Development of an analysis method for a glycosylated protein using MALDI-MS and separation techniques

Sammanfattning: The antibody Immunoglobulin G (IgG) main function is to protect and prevent the body from infections, and it is normally found in human serum. This study is about IgG glycosylation, which is associated with different types of diseases such as neurological diseases, cancers and immunodeficiency etc. This study attempts to optimize IgG glycopeptide enrichment in a 100 μL micropipette tip set up, and to separate the enriched glycopeptides using capillary electrophoresis (CE). Matrix-assisted laser desorption/ionization – mass spectrometry (MALDI-MS) was used for data acquisition and glycopeptide profiling.  In this study, loading solutions with different combinations of acetonitrile (ACN) and trifluoroacetic acid (TFA), together with various precondition and sample preparation procedures were evaluated on IgG digest samples. Best enrichment performance, particularly regarding the selectivity, was achieved using the parameters as follows: loading solution of 83% ACN/16% H2O/1% TFA, sample solution in H2O containing 83% ACN, using a 100 μL micropipette tip packed with 1 mg cotton wool. A re-enrichment step was carried out on enriched glycopeptide samples, and improved selectivity of glycopeptides could be observed. Enriched glycopeptides could be separated into three major groups by CE using an acidic background electrolyte of 50 mM formic acid and 50 mM acetic acid, pH 2.5.