Cancer evolves as a result of an accumulation of mutations and chromosomal aberrations. Developments in sequencing technologies have enabled the discovery and cataloguing of millions of such mutations. The identification of protein-level...
moreCancer evolves as a result of an accumulation of mutations and chromosomal aberrations. Developments in sequencing technologies have enabled the discovery and cataloguing of millions of such mutations. The identification of protein-level alterations, typically by using reversed-phase protein arrays or mass spectrometry, has lagged, however, behind gene and transcript-level observations. In this study, we report the use of mass spectrometry for detecting the presence of mutations-missense, indels and frame shifts-in MCF7 and SKBR3 breast cancer, and non-tumorigenic MCF10A cells. The mutations were identified by expanding the database search process of raw mass spectrometry files by including an in-house built database of mutated peptides (XMAn-v1) that complemented a minimally redundant, canonical database of Homo sapiens proteins. The work resulted in the identification of nearly 300 mutated peptide sequences, of which ~50 were characterized by quality tandem mass spectra. We describe the criteria that were used to select the mutated peptide sequences, evaluate the parameters that characterized these peptides, and assess the artifacts that could have led to false peptide identifications. Further, we discuss the functional domains and biological processes that may be impacted by the observed peptide alterations, and how protein-level detection can support the efforts of identifying cancer driving mutations and genes. Mass spectrometry data are available via ProteomeXchange with identifier PXD014458. Cancer develops as a result of genetic alterations that include mutations and chromosomal abnormalities. Mutations include alterations such as single nucleotide substitutions (missense, nonsense and silent) and small insertions, deletions or frameshifts. Chromosomal abnormalities can be numerical, i.e., manifest themselves either as gains or losses in chromosome numbers (aneuploidy), or structural, i.e., lead to rearrangements such as translocations, inversions, deletions, insertions or duplications. Comprehensive high-throughput sequencing efforts have revealed a complex interplay of aberrations that define the heterogeneous nature of cancer genomes. Earlier studies have identified ~140 genes that drive tumorigenesis, when altered by mutations 1. These genes regulate three essential cellular processes through twelve signaling pathways, i.e., genome maintenance, cell survival and cell fate. Depending on type, tumors can harbor from 1-2 to 8 driver mutations that generally contribute to the development of cancer over 20-30 years in adults 1. The field of cancer genomics, supported by the advance of sequencing technologies, has witnessed, however, a remarkable growth in the past decade, leading to joint multi-institutional efforts aimed at creating comprehensive resources that encompass hundreds to thousands of gene alterations with potential diagnostic and prognostic significance. Some of these resources include the Wellcome Sanger Institute's COSMIC (Catalogue of Somatic Mutations in Cancer) and CGC (Cancer Gene Census) databases 2 , the cancer-specific projects supported by the International Cancer Genome Consortium (ICGC) 3 , the Cancer Cell Line Encyclopedia (CCLE) 4 , and the PanCancer Atlas project conducted by The Cancer Genome Atlas (TCGA) collaboration 5. With millions of documented mutations, efforts have shifted towards advancing lists of specific cancer driver genes and mutations. On the line of such efforts, the OncoKB-a precision oncology knowledge database-has expanded the number of cancer genes to 1,019, highlighting actionable alterations 6 , while the CGC has catalogued 719 genes matched to cancer hallmarks and grouped into two tiers (tier 1 with documented activity in cancer, and tier 2 with strong indication of having a role in cancer). Recently, the PanCancer Atlas project reported results that were generated through an effort that encompassed the analysis of