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

Abstract

High‐performance liquid chromatography–mass spectrometry (HPLC‐MS) is one of the most powerful tools in peptide and protein analysis today. The success of HPLC‐MS has resulted from the advancements in both liquid chromatography and mass spectrometry instrumentation, and new methodologies in data acquisition, data analysis algorithms, and computational methods. To meet the demand for greater analytical depth, sensitivity, and higher throughput capabilities, liquid chromatography has seen the adoption of HPLC and then the transition to ultra‐high‐performance liquid chromatography (UHPLC), decreasing particle size to sub‐2 μm, and the availability of a wide range of stationary phases, including reversed‐phase, ion exchange, hydrophilic interaction, and size exclusion. The advances in soft ionization in mass spectrometry, in particular, matrix‐assisted laser desorption/ionization (MALDI) 1 and electrospray ionization (ESI),2 have transformed biomolecule mass spectrometry. ESI operates with a continuous flow, is an ideal interface to couple HPLC to mass spectrometry. A wide range of mass‐spectrometry instrumentations have emerged, one of the most significant among them is the advent of high‐resolution mass spectrometers, particularly the quadrupole time‐of‐flight (Q‐TOF) and orbitrap mass spectrometers. The HPLC‐MS analytical platform has been applied in many areas of peptide and protein analyses, including natural and synthetic peptides, protein posttranslational modifications, and antibodies. As a result of genome projects in the 1990s, there is an increasing need to develop analytical tools for proteomics. The combination of nano LC, tandem mass spectrometry, and database searching has become a standard analytical platform for large‐scale high‐throughput protein identification. 3 Many biological problems increasingly require the quantification of proteins. The techniques used for small molecules, such as stable isotope labeling, isotope dilution mass spectrometry, and selected reaction monitoring are now part of the repertoire for absolute quantitation of proteins.