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Rationale: A general N‐glycoproteomics analysis pipeline has been established for characterization of mutation‐related gain‐of‐glycosylation (GoG) at intact N‐glycopeptide molecular level, generating comprehensive site and structure information of N‐glycosylation. Methods: This study focused on mutation‐originated GoG using a mass spectrometry‐based N‐glycoproteomics analysis workflow. In brief, GoG intact N‐glycopeptide databases were built, consisting of 2701 proteins (potential GoG N‐glycosites and amino acids derived from MUTAGEN, VARIANT and VAR_SEQ in UniProt) and 6709 human N‐glycans (≤50 sequence isomers per monosaccharide composition). We employed the site‐ and structure‐specific N‐glycoproteomics workflow utilizing intact N‐glycopeptides search engine GPSeeker to identify GoG intact N‐glycopeptides from parental breast cancer stem cells (MCF‐7 CSCs) and adriamycin‐resistant breast cancer stem cells (MCF‐7/ADR CSCs). Results: With the criteria of spectrum‐level false discovery rate control of ≤1%, we identified 87 and 94 GoG intact N‐glycopeptides corresponding to 37 and 35 intact N‐glycoproteins from MCF‐7 CSCs and MCF‐7/ADR CSCs, respectively. Micro‐heterogeneity and macro‐heterogeneity of N‐glycosylation from GoG intact N‐glycoproteins with VAR_SEQ and VARIANT were found in both MCF‐7 CSCs and MCF‐7/ADR CSCs systems. Conclusions: The integration of site‐ and structure‐specific N‐glycoproteomics approach, conjugating with GoG characterization, provides a universal workflow for revealing comprehensive N‐glycosite and N‐glycan structure information of GoG. The analysis of mutation‐originated GoG can be extended to GoG characterization of other N‐glycoproteome systems including complex clinical tissues and body fluids. [ABSTRACT FROM AUTHOR]

Rationale: The functional study and application of an intact glycoprotein require the structural characterization of both the protein backbone and the glycan moiety; the former has been successfully demonstrated with selective fragmentation of the protein backbone in CID and ExD; whether the latter can be achieved with selective fragmentation of the glycan moiety remains to be explored. Methods: RNase B solution was electrosprayed and its intact glycoforms of GlcNAc2Mann (n = 5–9) with the highest abundance (charge state z = 16) were isolated individually and fragmented using CID, ETD, HCD, ETciD, and EThcD on the Orbitrap Fusion Lumos Tribrid mass spectrometer; the dissociation parameters were optimized for selective fragmentation of the N‐glycan moiety and protein backbone as well as high sequence coverage. The obtained spectra were interpreted using the protein and N‐glycan database search engines ProteinGoggle and GlySeeker, respectively. Results: With exploration of different dissociation parameters for all the five methods, selective fragmentation of the N‐glycan moiety (the protein backbone staying intact) was observed in both HCD and EThcD at low collisional energies, but only a few matched product ions were observed; more comprehensive fragmentation was observed at high collisional energies (the protein backbone lost). Selective protein backbone fragmentation was observed in all the five dissociation methods. Conclusions: For comprehensive structural characterization of intact N‐glycoproteins using tandem mass spectrometry, the composition and topology of the N‐glycan moiety can be identified using HCD and EThcD complementarily at low and high energies; while the amino acid sequence and glycosite can be identified using CID, ETD, HCD, ETciD, and EThcD with their optimal dissociation parameters. [ABSTRACT FROM AUTHOR]

Aberrant glycosylation has been commonly observed in various physiological and pathological disorders (including cancers), and quite a few glycoproteins have been approved by the US Food and Drug Administration (FDA) as markers for early diagnosis. Each glycoprotein may have multiple glycoforms, and cancer-related ones can be only some specific glycoforms which have much higher sensitivity and specificity; for example, AFP glycoform AFP-L3 with N-glycan of 01Y(61F)41Y41M(31M41Y41L41S61M41Y41L41S is of bigger diagnostic value for hepatocellular carcinoma than total AFP (i.e., combination of all glycoforms). Mass spectrometry-based glycomics is currently the state-of-the-art instrumental analytical pipeline for high-throughput characterization of various glycoforms, where not only monosaccharide composition but also comprehensive structural information (sequence and linkage) of N-glycans are now reported thanks to our recently developed N-glycan database search engine GlySeeker. With this new capability, here, we report our large-scale characterization of human liver N-glycome with primary structures; 214 unique N-glycans with unique primary structures were identified and visualized with spectrum-level false discovery rate ≤ 1% and number of best hits of 1. The LO2 N-glycans reported here serve as a basic reference for future liver N-glycome study, and further quantitative analysis will enable characterization of differentially expressed N-glycans and discovery of more effective markers for liver and other diseases. Data are available via ProteomeXchange with identifier PXD008158. [ABSTRACT FROM AUTHOR]

