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Exploring the Capabilities of the Protein Identification by Unconventional Sample Preparation Approaches: LC/MALDI/On-Target Digestion Approach and High Pressure-Assisted In-Gel Tryptic Digestion

  • Melkamu Getie-Kebtie
  • Michail A. AltermanEmail author
Chapter

Abstract

The rapid and comprehensive separation, identification, and characterization of proteins from complex biological samples are formidable challenges that the growing field of proteomics faces. A robust and efficient digest of separated proteins is central to the development of any quantitative and/or qualitative proteomic approach. We describe here two unconventional approaches to a trypsin digestion of biological samples. One of those approaches integrates separation of proteins using RP-HPLC with on-target proteolytic digestion of the proteins for subsequent MALDI-MS analysis. This approach allows the combined information from peptide mass fingerprinting (PMF), MS/MS peptide fragment fingerprinting (PPF) and whole protein MS to increase confidence in protein identification and structural analysis of proteins. The other approach is based on the application of high-pressure cycling technology (PCT) for in-gel trypsin digest of 1D electrophoretically separated proteins. That approach was used in our laboratory to develop a label-free mass spectrometry based method for a relative quantification of proteins after SDS PAGE separation, including co-migrating proteins.

Keywords

LC MALDI PCT Tryptic Digestion On-target 

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Copyright information

© Springer Science+Business Media B.V. 2011

Authors and Affiliations

  1. 1.Tumor Vaccines and Biotechnology Branch, Division of Cellular and Gene TherapiesCenter for Biologics Evaluation and Research, Food and Drug AdministrationBethesdaUSA

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