For antibody analysis, MALDI-MS ion abundances give a better semi-quantitative estimate of sialylation levels for esterified than for unreacted glycopeptides. The method is simple to use and helps to differentiate the branching patterns of sialic acids in antibodies.
Immunoglobulins, such as immunoglobulin G (IgG), are of prime importance in the immune system. Polyclonal human IgG comprises four subclasses, of which IgG1 and IgG2 are the most abundant in healthy individuals. In an effort to develop an absolute MALDI-ToF-MS quantitative method for these subclasses and their Fc N-glycoforms, (glyco)peptides were synthesized using a solid-phase approach and used as internal standards. Tryptic digest glycopeptides from monoclonal IgG1 and IgG2 samples were first quantified using EEQYN(GlcNAc)STYR and EEQFN(GlcNAc)STFR standards, respectively. For IgG1, a similar glycopeptide where tyrosine (Y) was isotopically labelled was used to quantify monoclonal IgG1 that had been treated with the enzyme Endo-F2, i.e., yielding tryptic glycopeptide EEQYN(GlcNAc)STYR. The next step was to quantify single subclasses within polyclonal human IgG samples. Although ion abundances in the MALDI spectra often showed higher signals for IgG2 than IgG1, depending on the spotting solvent used, determination of amounts using the newly developed quantitative method allowed to obtain accurate concentrations where IgG1 species were predominant. It was observed that simultaneous analysis of IgG1 and IgG2 yielded non-quantitative results and that more success was obtained when subclasses were quantified one by one. More experiments served to assess the respective extraction and ionization efficiencies of EEQYNSTYR/EEQFNSTFR and EEQYN(GlcNAc)STYR/EEQFN(GlcNAc)STFR mixtures under different solvent and concentration conditions. Graphical Abstract ᅟ.
The characterization of the N-glycan portion of antibodies has been the subject of several studies involving mass spectrometry. In this article, a workflow is presented that starts with the expression of a monoclonal antibody (EG2-hFc) in Chinese hamster ovary cells and continues with Protein A purification of the antibody. Then the protocol continues with gel electrophoresis. Bands containing the heavy chain are cut and isolated from the gel followed by tryptic digestion to obtain peptides and glycopeptides. The enrichment of glycopeptides by C18 chromatography is described followed by characterization using positive and negative modes MALDI-MS and MS/MS. An exoglycosidase, beta-galactosidase, is used to verify anomericity of linkages in the glycan portion of glycopeptides. In the last step, glycans are detached from glycopeptides using PNGase F labelled with phehylhydrazine and characterized by MALDI-MS/MS. This workflow is reported for the first time for this particular antibody and presents a valuable approach for the analysis of N-glycans on most antibodies and glycoproteins.Résumé : La caractérisation de la portion N-glycane des anticorps a fait l'objet de nombreuses études faisant appel à la spectrométrie de masse. Dans le présent article, nous présentons un protocole méthodologique qui débute par l'expression d'un anticorps monoclonal (EG2-hFc) dans des cellules ovariennes de hamster et se poursuit avec la purification de la protéine A de l'anticorps suivie de l'électrophorèse en gel. Les bandes contenant les chaînes de masse élevée sont découpées et isolées du gel, et une digestion tryptique est effectuée en vue d'obtenir des peptides et des glycopeptides. Nous décrivons l'enrichissement des glycopeptides par chromatographie sur colonne C18, puis la caractérisation au moyen de la MALDI-MS et SM/SM en modes positif et négatif. Nous avons utilisé la bêta-galactosidase, une exoglycosidase, afin de vérifier l'anoméricité des liaisons dans la portion glycane des glycopeptides. En dernière étape, nous avons détaché les glycanes des glycopeptides à l'aide de la PNGase F, les avons marqués à l'aide de la phénylhydrazine et les avons caractérisés par spectroscopie de masse MALDI-SM/SM. Ce protocole pour cet anticorps précis est publié ici pour la première fois et présente une approche intéressante permettant d'effectuer l'analyse des N-glycanes pour la plupart des anticorps et des glycoprotéines. [Traduit par la Rédaction]
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