Combined use of 2,4,6‐trihydroxyacetophenone as matrix and enzymatic deglycosylation in organic‐aqueous solvent systems for the simultaneous characterization of complex glycoproteins and N‐glycans by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry

Fenaille, François; Mignon, Maxime Le; Groseil, Catherine; Siret, L.; Bihoreau, Nicolas

doi:10.1002/rcm.2886

Cited by 12 publications

(8 citation statements)

References 13 publications

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“…Signal intensity was also increased if 10 mM sodium chloride was added to the matrix . Fenaille et al (2007a) have shown that THAP is a more efficient matrix than the more commonly used sinapinic acid for ionization of glycoproteins, even those with masses as high as 150 kDa. Singly charged ions were the major species produced but the larger glycoproteins yielded both doubly and triply charged ions in addition.…”

Section: B Simple Matricesmentioning

confidence: 99%

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Analysis of carbohydrates and glycoconjugates by matrix‐assisted laser desorption/ionization mass spectrometry: An update for 2007–2008

Harvey

2011

Mass Spectrometry Reviews

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This review is the fifth update of the original review, published in 1999, on the application of MALDI mass spectrometry to the analysis of carbohydrates and glycoconjugates and brings coverage of the literature to the end of 2008. The first section of the review covers fundamental studies, fragmentation of carbohydrate ions, use of derivatives and new software developments for analysis of carbohydrate spectra. Among newer areas of method development are glycan arrays, MALDI imaging and the use of ion mobility spectrometry. The second section of the review discusses applications of MALDI MS to the analysis of different types of carbohydrate. Specific compound classes that are covered include carbohydrate polymers from plants, N- and O-linked glycans from glycoproteins, biopharmaceuticals, glycated proteins, glycolipids, glycosides and various other natural products. There is a short section on the use of MALDI mass spectrometry for the study of enzymes involved in glycan processing and a section on the use of MALDI MS to monitor products of the chemical synthesis of carbohydrates with emphasis on carbohydrate-protein complexes and glycodendrimers. Corresponding analyses by electrospray ionization now appear to outnumber those performed by MALDI and the amount of literature makes a comprehensive review on this technique impractical. However, most of the work relating to sample preparation and glycan synthesis is equally relevant to electrospray and, consequently, those proposing analyses by electrospray should also find material in this review of interest.

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Section: B Simple Matricesmentioning

confidence: 99%

“…Under these conditions, deglycosylation was much more efficient than when reactions were conducted in water or buffers. Most glycoproteins could be deglycosylated in 1 hr at 378C and, furthermore, because of the absence of salts, etc., the mixture could be examined directly by MALDI (Fenaille et al, 2007a).…”

Section: Analysis Of Carbohydrates and Glycoconjugatesmentioning

confidence: 99%

Analysis of carbohydrates and glycoconjugates by matrix‐assisted laser desorption/ionization mass spectrometry: An update for 2007–2008

Harvey

2011

Mass Spectrometry Reviews

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“…FVIIa was enzymatically digested using PNGase F and Endo H in organic-aqueous solvent systems [22]. Briefly, 1 μL of a FVIIa aqueous solution (i.e.…”

Section: Estimation Of N-glycan Composition By Endoglycosidase Digestmentioning

confidence: 99%

Mass spectrometric characterization of N- and O-glycans of plasma-derived coagulation factor VII

et al. 2008

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Factor VII (FVII) is a vitamin K-dependent glycoprotein which, in its activated form (FVIIa), participates in the coagulation process by activating factor X and factor IX. FVII is secreted as single peptide chain of 406 residues. Plasma-derived FVII undergoes many post-translational modifications such as gamma-carboxylation, N- and O-glycosylation, beta-hydroxylation. Despite glycosylation of recombinant FVIIa has been fully characterized, nothing is reported on the N- and O-glycans of plasma-derived FVII (pd-FVII) and on their structural heterogeneity at each glycosylation site. N- and O-glycosylation sites and site specific heterogeneity of pd-FVII were studied by various complementary qualitative and quantitative techniques. A MALDI-MS analysis of the native protein indicated that FVII is a 50.1 kDa glycoprotein modified on two sites by diantennary, disialylated non-fucosylated (A2S2) glycans. LC-ESIMS/MS analysis revealed that both light chain and heavy chain were N-glycosylated mainly by A2S2 but also by triantennary sialylated glycans. Nevertheless, lower amounts of triantennary structures were found on Asn(322) compared to Asn(145). Moreover, the triantennary glycans were shown to be fucosylated. In parallel, quantitative analysis of the isolated glycans by capillary electrophoresis indicated that the diantennary structures represented about 50% of the total glycan content. Glycan sequencing using different glycanases led to the identification of triantennary difucosylated structures. Last, MS and MS/MS analysis revealed that FVII is O-glycosylated on the light chain at position Ser(60) and Ser(52) which are modified by oligosaccharide structures such as fucose and Glc(Xyl)(0-1-2), respectively. These latter three O-glycans coexist in equal amounts in plasma-derived FVII.

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“…Analyses were carried out using different matrix solutions: (i) HCCA, 0.3 mg/mL in ethanol/acetone (2:1, v/v); (ii) DHB, 20 mg/mL in acetonitrile; (iii) THAP, 10 mg/mL in acetonitrile/water (1:1, v/v) containing 0.1% TFA. HCCA and DHB matrices were used for the analysis of proteolytic peptides and glycopeptides, respectively; THAP was used for the analysis of native and deglycosylated proteins (Fenaille et al 2007). For each sample, 0.5 µL of analyte solution and 0.5 µL of matrix solution were deposited and thoroughly mixed on the target.…”

Section: Maldi-tofms Analysesmentioning

confidence: 99%

“…CFH was enzymatically digested using PNGase F and Endo H in organic-aqueous solvent systems (Fenaille et al 2007). Briefly, 1 µL of a CFH aqueous solution (i.e., 2 µg) was mixed with 6 µL of acetonitrile/water (75:25, v/v) and 0.5 µL of a PNGase F (i.e., 1.25 mU) or Endo H solution (i.e., 2.5 mU).…”

Section: Deglycosylation Of Cfh For Maldi-tofms Analysismentioning

confidence: 99%

Site-specific N-glycan characterization of human complement factor H

et al. 2007

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Human complement factor H (CFH) is a plasma glycoprotein involved in the regulation of the alternative pathway of the complement system. A deficiency in CFH is a cause of severe pathologies like atypical haemolytic uraemic syndrome (aHUS). CFH is a 155-kDa glycoprotein containing nine potential N-glycosylation sites. In the current study, we present a quantitative glycosylation analysis of CFH using capillary electrophoresis and a complete sitespecific N-glycan characterization using matrix-assisted laser desorption/ ionization time-of-flight (MALDI-TOF) and liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESIMS/MS

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Combined use of 2,4,6‐trihydroxyacetophenone as matrix and enzymatic deglycosylation in organic‐aqueous solvent systems for the simultaneous characterization of complex glycoproteins and N‐glycans by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry

Cited by 12 publications

References 13 publications

Analysis of carbohydrates and glycoconjugates by matrix‐assisted laser desorption/ionization mass spectrometry: An update for 2007–2008

Analysis of carbohydrates and glycoconjugates by matrix‐assisted laser desorption/ionization mass spectrometry: An update for 2007–2008

Mass spectrometric characterization of N- and O-glycans of plasma-derived coagulation factor VII

Site-specific N-glycan characterization of human complement factor H

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