Synthesis of Fc peptides and glycopeptides to be used as internal standards for absolute quantitation of glycoforms of two human IgG subclasses by MALDI-MS analysis
Abstract
Immunoglobulins (IgGs) play a central role in the immune system of living organisms. Human IgG comprises of 4 subclasses, ranging from IgG1 to IgG4, of which IgG1 and IgG2 are the most abundant in healthy individuals. In an effort to develop an absolute MALDI-ToF-MS quantitation method for these subclasses and their Fc N-glycoforms, peptides and (glyco)peptides were synthesized using a solid phase approach and used as internal standards for enriched fractions from human IgG tryptic digests. Mass spectrometry was used to analyze biomolecules quantitatively throughout the experiment. Tryptic digest glycopeptides from monoclonal IgG1 and IgG2 samples were first quantified using EEQYN(GlcNAc)STYR and EEQFN(GlcNAc)STFR, respectively. It was observed that simultaneous quantitation of IgG1 and IgG2 yielded non-quantitative results, and more success was obtained when subclasses were quantified one by one. The study also explored the relative sensitivities of two synthetic Fc tryptic peptides of IgG1 and IgG2 under MALDI conditions.
Description
Keywords
Immunoglobulins, Internal Standards, Mass Spectrometry