell lines 
 using rneasy kit ( qiagen ) according to the instructions provided by 
 the manufacturer .
cdna libraries with paired - end , indexed adapters 
 were created using the illumina truseq rna sample prep kit .
two libraries 
 were pooled and sequenced on a single lane of a hiseq2000 machine . 

rna - seq reads were aligned to the human genome ( hg19 ) using tophat 
 ( version v2.0.4 ) , and transcriptome assembly was performed using cufflinks 
 ( version v2.0.2 ) .
sequences 
 for seps were submitted in fasta format to skyline ( version 2.1.0.4936 ) for analysis .
the goal was to identify peptides 
 from these sequences that are most amenable for targeted proteomics 
 using multiple - reaction monitoring ( mrm ) .
skyline predicted transitions 
 for each peptide , and we used all of transitions in a targeted mrm 
 experiment to identify the presence or absence of the peptide .
we 
 must detect at least three transitions for a given peptide to determine 
 that it is present in the sample .
the output from skyline is imported 
 directly into a targeted method for analysis with a tsq quantum ultra 
 triple stage quadrupole mass spectrometer ( i.e. , a triple quad ( qqq ) , 
 thermo fisher scientific ) .
peptide samples were analyzed using the 
 tsq with a 90 min gradient and targeted mrm tandem mass spectrometry 
 using the aforementioned skyline method .
samples were trapped for 
 15 min with flow rate of 2 l / min on a trapping column 100 m 
 i d packed for 5 cm in - house with 5 m magic c18 aq beads ( waters ) 
 and eluted with a gradient to 20 cm  75 m i d analytical 
 column ( new objective ) packed in - house with 3 m magic c18 aq 
 beads ( waters ) .
peptides were eluted with 300 nl flow rate using a 
 nanoacquity pump ( waters ) using a binary gradient of 232% 
 b over 180 min ( a : 0.1% formic acid in water ; b : 0.1% formic acid 
 in acetonitrile ) .
our 
 first goal was to determine whether changes to the reported sep - discovery 
 workflow would lead to the identification of additional seps in k562 
 cells , and whether any particular workflow is superior to others . 
 in the reported workflow
, seps are separated 
 from larger proteins with a 30 kda molecular weight cutoff ( mwco ) 
 filter , and the 30 kda fraction then undergoes electrostatic 
 repulsion hydrophilic interaction chromatography ( erlic ) , followed by lc 
this workflow led to the identification of 90 seps , 86 of which were 
 novel , in the commonly used k562 leukemia cell line .
more recently ( 2013 ) , vanderperre and colleagues have identified 1259 
 nonannotated polypeptides .
below , total 
 seps refer to the total number of seps discovered and novel seps refer 
 to any seps from these groups that were not identified in the slavoff 
 et al . or vanderperre et al . manuscripts .
( a ) schematic 
 of the four different sep discovery workflows used : mwco+lc  ms ; 
 mwco+erlic+lc  ms ; page+erlic+lc  ms ; and page+lc  ms . 
 the k562 peptidome is separated by size using a 30 kda mwco filter 
 ( mwco ) or polyacrylamide gel electrophoresis ( page ) and then analyzed 
 directly by lc  ms ( first and last lane ) or fractionated by 
 erlic prior to lc  ms analysis ( middle lanes ) .
( b ) number of 
 total sep and novel seps identified in k562 cells using each of the 
 four different sep discovery workflows .
three additional workflows were tested here : mwco+lc  ms / ms , 
 page+erlic+lc  ms / ms , and page+lc  ms / ms . in these workflows , 
 mwco indicates fractionation using a 30 kda mwco filter , while page 
 refers to molecular weight fractionation using a 16% tricine polyacrylamide 
 gel , where the region between 2 and 15 kda is analyzed by lc 
all of these workflows led 
 to the discovery of novel human seps , though the number of seps , and 
 the ease of the different methods varied .
we began by comparing 
 the mwco+lc  ms / ms and the page+lc  ms / ms 
 workflows .
these workflows differ in their approach to peptidome isolation 
 by using a 30 kda mwco filter or the excising the 215 kda 
 portion of a 16% tricine gel .
after separation , the 30 kda 
 fraction is treated with trypsin and then analyzed by lc 
the 215 kda gel slice undergoes an in - gel trypsin digest , 
 followed by lc 
seps are identified using a 
 custom k562 database generated from rna - seq data that will account 
 for polypeptides produced from previously nonannotated protein - coding 
 sorfs .
we identified 13 seps using the mwco+lc  ms / ms workflow 
 with a single lc 
six were novel , 
 while seven were previously identified ( figure 1b ) . in comparison ,
the page+lc  ms / ms workflow identified 19 
 seps , with seven of these being novel .
these results indicate that 
 both mwco and page fractionation are able to identify similar number 
 of total seps ( 13 vs 19 ) per lc 
none of the novel seps discovered by these two 
 methods overlapped , resulting in the discovery of an additional 13 
 human seps ( six from mwco and seven from page ) .
this analysis led to the identification 
 of 94 seps and 80 novel seps ( figure 1b ) .
thus , 
 the two workflows that utilize erlic identify 9094 seps per 
 run , while workflows without erlic identified 1319 seps per 
 run .
as expected , increased fractionation results in better coverage , 
 and there is no substantial difference between different methods of 
 peptidome size fractionation ( i.e. , page or mwco ) .
we hypothesize that the low 
 natural abundance of seps and shotgun peptidomics , which is inherently 
 stochastic , results in the low overlap 
 among samples . indeed , the yates lab has demonstrated that in complex 
 mixtures data - dependent data acquisition does not completely sample 
 all peptides in a sample and therefore does not provide information 
 on all ions . on the basis of models of 
 this process
, they determined that for yeast - cell - soluble lysate 10 
 replicates are required to achieve 95% saturation of the proteome .
in addition , most seps are short ( < 100 amino 
 acids ) such that they do not generate many tryptic peptides that can 
 be used to identify a sep . in most cases
, we detect only a single 
 peptide for each sep identified , and if this peptide is missed due 
 to inefficient ionization or low abundance then the entire sep and 
 sorf is overlooked .
together , these factors contribute to the variable 
 detection of seps . if sep detection was stochastic
, then biological 
 and technical replicates would be expected to show little overlap 
 in the seps identified per lc 
we repeated the page+lc  ms / ms 
 for three additional k562 samples for a total of four biological replicates 
 ( which includes the sample from figure 1 ) . 

