one 
 important goal of the chromosome - centric human proteome project 
 ( c - hpp ) , an international consortium , is to completely map the human 
 proteome by identification of proteins in selected tissues and cells . as an articulated goal
, the c - hpp is also determined to capture 
 biological features of gene variation , gene regulation , and protein 
 expression mapped by chromosome localization .
consequently , all c - hpp 
 projects are generating and reporting protein data in a format that 
 is aligned with the dna sequence of individual chromosomes and with 
 the output of transcriptome data ( rna sequencing ) .
in addition to 
 sequential data derived from the most frequent proteoforms ( consensus 
 or canonical sequences ) , it is desirable to characterize additional 
 major proteoforms , such as alternative splicing transcript ( ast ) , 
 single amino acid variants ( sav ) , and post - translational modifications 
 ( ptms ) .
a complementary hpp activity , the biology / disease - driven hpp 
 ( b / d - hpp ) , is focused on generation of knowledge from studies of cellular 
 mechanisms and biochemical processes , analyzing proteomes associated 
 with human diseases .
the results are expected 
 to facilitate routine determinations of processes and disease relevant 
 proteins in life science research . at present ,
human protein 
 sequence data is collected at a rapid 
 pace , predominantly due to the technological advances in mass spectrometry 
 ( ms ) .
however , roughly 10% is still missing , due to the lack of quality 
 observations of certain proteins , incorrect gene annotation , very 
 low abundance or absence of expression in most tissues , or unfavorable 
 structure ( or cleavage sites ) for bottom - up ms studies . on the other 
 hand
, two recent publications have set new milestones in providing 
 the most complete draft of the human proteome to date . according to the proteomics db ( http://www.proteomicsdb.org ) ,
the current state of the chromosome 19 is at 96.4% completion 
 ( 1352 genes and 1304 proteins ) and includes details about a high number 
 of asts (  isoforms  ) .
nevertheless , 
 if all proteoforms with different sequences that have biological functions 
 are considered , then the exact size of the human proteome is still 
 unknown today and may reach extremely high numbers , up to several 
 million . because the hpp s directive is to 
 identify at least one ast and one sav of each consensus proteins as 
 well as three major ptms ( phosphorylation , glycosylation and acetylation ) , the number of proteoform entries in the complete 
 human proteome is estimated in the range of 100  000 to 1  000  000 .
the latest downloadable version of the nextprot database ( http://www.nextprot.org ) includes 20  055 protein entries 
 ( released on 19 september 2014 ) .
there are 1430 genes and 1426 protein 
 entries reported for chromosome 19 , which are identified at the protein 
 level ( 1129 entries ) , transcript level ( 248 entries ) , uncertain ( 36 
 entries ) , homology ( 10 entries ) , and predicted ( 7 entries ) .
although 
 this database fasta file does not include any information about savs 
 ( nor do the uniprot databases ) , references to mutant proteoforms are 
 listed on the nextprot web site when certain proteins are selected . 
 however , the level of identification for these savs is not indicated 
 otherwise .
genomic databases are typically more detailed , providing 
 a list of missense single nucleotide variations ( missense snvs ) of 
 each human gene .
databases such as 1000 genomes ( http://www.1000genomes.org ) provide population - based frequency information as well .
other genomic 
 databases such as the catalogue of somatic mutations in cancer ( cosmic 
 at http://cancer.sanger.ac.uk/cancergenome/projects/cosmic/ ) have accumulated gene expression variants and revealed disease 
 relation .
genome investigations have identified a large number 
 of gene mutations , 
 including missense snvs that are currently 
 used for risk analysis of cancer , for example , brca1 and brca2 . however , it is important to underline 
 that not the genes but the proteins have biological function and are 
 the working units of cell machinery . consequently , their expression 
 patterns in both qualitative and quantitative aspects should be characterized 
 when the altered biology of diseases are in the focus of study . despite 
 this fact , the majority of protein identifications are based upon 
 ms / ms data from shotgun proteomics experiments searched against protein 
 databases such as uniprotkb and nextprot , which are typically restricted 
 to proteoforms with consensus and ast sequences only . thus , direct 
 searches of savs can not be performed with tandem spectra and such 
 data is seldom reported , despite the fact that savs may play important 
 role in disease biology .
some search 
 engines are constructed by use of various approaches 
 and algorithms in order to provide tools to recognize savs . as an 
 example , peaks ( bioinformatics solutions inc . ,
waterloo , on , canada ) 
 offers an algorithm ( spider ) that can 
 utilize the match of peptide spectrum matches ( psms ) with replaced 
 amino acids .
