Sequential Immunoaffinity Purification of Post-Translationally Modified Peptides for Improved Enrichment Specificity

摘要

Post-translational modification (PTM) of proteins, including phosphorylation, acetylation, methylation, and ubiquitination, is critical to all aspects of cellular signaling. Antibody-based and enrichments of post-translationally modified peptides combined with LCMS have proven to be powerful methods for the study of PTMs in a wide variety of cells and tissues, and in profiling various disease states. Here, PTM peptides were sequentially enriched from samples twice with the same antibody. This sequential enrichment both qualitatively and quantitatively reduced the number of nonspecific, unmodified peptides identified in the samples with minimal loss of PTM peptides of interest. This increase in enrichment specificity (% modified peptides) allows more instrument time for identification of peptides of interest as well as improving quantitative accuracy of the method.