an average of 22 seps were detected per run with a range between 11 
 and 37 seps in each sample ( figure 2a ) .
of 
 the 87 total seps identified , 26 overlapped with previously detected 
 seps and 61 were novel , for an average of 15 new human seps per run . 

these 
 results bring the total number of novel seps detected here to 147 
 ( 80 from page+erlic+lc  ms ( figure 1 ) , 
 6 from mwco + lc  ms ( figure 1 ) , and 
 61 from four page+lc  ms biological replicates ( figure 2 ) ) .
the lack of overlap between samples is consistent 
 with our previous observations and supports the idea that sep detection 
 is variable , as predicted from proteomics studies .
( a ) number of seps detected in four biological replicates of 
 k562 cells .
the detected seps include 
 the total number of seps identified as well as the novel seps that 
 were characterized for the first time .
( b ) three technical replicates 
 of biological replicate # 4 from panel a were performed using the page+lc 
the total number of seps detected in 
 each run ( black ) , nonoverlapping seps ( gray ; seps that were not present 
 in either of the other two technical replicates ) , and novel seps ( light 
 gray ; seps that were not detected in any other analysis ) .
we analyzed biological replicate # 4 ( figure 2a )  where we identified 37 total seps in the first run  
 two more times to provide a total of three technical replicates . in 
 the three runs , we identified 37 , 29 , and 29 seps in each run ( figure 2b ) .
of the 29 seps identified in the second run , 
 25 were not detected in the first run ( i.e. , nonoverlapping seps ) , 
 and of the 29 detected in the third run , 15 were not detected in the 
 first or second runs ( figure 2b ) .
the number 
 of novel seps identified per run decreased from 28 to 12 as more runs 
 were performed , but there was still a substantial number of novel 
 seps discovered even after three technical replicates .
this result 
 affirms the hypothesis that sep detection is stochastic and demonstrates 
 the value in performing biological or technical replicates to increase 
 the number of seps discovered .
additionally , we also performed five 
 more technical replicates ( using biological replicate # 3 from figure 2a ) and detected 48 seps ( with 32 of these being 
 novel seps ) ( supporting information , figure 
 1 ) . at this point , we had identified a total of 195 novel seps ( i.e. , 
 not identified in slavoff et al . or vanderperre et al . ) in k562 cells 
 through a combination of different workflows and biological and technical 
 replicates .
three to four biological / technical replicates matched 
 the total 
 number and novel seps identified through an erlic fractionation ; however , 
 we analyzed a total of 25 erlic fractions by lc 
thus , 
 it seems more efficient to perform multiple technical and or biological 
 replicates when wanting to identify more seps , as predicted from similar 
 conclusions made with data - dependent proteomics experiments .
finally , a handful of seps was detected among 
 biological or technical replicates repeatedly , such as asnsd1-sep 
 and cir1-sep .
asnsd1-sep is the most frequently sep and therefore 
 is likely to have high cellular concentration and stability .
asnsd1-sep 
 also shows an unmistakable evolutionary signature of protein coding 
 regions ( supporting information , figure 
 2 ) , as measured across 29 eutherian mammals by phylocsf .
in total , 195 novel seps represent a > 200% 
 increase 
 from the previous study and also the largest number of seps ever reported 
 from a single cell line . in the majority of cases
analysis of our data showed that only 7 out of the 195 novel 
 seps had more than one unique peptide to support the protein - coding 
 potential of the sorf . to obtain additional data to support the identification 
 of a novel protein - coding sorf , we previously relied on molecular 
 biology .
we cloned the candidate protein - coding 
 sorfs and tested whether they produce seps in mammalian cells to ensure 
 that the newly identified sorf actually coded for proteins . while 
 successful , this approach is time - consuming and does not provide the 
 necessary throughput to validate large numbers of seps easily .
we 
 decided to use mass spectrometry instead of molecular biology to increase 
 the throughput and provide more evidence of the endogenous detection 
 of seps .
this approach would 
 afford more than one peptide from the sorf and in doing so would provide 
