Trends in oligomannosylation and α1,2-mannosidase expression in human cancers.

Aberrant protein glycosylation is a prominent cancer feature. While many tumour-associated glycoepitopes have been reported, advances in glycoanalytics continue to uncover new associations between glycosylation and cancer. Guided by a comprehensive literature survey suggesting that oligomannosylation (Man5-9 GlcNAc2) is a widespread and often regulated glycosignature in human cancers, we here revisit a valuable compilation of nearly 500 porous graphitized carbon LC-MS/MS N-glycomics datasets acquired across 11 human cancer types to systematically test for oligomannose-cancer associations. Firstly, the quantitative glycomics data obtained across 34 cancerous cell lines demonstrated that oligomannosylation is a pan-cancer feature spanning in a wide abundance range. In keeping with literature, our quantitative glycomics data of tumour and matching control tissues and new MALDI-MS imaging data of tissue microarrays showed a strong cancer-associated elevation of oligomannosylation in both basal cell (p = 1.78 × 10-12) and squamous cell (p = 1.23 × 10-11) skin cancer and colorectal cancer (p = 8.0 × 10-4). The glycomics data also indicated that some cancer types including gastric and liver cancer exhibit unchanged or reduced oligomannose levels, observations also supported by literature and MALDI-MS imaging data. Finally, expression data from public cancer repositories indicated that several α1,2-mannosidases are regulated in tumour tissues suggesting that these glycan-processing enzymes may contribute to the cancer-associated modulation of oligomannosylation. This omics-centric study has compiled robust glycomics and enzyme expression data revealing interesting molecular trends that open avenues to better understand the role of oligomannosylation in human cancers.

Protein Paucimannosylation Is an Enriched N-Glycosylation Signature of Human Cancers.

While aberrant protein glycosylation is a recognized characteristic of human cancers, advances in glycoanalytics continue to discover new associations between glycoproteins and tumorigenesis. This glycomics-centric study investigates a possible link between protein paucimannosylation, an under-studied class of human N-glycosylation [Man1-3 GlcNAc2 Fuc0-1 ], and cancer. The paucimannosidic glycans (PMGs) of 34 cancer cell lines and 133 tissue samples spanning 11 cancer types and matching non-cancerous specimens are profiled from 467 published and unpublished PGC-LC-MS/MS N-glycome datasets collected over a decade. PMGs, particularly Man2-3 GlcNAc2 Fuc1 , are prominent features of 29 cancer cell lines, but the PMG level varies dramatically across and within the cancer types (1.0-50.2%). Analyses of paired (tumor/non-tumor) and stage-stratified tissues demonstrate that PMGs are significantly enriched in tumor tissues from several cancer types including liver cancer (p = 0.0033) and colorectal cancer (p = 0.0017) and is elevated as a result of prostate cancer and chronic lymphocytic leukaemia progression (p < 0.05). Surface expression of paucimannosidic epitopes is demonstrated on human glioblastoma cells using immunofluorescence while biosynthetic involvement of N-acetyl-ß-hexosaminidase is indicated by quantitative proteomics. This intriguing association between protein paucimannosylation and human cancers warrants further exploration to detail the biosynthesis, cellular location(s), protein carriers, and functions of paucimannosylation in tumorigenesis and metastasis.

Mycobacterium tuberculosis Infection Manipulates the Glycosylation Machinery and the N-Glycoproteome of Human Macrophages and Their Microparticles.

