Recent Advances and Special Considerations for the Analysis of Phosphorylated Peptides by LC-ESI-MS/MS

Liquid chromatography electrospray tandem mass spectrometry (LC-ESI-MS/MS) is the primary method for sample analysis used in proteomics. The high-resolution separation of liquid chromatography (LC), coupled with the sensitivity and specificity of mass spectrometry (MS) permits the identification of thousands of peptides in a single analysis of protease digested samples. Although numerous advances have been made in recent years, there is still a long way to go to achieve the ultimate goal of proteomics-----the comprehensive characterization of all proteins, including post-translational modifications (PTM), irrespective of sample type and complexity. Due to the importance of protein phosphorylation in biological processes, its identification and quantification have become an intensely studied subset of proteomics. Phosphorylated peptides exhibit increased acidity and high affinity towards metal ions in comparison to other peptides. Therefore, special considerations should be taken when developing an LC-MS technique for phosphorylated peptide analysis. While advancements in instrumentation continue to increase the sensitivity, speed, resolution, and dynamic range of current mass spectrometers, the development of complementary LC methods has received less attention. In this communication, we will review the advancements in LC technology in proteomics, with a focus on the analysis of phosphorylated peptides.