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One step carboxyl group isotopic labeling for quantitative analysis of N-glycopeptide by Mass Spectrometry

ABSTRACT: Glycosylation is one of the most common and important post-translational modifications. Quantitative analysis of intact N-glycopeptides is critical to understand the role of protein glycosylation in physiological and pathological processes. In this work, we developed a novel approach called methylamine stable isotope labeling (MeSIL) to relatively quantify intact N-glycopeptides through one step isotopic labeling. Isotopic methylamine was employed to label both the sialic acid residues of glycans and the carboxyl groups on the peptide moiety, and followed by the mixing of samples for mass spectrometric analysis. The relative abundance of intact N-glycopeptides between two samples was obtained by comparing the signal of the particular peak pairs with a 3*N Da mass shift (where N is an integer correlating with the number of carboxylic acids within the glycopeptides). Additionally, the number of sialylated glycopeptides can be distinguished simultaneously through the mass difference after labeling. The MeSIL str

ORGANISM(S): Homo Sapiens

SUBMITTER: Haojie Lu

PROVIDER: PXD021104 | iProX | Tue Aug 25 00:00:00 BST 2020

REPOSITORIES: iProX

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One step carboxyl group isotopic labeling for quantitative analysis of intact <i>N</i>-glycopeptides by mass spectrometry.

Sun Zhenyu Z Ji Guanghui G Wang Guoli G Wei Lei L Zhang Ying Y Lu Haojie H

Chemical communications (Cambridge, England) 20210401 34

Here, we have developed an approach termed methylamine stable isotope labeling (MeSIL) to relatively quantify N-glycopeptides through one step labeling. It is the first time that this approach is applied to measure N-glycopeptide changes in huh7 cells after Zika virus infection and it has been revealed that differentially expressed N-glycopeptides played important roles in virus infection at the glycosylation site-specific level. ...[more]

PMID: 33908447

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Project description:To unravel molecular targets involved in glycopeptide resistance, three isogenic strains of Staphylococcus aureus with different susceptibility levels to vancomycin or teicoplanin were subjected to whole-genome microarray-based transcription and quantitative proteomic profiling. Quantitative proteomics performed on membrane extracts showed exquisite inter-experimental reproducibility permitting the identification and relative quantification of >30% of the predicted S. aureus proteome. In the absence of antibiotic selection pressure, comparison of stable resistant and susceptible strains revealed 94 differentially expressed genes and 178 proteins. As expected, only partial correlation was obtained between transcriptomic and proteomic results during stationary-phase. Application of massively parallel methods identified one third of the complete proteome, a majority of which was only predicted based on genome sequencing, but never identified to date. Several over‑expressed genes represent previously reported targets, while series of genes and proteins possibly involved in the glycopeptide resistance mechanism were discovered here, including regulators, global regulator attenuator, hyper‑mutability factor or hypothetical proteins. Gene expression of these markers was confirmed in a collection of genetically unrelated strains showing altered susceptibility to glycopeptides. Our proteome and transcriptome analyses have been performed during stationary‑phase of growth on isogenic strains showing susceptibility or intermediate level of resistance against glycopeptides. Altered susceptibility had emerged spontaneously after infection with a sensitive parental strain, thus not selected in vitro. This combined analysis allows the identification of hundreds of proteins considered, so far as hypothetical protein. In addition, this study provides not only a global picture of transcription and expression adaptations during a complex antibiotic resistance mechanism but also unravels potential drug targets or markers that are constitutively expressed by resistant strains regardless of their genetic background, amenable to be used as diagnostic targets. Keywords: Molecular markers, antibiotic resistance, glycopeptides, growth-phase

Project description:Glycosylation influences diverse life processes and changes in pathological processes. The glycoproteomic has been recognized as a hot area to screen markers as altered glycosylation is a universal feature of cancer cells. However, the high throughput screening of glycosylation markers from a large number of clinical samples faces greater technical challenges than peptides without or with other modifications. The most prominent problem in tandem mass tag-based high throughput quantification of intact glycopeptides is the inefficient cleavage of peptide backbone, glycosidic bonds and tandem mass tag concurrently to offer successful peptide and glycan composition determination as well as good quantification. Here, we managed to implement the tandem mass tag labeling into quantitative glycoproteomics by developing a chemical labeling-assisted complementary dissociation method for multiplexed analysis of intact N-glycopeptides. Chemical labeling of intact N-glycopeptides with charge-increasing reagent improved the efficiency of electron transfer dissociation (ETD) significantly for their identification and also increased the efficiency of higher-energy collision dissociation (HCD) in generating reporter ion for quantification, thereby capitalizing on the complementary nature of these two dissociation methods in quantification of intact N-glycopeptides. By using this new strategy, we investigated the site-specific glycosylation of IgG collected from 90 human serum including three stages of liver diseases, hepatitis B virus (HBV), cirrhosis (CIR), hepatocellular carcinoma (HCC) and healthy controls. As a result, 313 intact N-glycopeptides from IgG were identified in total, which represents the most comprehensive site-specific and subclass-specific N-glycosylation of human serum IgG to date. Quantitative glycoproteomics revealed that the combination of IgG1-H3N5F1 and IgG4-H3N4 displayed powerful prediction capability for distinguishing different stages of liver disease patients, which has potential for non-invasive monitoring and pre-stratiﬁcation of liver disease diagnosis. Moreover, we analyzed the human serum using this newly developed method and identified near 2000 intact N-glycopeptides from only 10 L serum and showed this method has applicability for quantification of intact N-glycopeptides in different studies.

Dataset Information

One step carboxyl group isotopic labeling for quantitative analysis of N-glycopeptide by Mass Spectrometry

Publications

One step carboxyl group isotopic labeling for quantitative analysis of intact <i>N</i>-glycopeptides by mass spectrometry.

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