 the necessary data to validate the sorf and should increase throughput .
skyline , a program designed to identify the best tryptic peptides 
 from an orf for targeted mrm experiments , was used to define the mrm transitions for peptides derived from 
 105 seps .
these seps included the 81 from the page+erlic+lc  ms 
 and 7 from mcwo+lc  ms ( figure 1 ) as 
 well as 17 seps from the biological replicates # 1 and # 2 , which were 
 identified by page+lc 
trypsin digestion of these 105 seps resulted 
 in 224 tryptic peptides , and over 700 transitions were predicted by 
 skyline and monitored by targeted mrm lc 
the total number 
 of seps was capped at 105 in this targeted mrm lc 
ms experiment 
 due to the total number of mrm transitions that could be easily monitored 
 per run .
this experiment confirmed the presence of 62 peptides out 
 of the possible 224 ( 27% ) , and the identification of these peptides 
 resulted in having at least two peptides identified for 36 out of 
 the 105 seps ( 34% ) ( supporting information , table s1 ) .
skyline analysis of prr3-sep ( figure 3 ) , for example , identified mrm transitions for four tryptic 
 peptides , and a targeted lc  ms / ms using these transitions identified 
 the existence of two out of four of these peptides ( supporting information , figure 3 for ms / ms of prr3-sep peptide 
 that we detected ) .
along , with the prr3-sep peptide identified during 
 shotgun peptidomics , we now have a total of three peptides identified 
 from the prr3-sep , which provides the necessary confirmation that 
 the prr3 sorf is protein - coding . validating seps with targeted mass spectrometry . 

ms identifies additional 
 peptides from this sep . the tryptic peptide ( blue box ) that was detected 
 in the original shotgun proteomics experiment led to the initial identification 
 of the prr3-sep .
to identify additional peptides from prr3-sep , we 
 used skyline to predict mrm transitions for four tryptic peptides 
 from prr3-sep , and this information is fed into a targeted lc 
this experiment identified peptides for two out of the 
 four peptides and provided an additional two peptides ( red and purple 
 boxes ) to validate this prr3-sep . using molecular biology and peptide synthesis
we had previously 
 validated 17 out of 86 novel seps ( 20% ) by expression or coelution 
 over several weeks .
here we validated 
 36 out of a 105 seps ( 34% ) by identifying a second peptide in approximately 
 1 week .
thus , using 
 skyline to define mrm transitions for 
 sep tryptic peptides and targeted mrm lc  ms / ms to validate 
 seps provides a much more facile and efficient approach .
we analyzed 
 the length distribution , codon usage , and source of rna to determine 
 whether the 195 newly identified seps in k562 cells differ substantially 
 from the 86 seps we had previously identified .
the length distribution for the seps was determined by 
 using aug - to - stop or upstream stop - to - stop ( i.e. , distance between 
 two in frame stop codons that encompass the sorf ) .
we did not try 
 to predict alternative start codons for the length distribution because 
 we did not want to bias the analysis toward shorter lengths .
the seps 
 range between 8 and 134 amino acids long , with the majority ( > 90% ) 
 of new seps being < 100 amino acids long ( figure 4a ) . overview of 195 novel seps identified in k562 cells .
( a ) length 
 of each sep was determined using a defined set of criteria ( see methods ) , and the length distribution reveals that 
 the majority ( > 90% ) of seps discovered are between 8 and 100 amino 
 acids .
( b ) seps utilize aug , near cognate codons ( i.e. , one base away 
 from aug ) , and unknown codons to initiate translations .
( c ) seps are 
 primarily derived from nonannoated rnas ( i.e. , not found in refseq 
 database ) , but refseq rnas do account for the production of 24% of 
 these seps . for the refseq - rnas ,
the sorfs are found on coding rnas 
 at the 3-utr and cds and on noncoding rnas such as antisense 
 rnas and noncoding rnas .
an upstream in - frame aug was assumed to be the start if present ; otherwise , 
 the initiation codon was assigned to an in - frame near - cognate codon 
 ( ncc ) , which differs from aug by a single base .
nccs were commonly 
 found in ribosome - profiling experiments and our previous sep discovery effort , so this result is consistent with what has already been observed . 