Tuberculosis (TB) remains a prevalent and lethal infectious disease. The glycobiology associated with Mycobacterium tuberculosis infection of frontline alveolar macrophages is still unresolved. Herein, we investigated the regulation of protein N-glycosylation in human macrophages and their secreted microparticles (MPs) used for intercellular communication upon M. tb infection. LC-MS/MS-based proteomics and glycomics were performed to monitor the regulation of glycosylation enzymes and receptors and the N-glycome in in vitro-differentiated macrophages and in isolated MPs upon M. tb infection. Infection promoted a dramatic regulation of the macrophage proteome. Most notably, significant infection-dependent down-regulation (4-26 fold) of 11 lysosomal exoglycosidases, e.g., ß-galactosidase, ß-hexosaminidases and α-/ß-mannosidases, was observed. Relative weak infection-driven transcriptional regulation of these exoglycosidases and a stronger augmentation of the extracellular hexosaminidase activity demonstrated that the lysosome-centric changes may originate predominantly from infection-induced secretion of the lysosomal content. The macrophages showed heterogeneous N-glycan profiles and displayed significant up-regulation of complex-type glycosylation and concomitant down-regulation of paucimannosylation upon infection. Complementary intact N-glycopeptide analysis supported a subcellular-specific manipulation of the glycosylation machinery and altered glycosylation patterns of lysosomal N-glycoproteins within infected macrophages. Interestingly, the corresponding macrophage-derived MPs displayed unique N-glycome and proteome signatures supporting a preferential packaging from plasma membranes. The MPs were devoid of infection-dependent N-glycosylation signatures, but interestingly displayed increased levels of the glyco-initiating oligosaccharyltransferase complex and associated α-glucosidases that correlated with increased formation, N-glycan precursor levels and N-glycan density of infected MPs. In conclusion, this system-wide study provides new insight into the host- and pathogen-driven N-glycoproteome manipulation of macrophages in TB.

Toward Automated N-Glycopeptide Identification in Glycoproteomics.

Advances in software-driven glycopeptide identification have facilitated N-glycoproteomics studies reporting thousands of intact N-glycopeptides, i.e., N-glycan-conjugated peptides, but the automated identification process remains to be scrutinized. Herein, we compare the site-specific glycoprofiling efficiency of the PTM-centric search engine Byonic relative to manual expert annotation utilizing typical glycoproteomics acquisition and data analysis strategies but with a single glycoprotein, the uncharacterized multiple N-glycosylated human basigin. Detailed site-specific reference glycoprofiles of purified basigin were manually established using ion-trap CID-MS/MS and high-resolution Q-Exactive Orbitrap HCD-MS/MS of tryptic N-glycopeptides and released N-glycans. The micro- and macroheterogeneous basigin N-glycosylation was site-specifically glycoprofiled using Byonic with or without a background of complex peptides using Q-Exactive Orbitrap HCD-MS/MS. The automated glycoprofiling efficiencies were assessed against the site-specific reference glycoprofiles and target/decoy proteome databases. Within the limits of this single glycoprotein analysis, the search criteria and confidence thresholds (Byonic scores) recommended by the vendor provided high glycoprofiling accuracy and coverage (both >80%) and low peptide FDRs (<1%). The data complexity, search parameters including search space (proteome/glycome size), mass tolerance and peptide modifications, and confidence thresholds affected the automated glycoprofiling efficiency and analysis time. Correct identification of ambiguous peptide modifications (methionine oxidation/carbamidomethylation) whose mass differences coincide with several monosaccharide mass differences (Fuc/Hex/HexNAc) and of ambiguous isobaric (Hex1NeuAc1-R/Fuc1NeuGc1-R) or near-isobaric (NeuAc1-R/Fuc2-R) monosaccharide subcompositions remains challenging in automated glycoprofiling, arguing particular attention paid to N-glycopeptides displaying such "difficult-to-identify" features. This study provides valuable insights into the automated glycopeptide identification process, stimulating further developments in FDR-based glycoproteomics.

Integrated Proteomic and Genomic Analysis of Gastric Cancer Patient Tissues.

