Characterization of Glycosylation of Membrane Receptors at Low Levels using Extended Range Proteomic Analysis (ERPA)

摘要

Despite of the recent progress in mass spectrometry and sample preparation, characterization of protein posttranslational modifications (PTMs) still remains a challenge, especially at low protein levels. Most of the current strategies for identification PTMs rely on enrichment of PTM containing peptides or proteins followed by LC-MS analysis. However, due to the fact that enrichment efficiency often depends on properties of analyzed compounds, only incomplete or biased information is typically obtained. Recently, we developed a new strategy, termed Extended Range Proteomic Analysis (ERPA), that allows analysis of peptides up to 10 kDa, which significantly improves chances for observation of PTMs without the need for specific enrichment. We optimized this technique for characterization of limited amounts of protein (<100 ng) purified by immuno-precipitation from cell cultures and applied this approach for analysis of protein glycosylation and phosphorylation.