Variant peptide detection utilizing mass spectrometry: laying the foundations for proteogenomic identification and validation.
BACKGROUND
Proteogenomics is an emerging field at the intersection of genomics and proteomics. Many variant peptides corresponding to single nucleotide variations (SNVs) are associated with specific diseases. The aim of this study was to demonstrate the feasibility of proteogenomic-based variant peptide detection in disease models and clinical specimens.


METHODS
We sought to detect p53 single amino acid variant (SAAV) peptides in breast cancer tumor samples that have been previously subjected to sequencing analysis. Initially, two cancer cell lines having a cellular tumor antigen p53 (TP53) mutation and one wild type for TP53 were analyzed by selected reaction monitoring (SRM) assays as controls. One pool of wild type and one pool of mutated for TP53 cytosolic extracts were assayed with a shotgun proteogenomic workflow. Furthermore, 18 individual samples having a mutation in TP53 were assayed by SRM.


RESULTS
Two mutant p53 peptides were successfully detected in two cancer cell lines as expected from their DNA sequence. Wild type p53 peptides were detected in both cytosolic pools, however, none of the mutant p53 peptides were identified. Mutations at the protein level were detected in two cytosolic extracts and whole tumor lysates from the same patients by SRM analysis. Six thousand and six hundred and twenty eight non-redundant proteins were identified in the two cytosolic pools, thus greatly improving a previously reported cytosolic proteome.


CONCLUSIONS
In the current study we show the great potential of using proteogenomics for the direct identification of cancer-associated mutations in clinical samples and we discuss current limitations and future perspectives.
.
Lampros
L
Dimitrakopoulos
.
Ioannis
I
Prassas
.
Els M J J
EMJJ
Berns
.
John A
JA
Foekens
.
Eleftherios P
EP
Diamandis
.
George S
GS
Charames
D045744:Cell Line, Tumor; D002853:Chromatography, Liquid; D003600:Cytosol / Q000737:chemistry; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D013058:Mass Spectrometry; D009154:Mutation; D009369:Neoplasms / Q000235:genetics; D010455:Peptides / Q000032:analysis* / Q000737:chemistry / Q000235:genetics*; D000071696:Proteogenomics; D015203:Reproducibility of Results; D053719:Tandem Mass Spectrometry; D016159:Tumor Suppressor Protein p53 / Q000032:analysis / Q000737:chemistry / Q000235:genetics*; D054928:Validation Studies as Topic
D010455:Peptides; C495901:TP53 protein, human; D016159:Tumor Suppressor Protein p53
LC-MS/MS; SRM; p53; proteogenomics; variant peptides