V-erb-b2 erythroblastic leukemia viral oncogene homologue 2, known as ERBB2, is an important oncogene in the development of certain cancers. It can form a heterodimer with other epidermal growth factor receptor family members and activate kinase-mediated downstream signaling pathways. ERBB2 gene is located on chromosome 17 and is amplified in a subset of cancers, such as breast, gastric, and colon cancer. Of particular interest to the Chromosome-Centric Human Proteome Project (C-HPP) initiative is the amplification mechanism that typically results in overexpression of a set of genes adjacent to ERBB2, which provides evidence of a linkage between gene location and expression. In this report we studied patient samples from ERBB2-positive together with adjacent control nontumor tissues. In addition, non-ERBB2-expressing patient samples were selected as comparison to study the effect of expression of this oncogene. We detected 196 proteins in ERBB2-positive patient tumor samples that had minimal overlap (29 proteins) with the non-ERBB2 tumor samples. Interaction and pathway analysis identified extracellular signal regulated kinase (ERK) cascade and actin polymerization and actinmyosin assembly contraction as pathways of importance in ERBB2+ and ERBB2- gastric cancer samples, respectively. The raw data files are deposited at ProteomeXchange (identifier: PXD002674) as well as GPMDB.

Comparative studies of the proteome, glycoproteome, and N-glycome of clear cell renal cell carcinoma plasma before and after curative nephrectomy.

Clear cell renal cell carcinoma is the most prevalent of all reported kidney cancer cases, and currently there are no markers for early diagnosis. This has stimulated great research interest recently because early detection of the disease can significantly improve the low survival rate. Combining the proteome, glycoproteome, and N-glycome data from clear cell renal cell carcinoma plasma has the potential of identifying candidate markers for early diagnosis and prognosis and/or to monitor disease recurrence. Here, we report on the utilization of a multi-dimensional fractionation approach (12P-M-LAC) and LC-MS/MS to comprehensively investigate clear cell renal cell carcinoma plasma collected before (disease) and after (non-disease) curative nephrectomy (n = 40). Proteins detected in the subproteomes were investigated via label-free quantification. Protein abundance analysis revealed a number of low-level proteins with significant differential expression levels in disease samples, including HSPG2, CD146, ECM1, SELL, SYNE1, and VCAM1. Importantly, we observed a strong correlation between differentially expressed proteins and clinical status of the patient. Investigation of the glycoproteome returned 13 candidate glycoproteins with significant differential M-LAC column binding. Qualitative analysis indicated that 62% of selected candidate glycoproteins showed higher levels (upregulation) in M-LAC bound fraction of disease samples. This observation was further confirmed by released N-glycans data in which 53% of identified N-glycans were present at different levels in plasma in the disease vs non-disease samples. This striking result demonstrates the potential for significant protein glycosylation alterations in clear cell renal cell carcinoma cancer plasma. With future validation in a larger cohort, information derived from this study may lead to the development of clear cell renal cell carcinoma candidate biomarkers.

Comprehensive N-glycome profiling of cultured human epithelial breast cells identifies unique secretome N-glycosylation signatures enabling tumorigenic subtype classification.

The secreted cellular sub-proteome (secretome) is a rich source of biologically active glycoproteins. N-Glycan profiling of secretomes of cultured cancer cells provides an opportunity to investigate the link between protein N-glycosylation and tumorigenesis. Utilizing carbon-LC-ESI-CID-MS/MS of protein released native N-glycans, we accurately profiled the secretome N-glycosylation of six human epithelial breast cells including normal mammary epithelial cells (HMEC) and breast cancer cells belonging to luminal A subtype (MCF7), HER2-overexpressing subtype (SKBR3), and basal B subtype (MDA-MB157, MDA-MB231, HS578T). On the basis of intact molecular mass, LC retention time, and MS/MS fragmentation, a total of 74 N-glycans were confidently identified and quantified. The secretomes comprised significant levels of highly sialylated and fucosylated complex type N-glycans, which were elevated in all cancer cells relative to HMEC (57.7-87.2% vs 24.9%, p < 0.0001 and 57.1-78.0% vs 38.4%, p < 0.0001-0.001, respectively). Similarly, other glycan features were found to be altered in breast cancer secretomes including paucimannose and complex type N-glycans containing bisecting ß1,4-GlcNAc and LacdiNAc determinants. Subtype-specific glycosylation were observed, including the preferential expression of α2,3-sialylation in the basal B breast cancer cells. Pathway analysis indicated that the regulated N-glycans were biosynthetically related. Tight clustering of the breast cancer subtypes based on N-glycome signatures supported the involvement of N-glycosylation in cancer. In conclusion, we are the first to report on the secretome N-glycosylation of a panel of breast epithelial cell lines representing different subtypes. Complementing proteome and lipid profiling, N-glycome mapping yields important pieces of structural information to help understand the biomolecular deregulation in breast cancer development and progression, knowledge that may facilitate the discovery of candidate cancer markers and potential drug targets.

