Analyses of PCSK9 post-translational modifications using time-of-flight mass spectrometry.

Post-translational modification(s) can affect a protein's function - changing its half-life/stability, its protein-protein interactions, biological activity and/or sub-cellular localization. Following translation, a protein can be modified in several ways, including (i) disulfide bridge formation, (ii) chemical conversion of its constituent amino acids (for instance, glutamine can undergo deamidation to glutamic acid), (iii) sulfation, phosphorylation, de/acetylation, and glycosylation (to name a few), (iv) addition of other proteins as occurs during sumoylation and ubiquitination, and (v) proteolytic cleavage(s). There are several techniques available to identify and monitor post-translational modifications of proteins and peptides including mass spectrometry, two-dimensional sodium dodecyl sulfate polyacrylamide electrophoresis (2D-SDS-PAGE), radiolabeling, and immunoblotting. Ciphergen's surface-enhanced laser desorption/ionization time-of-flight mass spectrometer (SELDI-TOF-MS) has been used successfully for protein/peptide profiling in disease states and for the detection of protein/peptide biomarkers (1-4). In this chapter, the secreted proprotein convertase subtilisin/kexin 9 (PCSK9), which we study in our lab, is used to demonstrate coupling of immunoprecipitation with Ciphergen's time-of-flight mass spectrometer and its ProteinChip software to detect and analyze the common post-translational modifications of phosphorylation and glycosylation. The following topics are covered (1): preparation of cell extracts/samples/spent media (2), processing of samples by immunoprecipitation including optimization of conditions and (3) data acquisition by mass spectrometry and its subsequent analyses.