A New Delayed Extraction MALDI-TOF MS-MS for Characterization of Protein Digests

Abstract

Two-dimensional gel electrophoresis is commonly used to separate and visualize proteins present in complex mixtures such as cell lysates [1]. Digestion of selected spots by one or more endopeptidases followed by generation of peptide mass maps using MALDI-TOF MS is now widely accepted as the first step toward identifying and characterizing the proteins [2]. Following the development of delayed extraction techniques for MALDI [3–5], and improved sample preparation and clean-up methodology [6], this strategy is often successful at identifying proteins represented in a database [7] even when the proteins are present at low levels. In favorable cases protein identification is successful at the sub-femtomole level. When this approach fails, it is generally necessary to generate sequence data using either Edman degradation or MS-MS techniques. The MS-MS technique known as post-source decay (PSD) with MALDI-TOF [8] sometimes provides sufficient sequence information, but often the sensitivity and mass accuracy are inadequate and interpretation is difficult. Recent developments in electrospray ionization, such as nanospray [9] have dramatically improved the sensitivity of triple quadrupole and ion trap MS-MS, but these techniques are rather slow and tedious, and rapid, automated interpretation of data to provide reliable sequences is not yet routine. New instruments employing TOF analyzers in place of the third quadrupole in triple quadrupole MS-MS systems have very recently been described which provide improved resolution and mass accuracy in fragment ion measurements [10, 11].