Integrated Proteomic and Glycoproteomic Analyses of Prostate Cancer Cells Reveal Glycoprotein Alteration in Protein Abundance and Glycosylation*

Abstract

Prostate cancer is the most common cancer among men in the U.S. and worldwide, and androgen-deprivation therapy remains the principal treatment for patients. Although a majority of patients initially respond to androgen-deprivation therapy, most will eventually develop castration resistance. An increased understanding of the mechanisms that underline the pathogenesis of castration resistance is therefore needed to develop novel therapeutics. LNCaP and PC3 prostate cancer cell lines are models for androgen-dependence and androgen-independence, respectively. Herein, we report the comparative analysis of these two prostate cancer cell lines using integrated global proteomics and glycoproteomics. Global proteome profiling of the cell lines using isobaric tags for relative and absolute quantitation (iTRAQ) labeling and two-dimensional (2D) liquid chromatography-tandem MS (LC-MS/MS) led to the quantification of 8063 proteins. To analyze the glycoproteins, glycosite-containing peptides were isolated from the same iTRAQ-labeled peptides from the cell lines using solid phase extraction followed by LC-MS/MS analysis. Among the 1810 unique N-linked glycosite-containing peptides from 653 identified N-glycoproteins, 176 glycoproteins were observed to be different between the two cell lines. A majority of the altered glycoproteins were also observed with changes in their global protein expression levels. However, alterations in 21 differentially expressed glycoproteins showed no change at the protein abundance level, indicating that the glycosylation site occupancy was different between the two cell lines. To determine the glycosylation heterogeneity at specific glycosylation sites, we further identified and quantified 1145 N-linked glycopeptides with attached glycans in the same iTRAQ-labeled samples. These intact glycopeptides contained 67 glycan compositions and showed increased fucosylation in PC3 cells in several of the examined glycosylation sites. The increase in fucosylation could be caused by the detected changes in enzymes belonging to the glycan biosynthesis pathways of protein fucosylation observed in our proteomic analysis. The altered protein fucosylation forms have great potential in aiding our understanding of castration resistance and may lead to the development of novel therapeutic approaches and specific detection strategies for prostate cancer. Molecular & Cellular

Terminal Galactosylation and Sialylation Switching on Membrane Glycoproteins upon TNF-Alpha-Induced Insulin Resistance in Adipocytes

“…Mapping the extreme complexity of the mammalian glycoproteome remains challenging, but recent advances in mass spectrometry have dramatically aided this endeavor. While large-scale glycopeptide identification is now feasible and hundreds of glycopeptides have been identified (27,(43)(44)(45)(46)(47)(48), systems-wide quantification of the glycoproteome has had little attention (49). The glycopeptide identification has been advanced by the development of specific glycopeptide search algorithms or modification of existing proteomics pipelines (50 -53).…”

Maturing Glycoproteomics Technologies Provide Unique Structural Insights into the N-glycoproteome and Its Regulation in Health and Disease

“…Although engaging multiple dissociation modes is becoming more popular, some research- ers still opt for applying single fragmentation techniques i.e. HCD (46) and ETD (7) in glycoproteomics, for example because of the nature of the research question (e.g. only investigating the site-specific glycan monosaccharide composition), limited availability of instruments allowing multiple fragmentation modes or to avoid the longer MS duty cycle associated with approaches using alternating/hybrid dissociation methods.…”

“…Quantitative Glycoproteomics-As discussed in more detail below, the first studies performing isotope-assisted quantitative glycoproteomics using stable isotope labeling with amino acids in cell culture (SILAC) (105) and isobaric tags for relative and absolute quantitation (iTRAQ) (46,108) have recently been published. This has enabled accurate relative quantitation of single glycoforms on individual protein sites by comparing the precursor (SILAC) or the product (iTRAQ) ion intensities across two or more conditions.…”

“…Some glycoproteomics approaches even by-pass this step because of the increased capacity of modern LC-MS/MS instrumentation to handle the extreme complexity of biologically-relevant glycopeptide mixtures, and perhaps because the multiple LC fractions resulting from off-line separations dramatically increase the required LC-MS/MS instrument time, lower the overall sensitivity of the analysis and complicate the downstream quantitative data analysis (103,104). However, multiple glycoproteomics strategies, particularly in studies where the amounts of biological sample material are not a limiting factor, still opt to use off-line separation to increase the glycoproteome coverage (46,(105)(106)(107). The most exciting new developments in this area include the off-line use of neutral/high pH (pH 7-10) RP-LC (7,108), which displayed orthogonal glycopeptide retention behavior relative to the conventional acidic (ϳpH 2) RP-LC-(online) MS/MS.…”

“…These include site-specific analysis of N-glycoproteins isolated to relative purity rather than in complex mixtures (11, 12, 20, 34 -42), N-glycomics analyses of glycans released from glycoproteins (18,38,(43)(44)(45)(46)(47), and identification and quantification of previously glycosylated sites on de-N-glycosylated proteins ("deglycoproteomics") after removal of the entire glycan or with remnant N-glycan core remaining (48 -57). Although these studies per se do not qualify under our definition of glycoproteomics, (site-specific analysis of the glycoproteome at the intact glycopeptide level), they still provide useful information in conjunction with glycoproteomics for the glycobiologist, provided correct experimental design is applied (58).…”