2.2.1　Prediction of Tumor Risk

Mass spectrometry has been invaluable for the growth of proteomics, although detection of very low abundance proteins remains challenging.Techniques such as stable isotope labeling by amino acids in cell culture (SILAC), tandem mass tag (TMT) labeling and isobaric tag for relative and absolute quantification (iTRAQ) have tremendously improved sensitivity and protein detection by mass spectrometry.Additionally, these approaches enable simultaneous analysis and peptide quantification for multiple specimens and allow direct comparison across samples.For example,the high throughput capability of TMT LC-MS/MS enables uniform and rapid protein quantification of multiple specimens and comparison among distinct TMT sets when a specific reporter label is dedicated to a reference sample that is included with all LC-MS/MS analysis.The presence of a common reference sample permits direct comparison among specimens from different TMT sets when sample preparation and LC-MS/MS methods are standardized across analytical runs.This ability to analyze and compare large specimen cohorts is integral to harmonization among labs, and TMT labeling coupled with LC-MS /MS has become a prominent technique in multi-institution studies aimed at cancer biomarker discovery.Large-scale initiatives,such as the National Cancer Institute’s Clinical Proteomic Tumor Analysis Consortium (CPTAC),have successfully applied mass spectrometry methods for the identification of cancer-specific proteins and unique protein patterns leading to sub-classification of ovarian, breast and colorectal cancers [4-7].

Protein arrays and antibody-based proteomic methods are targeted approaches for protein detection and quantification with high sensitivity and multiplexing capabilities.These methods are not optimal for biomarker discovery as they use a directed approach, however they have great potential in a clinical setting for monitoring of validated disease biomarkers.Quantitative proteomic analysis is achieved through antibody labels that can be fluorophores, metal isotope tags, or nucleotide sequences.Recent efforts to improve multiplexing capabilities have focused on nucleotide barcode sequences that enable tag amplification and subsequent increase in signal [8].

Targeted proteomics is not limited to antibody methods.Selected Reaction Monitoring (SRM) and Multiple Reaction Monitoring (MRM) mass spectrometry techniques are emerging as reliable, high throughput assays for cancer biomarkers.Detection of target proteins is achieved with a triple quadrupole mass spectrometer, where specific peptides originating from the protein of interest are selected based on their mass to charge (m/z) ratio and subsequently fragmented into smaller components that are in turn quantified to assess protein abundance.The principle of protein detection is identical for SRM and MRM, with the primary difference that MRM is the application of SRM to multiple peptide fragments.Identifying and quantifying multiple peptides from one or more target proteins simultaneously, makes MRM capable of multiplexing with high sensitivity and the technique of choice for most directed assays.The primary advantage of MRM is the ability to detect multiple isoforms and post-translationally modified species for a given protein with high specificity in a single test run.In an effort to minimize invasive procedures, MRM assays are utilized for the detection of biomarker peptides in serum samples from breast and colorectal cancer patients among others [4, 9, 10].

The proteome is dynamic and regulated by post-translational modifications (PTM), which when aberrant can lead to uncontrolled proliferation and tumorigenesis.Prediction of impaired PTM cannot be achieved through genome and transcriptome analysis, it can only be detected through proteomic approaches that identify abnormal protein activity and/or abundance of the modified variant.Proteins exist in variable PTM states and detection of less abundant forms may be challenging.To improve sensitivity, it has become common practice, when possible, to incorporate an enrichment step in the sample preparation procedure that extracts peptides containing the targeted modification.Enriched peptides are subsequently analyzed through mass spectrometry techniques and the PTM of distinct proteins is quantified.Antibody-based techniques can also be used for PTM analysis when the modified epitope is recognized with high specificity and there is no background signal interference from other variants of the protein [11, 12].(https://www.daowen.com)

Impaired phosphorylation, usually hyperphosphorylation, is well-established as a cause for dysregulation of cell signaling and carcinogenesis.Therefore, it is not surprising that abnormal kinase activity and overexpression are frequently associated with an oncogenic phenotype.For example, genetic alterations and increased expression of the human epidermal growth factor receptor 2 (HER2) tyrosine kinase are implicated in certain breast and ovarian cancers.Although aggressive, HER2 positive breast cancer is susceptible to HER2 targeted therapies that block receptor signaling through antibody binding (trastuzumab and pertuzumab) or by inhibiting kinase activity (lapatinib and neratinib).Unfortunately, these HER2 targeted therapies are not efficacious in HER2 positive ovarian cancer patients, suggesting differences in kinase regulation and signaling.Similarly, genetic alterations in the epidermal growth factor receptor (EGFR) tyrosine kinase are observed in non-small cell lung carcinoma (NSCLC) and while treatment with EGFR-tyrosine kinase inhibitors shows promise in some patients, others develop resistance overtime due to selection for EGFR mutants such as T490M that are not susceptible to kinase inhibition [4, 13, 14].

Altered glycosylation impairs cell signaling, communication, and adhesion and is implicated in the onset and progression of cancer eventually leading to metastasis.Abnormal glycosylation can be due to dysregulation of glycosyltransferase transcription, expression, and activity or alterations in the sequence and conformation of the modified protein that prevents carbohydrate PTM.Glycoproteins represent the majority of clinically utilized serological biomarkers and are routinely used for monitoring disease progression and response to therapeutic intervention.The diagnostic utility of these glycoprotein cancer biomarkers, exception of prostate-specific antigen (PSA), is limited by lack of tissue specificity and they are used to monitor disease progression, but are not adequate for screening.For example, SLe tetrasaccharide (CA19-9) is elevated in patients with pancreatic, colorectal and gastric cancer and CA19-9 abundance serves as a prognostic factor as well as a measure of response to therapy.Similarly, carcinoem-bryonic antigen (CEA) is a cell surface glycoprotein, which is normally expressed during fetal development with protein levels decreasing afterbirth.Elevated CEA is observed in patients with colorectal cancer among other carcinomas and fluctuations in CEA blood levels are used to monitor disease recurrence after surgical resection [4].

Along with phosphorylation and glycosylation, other modifications receiving significant attention in cancer biomarker research include ubiquitination, methylation, and acetylation.Impaired ubiquitin-mediated degradation results in constitutive activity of oncoproteins such as transcription factor, which is observed across cancer types.Furthermore, mutations in ubiquitin ligase enzymes such as BRAC1 and Mdm2 are established causes of oncogenesis.Abnormal epigenetic methylation and acetylation impact transcriptional regulation resulting in impaired protein expression and dysregulated cell signaling.Hypermethylation patterns are associated with carcinogenesis and DNA methylation profiles can serve as cancer biomarkers [4].