Evaluation of Two Complementary Methods for Quantitative Profiling of PSA N-Glycans and N-Glycopeptides

摘要

The analytical methods for the structural characterization of protein glycosylation in glycomics and glycoproteomics are gradually becoming more suitable for biological applications. Over the past 10 years there has been a concerted effort to increase the sensitivity, speed and automation of the analysis of more complex glycoprotein mixtures at the level of biologically relevant dynamic ranges. However, these promising analytical tools, which predominantly are mass spectrometry based, needs thorough comparison and validation, in particular when quantitation is required. As a part of the ABRF Glycoprotein Research Group (gPRG) Quantitative Glycoprotein Study, we have profiled the single N -glycosylation of two sources of human prostate specific antigen (PSA) using two different, but commonly used, analytical methods allowing us to compare their qualitative and quantitative performance: i) Quantitative site-specific analysis of enriched PSA N -glycopeptides and ii) quantitative global analysis of released and reduced PSA N -glycans. For both approaches porous graphitized carbon LC connected directly with ion trap MS/MS (CID) was used for analyte separation and detection, respectively, but with different MS polarity mode detection. Using label-free relative quantitation, the two analytical methods produced very similar PSA glycoprofiles considering their different nature. Of the 40-50 monosaccharide compositions detected from each PSA variant, mostly minor quantitative differences in the glycoprofiles produced by the two approaches were observed, enhancing the confidence of the analysis. The pros and cons of the two glycoprofiling approaches and the intended near-future improvements will be discussed. Articles from Journal of Biomolecular Techniques : JBT are provided here courtesy of The Association of Biomolecular Resource Facilities.