others , like the freely available x!tandem ( http://www.thegpm.org ) employs another strategy that systematically changes each residue 
 in a peptide for all other possible amino acids and score the mutated 
 peptide ( and all potential modifications ) against all of the available 
 ms / ms spectra .
therefore , bioinformatic tools do exist that aid in 
 the process of identifying savs in shotgun proteomics data , and these 
 tools can prove to be valuable in the characterization of savs associated 
 with a particular disease .
glioblastomas are among the deadliest 
 of cancers ; fewer than 10% 
 of patients survive five years after diagnosis . even with standard - of - care 
 treatments , tumors nearly always recur .
recurrence is at least in 
 part due the existence of glioma stem cells ( gscs ) , which are resistant 
 to radiation and chemotherapy .
we have acquired comprehensive , quantitative 
 data sets from thirty - six gsc lines , including gene activity and protein 
 expression .
we have previously examined patterns of global as well 
 as chromosome-19-specific protein and gene expression in a subset 
 of the 36 gsc lines .
our hypothesis is that gscs , 
 as a rare type of diseased cell type , harbor protein savs due to germline 
 or somatic mutations .
those proteins may normally have cell protective 
 properties , but proteins with amino acid substitutions have the potential 
 to be transformed into promoters of genetic instability or invasivity , 
 as well as ineffective regulators of epigenetic or metabolic control . 
 through the use of a customized database and bioinformatic tools , 
 we provide the first comparative data of chromosome 19 snp products 
 on 36 glioma stem cell lines and compelling evidence for the expression 
 of a sav in branched - chain aminotransferase 2 ( bcat2 ) , a protein encoded 
 by chromosome 19 , in six of the gsc lines .
we have devised a 
 systematic workflow to address the second level 
 of protein expression complexity ( represented by the savs ) and have 
 applied this workflow in the identification of missense snv products 
 at the protein level in glioma samples based on high resolution ms / ms 
 data .
we used a custom protein database to widen the search window 
 for mutant proteoforms .
we have identified and further verified novel 
 mutant proteoforms that might be strongly associated with glioma .
isolation 
 of gscs from patient tumors was performed as previously described in accordance with the institutional review 
 board of the university of texas m.d .
downstream proteomic analyses were performed on identical cell culture 
 batches in order to reduce the influence of batch variance in the 
 comparative assays .
cell lysates from 2  10 gscs were reduced , alkylated , and analyzed in triplicate by lc 
ms / ms 
 on an orbitrap elite equipped with an easy nanolc 1000 pump ( thermo 
 fisher scientific , waltham , ma ) as described in a previous publication .
briefly , peptide mixtures were separated on 
 a c18 column ( proteopep ii , new objective , 10 cm  75 m ) 
 using a 240 min gradient ( solvent a , 0.1% formic acid in water ; solvent 
 b , 0.1% formic acid in acetonitrile ) .
data were acquired using high - resolution 
 data - driven analysis ( dda ) , with the survey scan ( ms ) acquired in 
 the orbitrap at 60  000 resolution ( at m / z 400 ) in profile mode .
the survey scan was followed by 
 ten hcd ms / ms spectra , acquired in centroid mode at 15  000 
 resolution in the orbitrap . for 
 database searching the technical replicates
were combined and searched 
 against a combination of the uniprotkb / swissprot - human database ( 2014_06 
 version , 40  548 protein entries ) with all known chromosome 
 19 sav sequences ( 132  264 ) together with 115 sequences of the 
 common repository of adventitious proteins ( crap ) contaminant database 
 ( http://www.thegpm.org/crap/index.html ) . searches were 
 performed using peaks 7.0 ( bioinformatics solutions ) with 10 ppm parent 
 mass error tolerance and 0.025 fragment mass tolerance , allowing for 
 a maximum of two missed cleavages and one nonspecific cleavage .
carbamidomethyl 
 cysteine was set as a fixed modification , and oxidation ( m ) and phosphorylation 
 ( sty ) were set as variable modifications .
peptides assigned as savs with a 10log p score > 30 were selected for further validation .
