Analysis of protein glycosylation by mass spectrometry

Morelle, Willy; Michalski, Jean Claude

doi:10.1038/nprot.2007.227

Cited by 367 publications

(362 citation statements)

References 47 publications

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“…The pooled chloroform phases (1.5 ml) were then washed 8 times with water. The chloroform phase containing the methylated derivatives was finally dried under a stream of nitrogen, and the extracted products were further purified on a C18 Sep-Pak (19). The C18 Sep-Pak was sequentially conditioned with methanol (5 ml) and water (10 ml).…”

Section: Methodsmentioning

confidence: 99%

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Sialylation levels of anti-proteinase 3 antibodies are associated with the activity of granulomatosis with polyangiitis (Wegener's)

Espy

Morelle²,

Kavian

et al. 2011

Arthritis & Rheumatism

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Objective. To investigate whether the glycosylation and sialylation levels of anti-proteinase 3 (anti-PR3) antibodies could affect their pathogenicity, and whether these levels could be correlated with the activity of granulomatosis with polyangiitis (Wegener's) (GPA).Methods. Forty-two serum samples positive for anti-PR3 antibodies from 42 patients with active or weakly active/inactive GPA were included. Anti-PR3 antibodies were assayed by enzyme-linked immunosorbent assay, and their levels of glycosylation and sialylation were assessed by enzyme-linked lectin assay. The glycosylation and sialylation levels of IgG purified from the serum of healthy donors and patients with active, remitted, or weakly active disease were assessed by permethylation and mass spectrometry analysis of glycans, following neuraminidase digestion. The neutrophil oxidative burst induced by purified IgG was assayed by spectrofluorimetry.Results. The mean sialylation ratio of anti-PR3 antibodies was significantly lower in patients with active disease than in patients with weakly active or inactive disease, and this was inversely correlated with the Birmingham Vasculitis Activity Score (BVAS) (P < 0.0001). Similar results were obtained using the BVAS/ GPA. The area under the receiver operating characteristic curve for the sialylation ratio of anti-PR3 antibodies, as a test to determine the activity of GPA, was 0.82 (P ‫؍‬ 0.0006). The characterization of N-glycans showed a decrease in 2,6-linked sialylated N-glycans and an increase in dHex 1 Hex 3 HexNAc 4 (mass/charge 1,836) agalactosylated structures in purified IgG from patients with active disease compared with controls. The anti-PR3 antibody-induced oxidative burst of neutrophils was inversely correlated with the sialylation levels of anti-PR3 IgG.

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Section: Methodsmentioning

confidence: 99%

“…Acetonitrile was evaporated under a stream of nitrogen and the samples were freeze-dried. Matrixassisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry and linkage analyses of permethylated glycans were performed as described elsewhere (19).…”

Section: Methodsmentioning

confidence: 99%

Sialylation levels of anti-proteinase 3 antibodies are associated with the activity of granulomatosis with polyangiitis (Wegener's)

Espy

Morelle²,

Kavian

et al. 2011

Arthritis & Rheumatism

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“…A variety of biochemical methods have been employed to study glycan modifications, especially involving mass spectrometric technology (Budnik et al, 2006;Bond and Kohler, 2007;Morelle and Michalski, 2007). Over the last few years, mass spectrometry-based glycoproteomics has established itself as an important analytical instrument for studying normal and pathological aspects of glycoproteins in a global way (Harvey, 2005;Geyer and Geyer, 2006;Wuhrer et al, 2007).…”

Section: Introductionmentioning

confidence: 99%

Lectin-based proteomic profiling of aged skeletal muscle: Decreased pyruvate kinase isozyme M1 exhibits drastically increased levels of N-glycosylation

O’Connell

Doran

Gannon

et al. 2008

European Journal of Cell Biology

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Since various neuromuscular diseases are associated with abnormal glycosylation, it was of interest to determine whether this key post-translational modification is also altered in aged skeletal muscle. Lectins represent highly versatile carbohydrate-binding proteins that are routinely employed for the characterization of glycoproteins. Here, we used the lectin wheat germ agglutinin (WGA) for the proteomic profiling of senescent fibers. WGA labeling of the soluble proteome from 3-month-versus 30-month-old rat gastrocnemius muscle, following two-dimensional gel electrophoretic separation, resulted in the identification of 13 distinct protein species. Analysis of WGA binding levels, in conjunction with mass spectrometric fingerprinting, revealed that one isoform of a major metabolic muscle protein exhibited a drastic alteration in the content of sialic acid and N-acetylglucosaminyl sugar residues. Pyruvate kinase isoform M1 with protein accession number gi|16757994|, exhibiting a pI of 6.6 and an apparent molecular mass of 57.8 kDa, showed a six fold increase in N-glycosylation and a three fold decrease in protein expression. In contrast to comparable levels of N-glycosylated proteins in young adult versus senescent muscle, as judged by fluoresceinconjugated WGA labeling of transverse muscle cryosections, staining with antibodies to the M1 isoform of pyruvate kinase showed reduced expression of this cytosolic element. Furthermore, activity assays demonstrated a reduced activity of this glycolytic enzyme in senescent muscle. This agrees with the idea that abnormal post-translational modifications in key metabolic enzymes may be involved in the conversion of aged muscle to slower twitch patterns and a drastic shift to more aerobic-oxidative metabolism.

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“…Analysis of N-and O-linked glycans from glycoproteins using MALDI-TOF mass spectrometry is common manipulation. The clean-up profiles of native and permethylated oligosaccharides for an efficient MALDI-TOF MS analysis have been described (Morelle et al, 2007;Zaia, 2010). There are less of native polysaccharides g i v i n g M A L D I -T O F M S a n a l y s i s w i t h o u t degradation and derivation.…”

Section: Maldi Mass Spectrometry Of Polysaccharidesmentioning

confidence: 99%

Tandem Mass Spectrometry of Tagged and Permethylated Polysaccharides

Yang¹

2012

Tandem Mass Spectrometry - Applications and Principles

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Analysis of protein glycosylation by mass spectrometry

Cited by 367 publications

References 47 publications

Sialylation levels of anti-proteinase 3 antibodies are associated with the activity of granulomatosis with polyangiitis (Wegener's)

Sialylation levels of anti-proteinase 3 antibodies are associated with the activity of granulomatosis with polyangiitis (Wegener's)

Lectin-based proteomic profiling of aged skeletal muscle: Decreased pyruvate kinase isozyme M1 exhibits drastically increased levels of N-glycosylation

Tandem Mass Spectrometry of Tagged and Permethylated Polysaccharides

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