High‐Performance Liquid Chromatography/Mass Spectrometry in Peptide and Protein Analysis

Abstract

AbstractSince the 1970s, high‐performance liquid chromatography/mass spectrometry (HPLC/MS) has gone through many stages of development. The main technical challenge has been the coupling of liquid chromatography (LC) systems with mass spectrometers. Soft ionization techniques that have contributed to HPLC/MS for peptide and protein study include fast atom bombardment (FAB), matrix‐assisted laser desorption/ionization (MALDI), and electrospray ionization (ESI). Among these techniques, FAB was developed first and used extensively in the 1980s; it lacked the sensitivity of the other two more recent techniques. MALDI and ESI are comparable in terms of sensitivity and the types of biomolecules that can be analyzed. However, MALDI has the slight advantage of being more tolerant to salts and able to analyze complex mixtures. On the other hand, the ability of ESI to interface mass spectrometry (MS) directly with LC makes it extremely useful for protein and peptide analysis. Another advantage that ESI offers is its ability to produce multiply charged protein ions, which allows the observation of large proteins with mass analyzers of limited mass range. In this article we discuss the experimental parameters that are important in HPLC/MS and the considerable progress made in ESI source design and capillary HPLC/MS.In the study of natural and synthetic peptides, HPLC/MS has been used to separate, characterize, and sequence the peptides being investigated. In protein analysis, HPLC/MS is most useful in characterizing protein post‐translation modification such as glycosylation and phosphorylation. We discuss specific HPLC/MS methods for detecting glycopeptides and phosphopeptides during chromatographic separation by monitoring marker ions: Hex+(m/z 163), HexNAc+(m/z 204), NeuAc+(m/z 292), HexHexNAc+(m/z 366) for glycopeptides, and PO2−(m/z 63), PO3−(m/z 79) for phosphopeptides. Another important application of HPLC/MS is in proteomic analysis. Here, the most significant development in the last decade has been the implementation of MS as the primary analytical technique to identify proteins separated by two‐dimensional polyacrylamide gel electrophoresis (2D/PAGE). Additionally, the need to identify gel‐separated proteins has promoted various MS‐based protein database search strategies. These include peptide mass mapping using proteolytic peptide masses for database search, and using peptide fragmentation data obtained from tandem mass spectrometry (MS/MS) to search protein sequence databases. Finally, we examine the recent advance in integrating HPLC/MS and database search strategy in proteomic analysis.