homology searching 
 was performed using the blast utility ( www.uniprot.org ) 
 against the uniprot - human database in order to confirm that each peptide 
 sequence was unique . for those peptides that passed these initial 
 filters , manual verification of ms / ms
spectral assignments of sav 
 peptides was performed by comparing m / z values for ions observed in the ms / ms spectrum with a theoretical m / z list generated using the ms - product 
 utility on the protein prospector web site ( http://prospector.ucsf.edu ) . for optimization of the assay , isotopically
labeled 
 peptides were mixed and diluted with 5% acetonitrile ( acn ) at a concentration 
 of 325 pmol/l for each synthetic peptide .
ms / ms using a tsq vantage triple quadrupole 
 mass spectrometer equipped with an easy n - lc ii pump ( thermo scientific , 
 waltham , ma ) . samples ( 2 l ) were injected onto an easy c18-a1 
 precolumn ( thermo scientific , waltham , ma ) , and following online desalting 
 and trapping at a pressure of 280 bar , the peptides were separated 
 on a 75 m  150 mm fused silica column packed with reprosil 
 c18 ( 3 m , 120  from dr .
separations 
 were performed in a 55 min linear gradient from 5 to 40% acn containing 
 0.1% formic acid at the flow rate of 300 nl / min .
the ms analysis was 
 conducted in positive ion mode at 1750 v applied spray voltage .
the 
 transfer capillary temperature was 270 c and tuned s - lens value 
 was used .
selected reaction monitoring ( srm ) transitions were acquired 
 in q1 and q3 operated at unit resolution ( 0.7 fwhm ) , the collision 
 gas pressure in q2 was set to 1.2 mtorr .
scheduled method was used 
 for data acquisition with 4 min time windows and the cycle time was 
 set to 1.5 s , whereas the maximum number of consecutive transitions 
 was 50 .
the srm assay optimization was done with the aid of 
 skyline v2.5.0.6079 software ( maccoss lab ) .
primarily , high numbers 
 of transitions , including b- and y - ion series , were chosen for each 
 peptide at both 2 + and 3 + charge states .
the five transitions , which 
 produced the most abundant signals without observed interferences 
 in the glioma samples , were selected for further analysis and utilized 
 for unambiguous identification .
for the sample analysis , the 
 same chromatographic conditions were 
 used as described above for the assay development .
identical srm parameters 
 were used for the heavy and native forms of each peptide .
all raw 
 data generated on the triple quadrupole mass spectrometer were imported 
 to skyline for data analysis .
paired - end 
 whole transcriptome sequencing was performed on the illumina hiseq 
 platform after random priming and rrna reduction .
transcript reads 
 were aligned to human reference transcriptome ( ensembl version 64 ) 
 using prada .
downstream data analyses 
 including variant calling were performed using burroughs - wheeler alignment , 
 samtools , and genome analysis toolkit .
more details on transcriptomic 
 data acquisition and analysis are available in lichti et al .
the bcat2 sav 
 was modeled using a combination of two crystal structures : the substrate , l - isoleucine covalently bound to the cofactor pyridoxamine phosphate 
 ( pmp ) , bound form of the enzyme ( pdb code : 1kt8 ) , and the pmp bound form ( pdb code : 1kta ) .
both were downloaded from www.pdb.org and prepared 
 using the visual molecular dynamics ( vmd ) software .
as 1kt8 is missing residues near the mutation site , it was used as a reference 
 structure onto which the coordinates of 1kta were superimposed in order to include 
 the coordinates of residues 175 to 191 .
residue 186 ( please , 
 note that our residue numbering scheme in this paper follows the recommendations 
 of human genome variation society and thus may differ by six from 
 other authors ) was then mutated from threonine to arginine , and hydrogens 
 were added .
the resulting coordinates were stored and from them 
 an additional starting structure was generated in which the arg186 
 backbone  dihedral was rotated from + 132 to 60 
 by rotating parts of both arg186 and pro187 , while  was held 
 constant at 80 . after rotation , residues 185188 were 
 minimized with the remainder of the protein fixed .
both these final 
 coordinates and the original unrotated structure were then minimized , 
 holding all atoms of the protein fixed except for those in residues 
 175191 .
the final coordinates of each were stored and used 
 as the basis for side chain rotamer generation . for 
 both
the original and rotated  backbone structures , 
 the side chain angles 1 and 2 of 
 arg186 were subsequently rotated in six increments of 60 , yielding 
 a total of 72 structures . from the original structure ,
the side chain 
 was rotated about the 1 bond without altering the 
 original 2 torsion , generating an additional six 
 structures .
two independent minimizations were performed on 
 each of the 78 structures : one in which the entire system was minimized , 
 and one in which only residues 177188 were minimized while 
 all else was held fixed .