Proteogenomic analysis of human colon carcinoma cell lines LIM1215, LIM1899, and LIM2405.

As part of the genome-wide and chromosome-centric human proteomic project (C-HPP), we have integrated shotgun proteomics approach and a genome-wide transcriptomic approach (RNA-Seq) of a set of human colon cancer cell lines (LIM1215, LIM1899 and LIM2405) that were selected to represent a wide range of pathological states of colorectal cancer. The combination of a standard proteomics approach (1D-gel electrophoresis coupled to LC/ion trap mass spectrometry) and RNA-Seq allowed us to exploit the greater depth of the transcriptomics measurement (∼ 9800 transcripts per cell line) versus the protein observations (∼ 1900 protein identifications per cell line). Conversely, the proteomics data were helpful in identifying both cancer associated proteins with differential expression patterns as well as protein networks and pathways which appear to be deregulated in these cell lines. Examples of potential markers include mortalin, nucleophosmin, ezrin, LASP1, alpha and beta forms of spectrin, exportin, the carcinoembryonic antigen family, EGFR and MET. Interaction analyses identified the large intermediate filament family, the protein folding network and adapter proteins in focal adhesion networks, which included the CDC42 and RHOA signaling pathways that may have potential for identifying phenotypic states representing poorly and moderately differentiated states of CRC, with or without metastases.

An improved method for the detection and enrichment of low-abundant membrane and lipid raft-residing proteins.

A high degree of optimisation is required in native co-immunoprecipitation (co-IP) experiments with added challenges for low-abundant membrane proteins and masking by IgG molecules. Although in vivo tagged-protein purification avoids the IgG masking problem, modifying the terminus of the protein may result in conformational and post-translational modification changes. In this paper, we propose a method which combines four key aspects to improve the solubility and enrichment of low-abundant plasma membrane proteins using the urokinase plasminogen activator receptor (uPAR) as an example. As this GPI-linked receptor predominantly resides in lipid rafts (LR), we used a modified RIPA lysis buffer containing the non-ionic detergent, octyl-glucoside which solubilizes LRs to extract uPAR. This is followed by a modified crosslinking co-IP which covalently crosslinks the antibodies to the beads. Crosslinking allowed for a significant increase in the detection of uPAR with minimal IgG contamination using on-bead digestion or acid elution followed by digestion and analysis on high-throughput one-dimensional (nanoLC) MS/MS instrument (AbSciex 5600). To the best of our knowledge, this method of isolation is the first to be done to increase the yield of a low-abundant membrane protein and may be useful for the purification of other non-raft and raft-residing membrane proteins.

An optimized approach for enrichment of glycoproteins from cell culture lysates using native multi-lectin affinity chromatography.

Lectins are capable of recognizing specific glycan structures and serve as invaluable tools for the separation of glycosylated proteins from nonglycosylated proteins in biological samples. We report on the optimization of native multi-lectin affinity chromatography, combining three lectins, namely, concanavalin A, jacalin, and wheat germ agglutinin for fractionation of cellular glycoproteins from MCF-7 breast cancer lysate. We evaluated several conditions for optimum recovery of total proteins and glycoproteins such as low pH and saccharide elution buffers, and the inclusion of detergents in binding and elution buffers. Optimum recovery was observed with overnight incubation of cell lysate with lectins at 4°C, and inclusion of detergent in binding and saccharide elution buffers. Total protein and bound recoveries were 80 and 9%, respectively. Importantly, we found that high saccharide strength elution buffers were not necessary to release bound glycoproteins. This study demonstrates that multi-lectin affinity chromatography can be extended to total cell lysate to investigate the cellular glycoproteome.