Abstract
Multi-dimensional proteomic analyses provide different layers of protein information, including protein abundance and post-translational modifications. Here, we report an integrated analysis of protein expression, phosphorylation, and N-glycosylation by serial enrichments of phosphorylation and N-glycosylation (SEPG) from the same tissue samples. On average, the SEPG identified 142,106 unmodified peptides of 8,625 protein groups, 18,846 phosphopeptides (15,647 phosphosites), and 4,019 N-glycopeptides (2,634 N-glycosites) in tumor and adjacent normal tissues from three gastric cancer patients. The combined analysis of these data showed that the integrated analysis additively improved the coverages of gastric cancer-related protein networks; phosphoproteome and N-glycoproteome captured predominantly low abundant signal proteins, and membranous or secreted proteins, respectively, while global proteome provided abundances for general population of the proteome. Therefore, our results demonstrate that the SEPG can serve as an effective approach for multi-dimensional proteome analyses, and the holistic profiles of protein expression and PTMs enabled improved interpretation of disease-related networks by providing complementary information.
Highlights
Extensive fractionation, high-resolution peptide separation, and high performance mass spectrometry significantly improved the proteome size as to be comparable to the transcriptome size detected by mRNA-sequencing
Isobaric tag for relative and absolute quantitation labeled peptides from human gastric cancer and adjacent normal tissues of a patient were subjected to fractionation by mid-pH reverse phase LC (RPLC) and to the immobilized-metal affinity chromatography (IMAC) method for the phosphopeptide enrichment, followed by a filter aided capture and elution (FACE) for the N-glycopeptide enrichment
To understand the characteristics of the multi-dimensional proteomes measured by the Serial enrichments of phosphorylation and N-glycosylation (SEPG) method, we first identified the peptides from the three proteomes using the Uniprot protein sequences (May, 2013; 90,191 entries) in the target-decoy setting by the MS-GF+ (v9387) search engine[38] (Methods)
Summary
Extensive fractionation, high-resolution peptide separation, and high performance mass spectrometry significantly improved the proteome size as to be comparable to the transcriptome size detected by mRNA-sequencing. The SEPTM method enabled quantitative analysis of about 8,000 proteins, and 20,000 phosphorylation, 15,000 ubiquitination and 3,000 acetylation sites per experiment in human leukemia cells, permitting a holistic view of cellular signal pathways Such advances in MS-based proteomic analysis have facilitated multi-dimensional proteomic analyses for understanding activities of multilayered protein networks. We present a MS-based method for multi-dimensional proteomic analyses of protein abundance, phosphorylation, and N-glycosylation by serial enrichments of phosphorylation and N-glycosylation from the same tissue samples In this method, isobaric tag for relative and absolute quantitation (iTRAQ) labeled peptides from human gastric cancer and adjacent normal tissues of a patient were subjected to fractionation by mid-pH reverse phase LC (RPLC) and to the immobilized-metal affinity chromatography (IMAC) method for the phosphopeptide enrichment, followed by a filter aided capture and elution (FACE) for the N-glycopeptide enrichment. The combined analysis of these data demonstrated that different types of data provided complementary coverages of gastric cancer-related protein networks, thereby facilitating improved interpretation of the networks
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