the final coordinates of each minimization 
 were used for electrostatic free energy calculations and structure 
 analysis .
electrostatic free energy calculations were conducted on the first 
 monomer ( chain a ) of each of the 156 bcat2 structures .
the pdb 2pqr software was used to generate pqr files for each structure . 
 here
, the charmm27 parameter set was used to obtain partial atomic 
 charges and atomic radii , and a ph of 7.0 was used in assigning protonation 
 states for titratable residues .
the total electrostatic free energy 
 of the system was calculated using the apbs software package .
the structures of each minimization set were 
 ranked by their corresponding energies and the best ten structures 
 of both sets were analyzed using vmd .
we addressed the determination of mutant proteoforms 
 in biological samples , a high priority within the c - hpp . a novel systematic 
 approach
was developed that is based on searching shotgun proteome 
 tandem spectra , collected via data dependent analysis , against a customized 
 protein sequence database containing chromosome 19 savs .
the new database 
 used for identification of mutant proteoforms was compiled with all 
 human consensus proteins together with a new set of missense snv - derived 
 sequences of chromosome 19 . to improve search quality , the frequently 
 used crap contaminant protein database was also included , resulting 
 in a database of 172  927 protein sequences in total . when search space is confined to short sequences derived from only 
 one chromosome , a problem
is expected that is similar to one observed 
 when a search is performed against a database for a partially sequenced 
 organism . in a standard target - decoy approach , a number of high quality 
 spectra will be wrongly matched to decoy sequences simply due to the 
 absence of correct peptide sequences in the target database .
this 
 makes adequate significance estimation , often represented in proteomics 
 as global false discovery rate , rather challenging .
because many proteins 
 exist in multiple isoforms , for example , structural proteins such 
 as myosin or tubulin , there is a high risk that a spectrum may be 
 also erroneously assigned to a sav - containing peptide .
those issues 
 can be addressed by creating a custom decoy database , a task that is not trivial in the case of short , low complexity 
 sequences .
alternatively , protein sequences from the other homologous 
 species , or in our case the rest of the canonical proteome from the 
 same species , could be appended to a database of interest , adding 
 more possibilities for the correct peptide - spectrum matches in a target 
 data set and , therefore , enabling target - decoy approach implemented 
 by standard search engines .
because 
 protein databases for shotgun proteomic searches do not 
 commonly include sav sequences , and different search engines may perform 
 differently with the same database ; three search engines ( sequest , 
 peaks , and mascot ) were employed and the corresponding search results 
 were compared .
triplicate data files for each cell line were used 
 as a single input for database searching , initially performed with 
 nearly identical search parameters .
an initial search and validation 
 on a subset of the gscs revealed 
 several savs .
manual verification of spectra containing savs led to 
 confirmation of a t186r substitution of branched - chain aminotransferase 
 2 ( bcat2 ) ( see figure 1 ) , a known natural variant 
 of the protein , in gsc28 .
this finding was further 
 validated at the transcript level and by srm ( see sections 3.2 and 3.3 , respectively ) .
hcd - ms / ms 
 spectrum of pvlignepslgvsqpr , supporting assignment of the p.thr186arg proteoform 
 of bcat2
. a complete list of theoretical and observed m / z values for ions observed in this spectrum 
 can be found in supporting information table 
 4 . due to the high confidence in 
 this
finding , identification of the 
 sav - containing peptide of bcat2 ( pvlignepslgvsqpr ) in gsc 28 was used as a positive control 
 for all database searches .
according to the snap database of gpmdb , 
 this tryptic peptide of bcat2 is less frequently observed compared 
 with its longer , miscleaved forms ( http://snap.thegpm.org/%7e/dblist_protein_mut/label=ensp00000322991 ) .
however , this may be a biased result of search algorithms that 
 consider such peptides as false .
interestingly , 
 peaks db was the only search engine that successfully identified the 
 peptide containing bcat2 p.thr186arg , with a 10log p score of 74 .
neither sequest nor mascot identified this 
 peptide , despite the high confidence of the assignment in peaks .
our 
 initial thought was that both failed to identify the peptide due to 
 trypsin cleavage specificity assigned in the search parameters , as 
 the n - terminus of the sav - containing peptide arises from cleavage 
 between arginine and proline .
