MALDI MS Peptide Mapping Performance by In-Gel Digestion on a Probe with Prestructured Sample Supports

摘要

Matrix-assisted laser desorption/ionization (tandem) mass spectrometry (MALDI MS) is widely used in protein chemistry and proteomics research for the identification and characterization of proteins isolated by polyacryl-amide gel electrophoresis.In an effort to minimize sample handling and increase sample throughput,we have developed a novel in-gel digestion protocol where sample preparation is performed directly on a MALDI probe with prestructured sample support.The protocol consists of few sample-handling steps and has minimal consumption of reagents,making the protocol sensitive,timesaving,and cost-efficient.Performance of the on-probe sample preparation protocol was demonstrated by analysis of a set of rat liver proteins obtained from a fluorescently stained (Cy3 and SyproRuby) two-dimensional polyacrylamide gel.The success rate of protein identification by on-probe tryptic digestion and MALDI peptide mass mapping was 89%.The on-probe in-gel digestion procedure provided superior sensitivity and peptide mass mapping performance as compared to our standard in-gel digestion protocol.The on-probe digestion technique resulted in significantly improved amino acid sequence coverage of proteins,mainly due to efficient recovery and detection of large (>1.5 kDa) hydrophobic peptides.These observations indicate that numerous tryptic peptides are lost when using the standard in-gel digestion methods and sample preparation techniques for MALDI MS.This study also demonstrates that the on-probe digestion protocol combined with MALDI tandem mass spectrometry provides a robust platform for proteomics research,including protein identification and determination of posttransla-tional modifications.