if neither of these criteria was met , then no start codon was assigned . 

many of these seps ( 70% ) do not appear to initiate with an 
 aug codon ( figure 4b ) .
lastly , we tried 
 to account for the rnas that are responsible for 
 producing these seps . first , we determined the rnas in the refseq 
 database that produce seps , and we refer to this pool of rnas as  annotated 
 rnas  .
these refseq rnas are primarily mrnas , which already 
 contain a protein - coding orf . slightly over a quarter of all seps 
 we discovered , 47 in total , are derived from refseq rnas ( figure 4c ) a breakdown of the distribution of these seps 
 on the rnas reveals that a majority are found on the 3-utr . 

we counted sorfs in the 3-utr only if there was an additional 
 stop codon between the start of the sorf and the stop codon of the 
 upstream orf and to avoid identifying read - through products .
in addition , we did not identify any splice acceptor sites at the 
 5-end of the 3-utr sorfs , indicating that these were not alternative exons .
seps are 
 also produced from sorfs regions that are frame - shifted 
 within the coding sequence ( cds ) of the longer orf .
these seps are 
 likely produced from rna splice forms that can only translate the 
 sorf to produce the sep because there is no plausible mechanism to 
 explain the production of the orf and sorf from the same mrna .
because splice forms are difficult to distinguish 
 by rna - seq , further experimentation is necessary to validate that 
 some seps are produced from a splice form of a known annotated rna . 

the remaining sorfs are found in the 5-utr of rnas ( two seps 
 in this study are generated from 5-utr of refseq annotated 
 rnas , and these seps were detected previously in the study by vanderperre 
 et al . ) , noncoding rnas , and antisense rnas ( i.e. , reverse - complement 
 of known rnas ) , which are produced at sites of transcription . the discovery of a protein - coding sequence within a rna that is 
 annotated as noncoding reveals a weakness in common algorithms that 
 assign protein - coding genes . the small 
 number of sorfs in the 5-utr of refseq rnas is the biggest 
 difference between this set of seps and the previously reported set , where the majority of refseq sorfs we found 
 were in the 5-utr
. there could be several reasons for this , 
 including the possibility that sorfs in the 5-utr produce 
 the most abundant seps and therefore we and others already discovered 
 the majority of them .
transcripts that are not part of the refseq 
 database are considered to be  non - annotated  .
we identified 
 148 seps that were generated from these nonannotated transcripts in 
 the k562 rna - seq database .
thus , there are still mrnas and protein - coding 
 genes that remain to be discovered .
we also measured the lengths , 
 initiation codon usage , and rna source 
 for the 36 mrm - validated seps from this set of 195 seps to determine 
 whether mrm targeting is enriching for a particular class of seps . 