therefore , we adjusted the trypsin specificity 
 in mascot to allow for cleavage n - terminal to proline and repeated 
 the search using gsc 28 as a positive control . because mascot again 
 failed to identify the peptide containing bcat2 p.thr186arg , we focused on peaks db to identify savs in the remaining cell lines 
 ( see table 1 for a verified list and supporting information table 1 for a complete 
 list of sav peptides identified by peaks ) . annotated hcd 
ms / ms spectra 
 and the corresponding ion tables can be found in supporting information figure 1 .
conflict in dbsnp and ensembl databases , 
 which indicates this mutation q  h. for those peptides that were confirmed to be truly 
 unique by homology 
 searching , manual verification of spectra was performed in order to 
 ensure that the assigned savs were correct . because our data were 
 acquired using high resolution ms in an orbitrap , mass error for the 
 precursor ions was used as an initial filter of quality . because the 
 mass errors for peptide assignments in each lc  ms / ms file fall 
 into a normal distribution , and 95% of correct assignments fall within 
 two standard deviations of the mean error , we removed peptides whose mass error was dramatically higher than 
 for other peptides within the same analytical run .
in a similar manner , 
 high mass accuracy ms / ms data facilitates spectral verification .
leucine 
 and isoleucine are indistinguishable from one another by exact mass 
 and chemical deamidation can convert glutamine and asparagine to glutamic 
 acid and aspartic acid , respectively .
furthermore , the carbamidomethyl 
 group ( 57.02146 ) has the same exact mass as a glycine residue .
therefore , 
 species formed due to overalkylation ( addition to nucleophilic amino 
 acid side chains or the peptide n - terminus ) can be assigned incorrectly 
 as being due to savs .
therefore , it is often critical to have orthogonal 
 validation of savs at the transcript level .
ms / ms is presented in table 1 . a further list of sav - containing peptides that 
 require additional transcript level validation is provided in supporting information table 2 . each identified peptide containing
, 
 we evaluated the individual reads from the transcriptome sequencing 
 to quantitate the frequency of c ( acg coding thr ) and g ( agg coding 
 arg ) alleles .
the c  g variant occurs at position 613 in the 
 reference mrna sequence of bcat2 nm_001190 ( rs11548193 in dbsnp ) .
a total of 8 gsc lines contained reads of > 50% 
 with c613 g variant , including gscs 28 , 274 , 711 , 275 , 17 , 
 280 , 289 , and 293 ( data not shown ) .
the successful identification of sav proteoforms 
 in gscs was further 
 validated by srm .
selected unique peptide sequences with a single 
 mutation were synthesized and srm assays were developed using multiple 
 transitions for each peptide ( supporting information table 3 ) .
a subset of ten gsc samples ( gscs 28 , 627 , 20 , 
 112 , 129 , 522 , 275 , 711 , 274 , and 72 ) was 
 tested for four newly identified mutant proteoforms .
the primary target 
 of srm confirmation was the bcat2 p.thr186arg mutation 
 that was unambiguously verified in samples gsc 28 , gsc 711 , 
 and gsc 275 by perfectly matching transitions and retention times 
 of heavy labeled internal standard and native peptides ( see figure 2a ) .
( a ) bcat2 p.thr186arg was confirmed by matching heavy - isotope - labeled synthetic peptide 
 ( blue ) with the endogenous pvlignepslgvsqpr ( red ) , whereas ( b ) the 
 endogenous signals of elongation factor 2 p.ile665leu and endophilin - a2 p.leu95ile could not agree with 
 the internal standards .
the specificity of srm assays was utilized in investigation 
 of 
 leu  ile and ile  leu mutations that were initially 
 identified in database search despite of the isobaric nature of these 
 savs . the difference in retention times observed in reversed phase 
 chromatography due to the lower hydrophobicity of peptides containing 
 isoleucine provided a utility .
a sav of elongation factor 2 ( p13639 - 1 p.ile665leu ) was expected with longer retention time but 
 the endogenous signal with identical transitions eluted earlier than 
 the synthetic isotope labeled standard .
a reversed case was also observed 
 when an endophilin - a2 mutation ( q99961 - 2 p.leu95ile ) was investigated ( see figure 2b ) . besides their validation power
, srm assays can be further utilized 
 in quantitative analysis of mutant allele expressions in biological 
 samples .
this information is necessary in the case of heterozygous 
 samples , in particular when the expression of the mutant allele has 
 a strong concordance with biological activity of cells .
the p.thr186arg mutation on bcat2 , confirmed at the rna level to be in eight of 
 the 36 gsc lines , is located along a flexible loop in close proximity 
 to the cxxc center ( figure 3 ) . to study the 
 possible effects of this mutation
, we generated 78 p.thr186arg bcat2 variant structures by rotating the arginine side chain about 
 the 1 and 2 and scanning the  
 backbond .