Rationale: Most of the current popular tandem mass spectrometers have the capability of resolving the primary structures (monosaccharide composition, sequence and linkage) of N-glycans; however, compositions or putative structures have mostly been reported so far. Identification and visualization tools of N-glycans are needed. Methods: The isotopic mass-to-charge ratio and envelope fingerprinting algorithm, which has been successfully used for intact protein database search and identification, was adapted for N-glycan database search, and a stand-alone N-glycan database search engine, GlySeeker, for automated N-glycan identification and visualization was developed and successfully benchmarked. Both pseudo 2D graph and one-line text formats with one-letter symbols for monosaccharides were proposed for representing N-glycans. N-glycans were identified with comprehensive interpretation of product ions and false discovery rate (FDR) control. Results: In a database search of reversed-phase liquid chromatography/tandem mass spectrometry (RPLC/MS/MS) datasets of the N-glycome enriched from OVCAR-3 ovarian cancer cells, with FDR ≤1% and number of best hits (NoBHs) = 1-30, 1525 N-glycans with comprehensive primary structural information (composition, sequence and linkage) were identified and visualized; among these 1525 N-glycans, 559 had NoBHs = 1, i.e. their structures were uniquely identified. This represents a large-scale identification and visualization of N-glycans with primary structures from tandem mass spectra. Conclusions: A stand-alone N-glycan database search engine called GlySeeker has been developed for large-scale identification and visualization of N-glycans with comprehensive interpretation of tandem mass spectra and FDR control. [ABSTRACT FROM AUTHOR]

N-linked glycosylation is one of the most common protein PTMs, and the topological structure (monosaccharide composition and sequence as well as glycosidic linkages) of N-glycans is vital information to understand their biological functions and roles. Tandem mass spectrometry has been widely used for topological structure characterization of N-glycans, where comprehensive understanding of fragmentation pathways and characteristics of product ions are essential to achieve best interpretation of MS/MS data and highest confidence of identification. Here, we report our glycomic study of N-glycome of mouse brain as well as fragmentation pathway analysis of the identified N-glycans. With LC-MS/MS analysis at both the positive and negative ESI modes together with our recently developed N-glycan database search engine GlySeeker, 221 unique N-glycans with putative topological structures were identified with target-decoy searches and number of best hits of 1. Analysis of fragmentation pathways and characteristics of product ions of permethylated N-glycans in the positive mode and native N-glycans in the negative mode were further carried out. The reported N-glycans serve as a basic reference for future glycosylation study of mouse brain; and in general database search of tandem mass spectra of N-glycans, B/Y/Z ions should be preferentially considered for the permethylated form in the positive mode and B/C/Z ions for the native form in the negative mode. [ABSTRACT FROM AUTHOR]

Glycosylation is a common protein PTM, and its aberrant regulation has been widely linked to various pathological conditions including cancers. Our recent development of intact N-glycopeptide search engine GPSeeker has enabled relatively quantitative structure-specific characterization of differentially expressed intact N-glycopeptides (DEGPs) with isotopic labeling of the peptide backbones and structure-specific fragment ions of the N-glycan moieties. Here we report our site- and structure-specific relatively quantitative N-glycoproteomics study of breast MCF-7 cancer cells (relative to epithelial MCF-10A cells) using RPLC-nanoESI-MS/MS and GPSeeker. With spectrum-level FDR ≤ 1%, 581 intact N-glycopeptides with comprehensive structural information of both the peptide backbones (amino acid sequences, N-glycosites) and the N-glycan moieties (monosaccharide composition, sequence and linkages) were identified from five technical replicates (TR1, TR2, TR3, TR4 and TR5). With the criteria of quantified at least thrice out of the five technical replicates with no <1.5-fold change, p ≤.05 and RSD ≤ 20%, 56 DEGPs were quantified from 23 N-glycosites on 19 intact N-glycoproteins. For the 19 intact N-glycoproteins observed with differential N-glycosylation expression, 14 (each with one or more DEGPs) were observed with uniform down regulation; 6 (each with one or more DEGPs) were observed with uniform up regulation; whereas one was observed with both up and down regulation. Differential N-glycosylation in breast MCF-7 cancer cells (relative to MCF-10A epithelial cells) were qualitatively and quantitatively characterized with site- and structure-specific N-glycoproteomics using RPLC-nanoESI-MS/MS (HCD with stepped NCEs) and intact N-glycopeptide search engine GPSeeker. With spectrum-level FDR ≤ 1%, 581 intact N-glycopeptides with comprehensive structural information of both the peptide backbones and the N-glycan moieties were identified; For the 248 putative N-glycosites, 248 were confirmed where 125 have not been annotated in UniProt as of July 25, 2019. For the 114 N-glycan putative linkage structures, 44 were confirmed with no less than one structure-diagnostic fragment ions. With the criteria of quantified at least thrice out of the five technical replicates with no < 1.5-fold change and p ≤.05, 56 DEGPs were quantified from 21 intact N-glycoproteins; 13 and 5 intact N-glycoproteins (each with one or more DEGPs) were observed with uniform down and up regulation; whereas one were observed with simultaneous up and down regulation. Unlabelled Image • Site- and structure-specific N-glycoproteomics study of breast cancer MCF-7 cells. • 1612 intact N-glycopeptides were identified with FDR ≤ 1%. • 122 differentially expressed intact N-glycopeptides were quantified. • 51 out of the 216 linkage structures were confirmed with diagnostic fragment ions. • 23 out of the 392 N-glycosites were found new to UniProt. [ABSTRACT FROM AUTHOR]