Enzymatic Tagging of Glycoproteins on the Cell Surface for Their Global and Site-Specific Analysis with Mass Spectrometry
Ontology highlight
ABSTRACT:
INSTRUMENT(S): LTQ Orbitrap Elite
ORGANISM(S): Homo Sapiens (ncbitaxon:9606)
SUBMITTER: 
Ronghu Wu
PROVIDER: MSV000087022 | MassIVE | Tue Mar 09 17:27:00 GMT 2021
REPOSITORIES: MassIVE
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Project description:The cell surface is normally covered with sugars that are bound to lipids or proteins. Surface glycoproteins play critically important roles in many cellular events, including cell-cell communications, cell-matrix interactions, and response to environmental cues. Aberrant protein glycosylation on the cell surface is often a hallmark of human diseases such as cancer and infectious diseases. Global analysis of surface glycoproteins will result in a better understanding of glycoprotein functions and the molecular mechanisms of diseases and the discovery of surface glycoproteins as biomarkers and drug targets. Here, an enzyme is exploited to tag surface glycoproteins, generating a chemical handle for their selective enrichment prior to mass spectrometric (MS) analysis. The enzymatic reaction is very efficient, and the reaction conditions are mild, which are well-suited for surface glycoprotein tagging. For biologically triplicate experiments, on average 953 N-glycosylation sites on 393 surface glycoproteins per experiment were identified in MCF7 cells. Integrating chemical and enzymatic reactions with MS-based proteomics, the current method is highly effective to globally and site-specifically analyze glycoproteins only located on the cell surface. Considering the importance of surface glycoproteins, this method is expected to have extensive applications to advance glycoscience.
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Project description:We report a strategy termed Bio-Orthogonal Cell line-specific Tagging of Glycoproteins (BOCTAG). Cells are equipped by transfection with an artificial biosynthetic pathway (mut-AGX1/NahK/Nh-GalNAc-T2) that transforms an alkyne tagged sugar into the corresponding nucleotide-sugar. Only transfected cells incorporate the bioorthogonal sugar into glycoproteins in the presence of non-transfected cells. The incorporation of alkyne-sugar in glycoproteins allows the installation, by copper(I)-catalyzed reaction, of an acid cleavable biotinylated tag. Glycoproteins are enriched using Streptavidin and. after on-bead digestion, glycopeptides are eluted in acid-conditions to be then analysed by mass spectrometry.
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Project description:We report a strategy termed Bio-Orthogonal Cell line-specific Tagging of Glycoproteins (BOCTAG). Cells are equipped by transfection with an artificial biosynthetic pathway (mut-AGX1/NahK/Nh-GalNAc-T2) that transforms an alkyne tagged sugar into the corresponding nucleotide-sugar. Only transfected cells incorporate the bioorthogonal sugar into glycoproteins in the presence of non-transfected cells. The incorporation of alkyne-sugar in glycoproteins allows the installation, by copper(I)-catalyzed reaction, of an acid cleavable biotinylated tag. Glycoproteins are enriched using Streptavidin and. after on-bead digestion, glycopeptides are eluted in acid-conditions to be then analysed by mass spectrometry.
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Project description:Recombinant proteins are of great interest in glycobiology and proteomics, known especially for their reproducibility and accessibility. However, variation in glycosylation among recombinant glycoproteins is not well understood and may depend on numerous conditions in the biomanufacturing process. In order to confidently assess variation in glycosylation measurements, it is vital to both optimize the measurement of, and determine the degree of variation between, distributions of glycosylation on specific sites of glycoproteins. This is especially important for glycoproteins that are known to have rapid sequence changes, such as with different influenza strains. In this study, eight strains of recombinant influenza hemagglutinin and neuraminidase produced from HEK293 cell line were obtained from four vendors and digestion was conducted using a series of complex multi-enzymatic methods designed to isolate glycopeptide sequons. Site-specific glycosylation profiles of intact glycopeptides were produced using mass spectrometric evaluation on an orbitrap system and compared using spectral similarity scores. Variation in glycan abundances and distribution was most pronounced between different strains of virus (similarity score = 383 out of 1000), whereas replicates resulted in low variation (similarity score = 957 out of 1000). Glycan variation was also measured based on differences between vendors, lots, batches, protease digestion, and intra-protein site. The most abundant glycans in all of these influenza glycoproteins were monofucosylated and complex, as reported by other laboratories. However, it was found that different vendors can produce very different glycan distributions for the same glycosylation site. Notably, it is demonstrated that glycan distributions are similar for conserved regions of influenza glycoproteins. Overall, these methods present a potential use in developing reproducible measurements of glycosylated biologics for quality control or making more informed decisions in biomanufacturing.
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Project description:We report a strategy termed Bio-Orthogonal Cell line-specific Tagging of Glycoproteins (BOCTAG). Cells are equipped by transfection with an artificial biosynthetic pathway (mut-AGX1/NahK) that transforms an alkyne tagged sugar into the corresponding nucleotide-sugar. Only transfected cells incorporate the bioorthogonal sugar into glycoproteins in the presence of non-transfected cells. The incorporation of alkyne-sugar in glycoproteins allows the installation, by copper(I)-catalyzed reaction, of a biotinylated tag. Glycoproteins are enriched using Streptavidin, and on-bead digestion yielded a peptide fraction that is analysed by mass spectrometry.
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Project description:We report a strategy termed Bio-Orthogonal Cell line-specific Tagging of Glycoproteins (BOCTAG). Cells are equipped by transfection with an artificial biosynthetic pathway (mut-AGX1/NahK) that transforms an alkyne tagged sugar into the corresponding nucleotide-sugar. Only transfected cells incorporate the bioorthogonal sugar into glycoproteins in the presence of non-transfected cells. The incorporation of alkyne-sugar in glycoproteins allows the installation, by copper(I)-catalyzed reaction, of a biotinylated tag. Glycoproteins are enriched using Streptavidin, and on-bead digestion yielded a peptide fraction that is analysed by mass spectrometry.
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