the structures that where highest 
 in electrostatic free energy or contained severe violations of the 
 chemical constrains were removed from the data set . of the structures 

lowest in electrostatic free energy , which satisfied the packing and 
 stereochemical constraints in minimization , four were grouped within 
 a few kilocalories per meters , whereas the rest were separated by 
 over 100 kcal / m in electrostatic free energy and so were not considered 
 for further analysis .
best four bcat2 p.thr186arg structures 
 are superimposed 
 with arg186 , glu229 , and the cxxc ( cys321 and cys324 ) highlighted . 
 among rotamers with residues 177188 minimized ,
the four overlaid 
 here were hundreds of kcal / m lower than the average of all rotamers . 

the arginine side chain is found in a salt bridge with glu229 and 
 toward the inside of the flexible arm , where the arginine side chain 
 has the most stable hydrogen bonding .
these lower energy positions 
 of the arginine side chain all greatly change the electrostatic environment 
 as compared with the thr186 wild type , and this may influence the 
 cxxc oxidation reaction that regulates enzyme activity . as seen by the four lowest energy structures ( figure 3 )
, the arg186 side chain gravitates toward the carboxylate 
 group of glu229 , as well as inward toward the cxxc site , both of which 
 provide stability through hydrogen bonding pairs . compared with the 
 thr186 wild type
, the arginine side chain significantly changes the 
 electrostatic environment near the cxxc , potentially shifting the 
 pka of the cysteine residues . the electrostatically 
 induced pka shift could influence the 
 cysteine protonation state and therefore the equilibrium of the oxidation 
 reaction ( figure 4 ) , consequently altering 
 enzyme activity . although direct interaction , for example , through 
 hydrogen bonding , is not sterically forbidden ,
it is also not required . 
 the electrostatic shift of the intrinsic pka of the cysteine would be sufficient to explain a change in activity . 

it has been demonstrated that the cxxc center ( cys321 and cys324 ) 
 contributes to enzyme activity through reversible disulfide bond formation 
 that prevents substrate from correctly orienting with the pyridoxal 
 phosphate ( plp ) cofactor .
reaction mechanism for 
 bcat2 , which catalyzes the transfer of an 
 amino group from leucine , isoleucine , and valine to -ketoglutarate 
 to form glutamate .
we demonstrated the utility of our custom protein database in the 
 identification of chromosome 19 savs in gscs . through a combination 
 of homology searching and manual verification of spectra identified 
 by peaks db
one of these savs , 
 bcat2 p.thr186arg , was validated orthogonally in 
 multiple cell lines by srm and by rna - seq future studies will be directed 
 toward verification of the remainder of these savs and a study of 
 the biological implications of these savs . the identification and 
 characterization of savs in glioma
has already yielded the identity 
 proteins that will be of further interest to study as potential modulators 
 of gliomagenesis , contributors to glioma pathology , and resistance 
 to standard of care treatments .
we expect that our methods are readily 
 transferrable to other cancer cells and tissues as well . to 
 improve visibility of savs , we strongly suggest extending the 
 registry of protein sequences in nextprot with information about the 
 mutant proteoforms pointing to identification levels as well as expression 
 sites .
 novel 
 proteoforms with single amino acid variations represent proteins 
 that often have altered biological functions but are less explored 
 in the human proteome . 
 we have developed an approach , searching high 
 quality shotgun proteomic data against an extended protein database , 
 to identify expressed mutant proteoforms in glioma stem cell ( gsc ) 
 lines . 
 the systematic search of ms / ms spectra using peaks 7.0 as the 
 search engine has recognized 17 chromosome 19 proteins in gscs with 
 altered amino acid sequences . 
 the results were further verified by 
 manual spectral examination , validating 19 proteoforms . 
 one of the 
 novel findings , a mutant form of branched - chain aminotransferase 2 
 ( p.thr186arg ) , was verified at the transcript level 
 and by targeted proteomics in several glioma stem cell lines . 
 the 
 structure of this proteoform was examined by molecular modeling in 
 order to estimate conformational changes due to mutation that might 
 lead to functional modifications potentially linked to glioma . based 
 on our initial findings 
 , we believe that our approach presented could 
 contribute to construct a more complete map of the human functional 
 proteome . 
Introduction
Experimental Section
Results
and Discussion
Conclusions