we find a similar distribution for sep length , start codon usage , 
 and sep mrna refseq annotation for the 36 mrm - validated seps ( supporting information , figure 4 ) as we do for 
 the 195 seps ( figure 3 ) , indicating that no 
 bias is introduced during the targeted mrm experiment and further 
 supporting the use of skyline - targeted mrm as a general , rapid approach 
 for the high - throughput validation of seps . to ascertain
whether seps are found in other 
 cell lines and whether some seps are specific to certain cell lines , 
 we profiled the mcf10a and mdamb231 cell lines .
these are breast cancer 
 cell lines that differ in their invasiveness , with mdamb231 being 
 invasive .
invasiveness is a measure of 
 the ability of a cell line to tunnel through a matrix in cell culture 
 and is thought to model the aggressiveness of the cancer cell line .
we obtained rna - seq data for these cell 
 lines , assembled these data into a transcriptome , and then translated 
 these sequences into a custom protein database .
ms / ms led to the identification of 
 12 and 17 seps , respectively ( figure 5a and supporting information , figure 5 ) .
ms / ms experiment 
 validated 14 of these seps ( out of 29 )  9 in the mcf10a cell 
 line and 5 in the mdamb231 cell line ( figure 5b ) .
( a ) steps in the 
 discovery and validation of seps from these cell lines .
( b ) total 
 of nine and five seps were validated using mrm in the mcf10a and mdamb231 
 cell lines , respectively .
( c ) these 14 validated seps were targeted 
 in mcf10a and mdamb231 , while 12 seps found in both cell lines , two 
 seps , tasp1-sep , and camd8-sep , were specific to the mdamb231 cell 
 line .
these 14 seps were targeted mrm 
 lc  ms in both cell lines 
 ( mcf10a and mdamb231 ) to determine whether any of these seps are specific 
 to either cell line . out of the 14 seps targeted ,
12 are present in 
 the mcf10a and mdamb231 cell lines , while two seps are found only 
 in the mdamb231 sample ( figure 5c ) .
together , 
 these experiments demonstrate that seps are found in additional ( i.e. , 
 not k562 ) cell lines and that some seps might be specific to particular 
 cell lines . to determine whether 
 we could find seps in human tissue , we used the protein database generated 
 from k562 cells ( this was the largest database we had ) and analyzed 
 a human breast cancer tissue biopsy by page+lc 
this 
 analysis yielded 25 seps , 22 of which were novel and 3 were also found 
 in k562 cells .
one sep found on the mybl2 rna ( mybl2-sep ) was found 
 in every sample we analyzed ( tumor sample , mcf10a , mdamb231 , and k562 
 cell lines ) , indicating that some seps are ubiquitous and may serve 
 broad biological roles .
these newly identified 25 tissue - derived 
 seps ( tdseps ) were then analyzed to estimate the lengths of the sorfs , 
 their initiation codon usage , and whether the rnas that produce these 
 seps were annotated or nonannotated .
the sep length for these tdseps 
 varied between 15 and 138 amino acids , the percentage of aug usage 
 was 24% , and most were derived from nonannotated rnas ( 80% ) , which 
 is consistent with data obtained from cell lines ( i.e. , k562 , mcf10a , 
 and mdamb231 ) ( figure 6 ) .
these data support 
 the idea that seps are ubiquitous and found in tissues as well , which 
 further enhances the interest in this class of polypeptides .
( a ) length distribution , 
 ( b ) initiation codon usage , and ( c ) rna source of tdseps were similar 
 to the distributions seen for seps derived from cell lines .
we 
 tested several parameters for our sep discovery workflow and 
 determined that running replicates ( technical / biological ) is the most 
 efficient way to detect more seps . in total , we describe the discovery 
 of an additional 237 human seps ( table 1 ) , 
 demonstrating the prevalence of this class of polypeptides . with an 
 increasing number of seps discovered through our shotgun profiling 
 it became obvious that our previous approach for validation would 
 not suffice and therefore we utilized a targeted mrm lc 
ms / ms 
 approach that relies on skyline to rapidly 
 identify multiple peptides from a single sep / sorf . through the analysis 
 of additional cell lines and a tumor biopsy
, we also find that seps 
 are ubiquitous and that at least some seps are specific to a cell 
 line .
this effort provides the necessary evidence for us to begin 
 to start large - scale sep profiling experiments .
these experiments 
 could be done by differentially profiling seps in disease models to 
 identify seps that might cause disease or can serve as a biomarker .
 the existence of nonannotated protein - coding 
 human short open reading 
 frames ( sorfs ) has been revealed through the direct detection of their 
 sorf - encoded polypeptide ( sep ) products . 
 the discovery of novel seps 
 increases the size of the genome and the proteome and provides insights 
 into the molecular biology of mammalian cells , such as the prevalent 
 usage of non - aug start codons . through modifications of the existing 
 sep - discovery workflow 
 , we discover an additional 195 seps in k562 
 cells and extend this methodology to identify novel human seps in 
 additional cell lines and human tissue for a final tally of 237 new 
 seps . 
 these results continue to expand the human genome and proteome 
 and demonstrate that seps are a ubiquitous class of nonannotated polypeptides 
 that require further investigation . 
Introduction
Materials
and Methods
Results and Discussion
Conclusions