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


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.


LC MALDI PCT Tryptic Digestion On-target 


  1. Alterman, M.A., Chaurasia, C.S., Lu, P., and Hanzlik R.P. (1995). Heteroatom substitution shifts regioselectivity of lauric acid metabolism from omega-hydroxylation to (omega-1)-oxidation. Biochem Biophys Res Commun 214, 1089–1094.CrossRefGoogle Scholar
  2. Harris, W.A., and Reilly, J.P. (2002) On-probe digestion of bacterial proteins for MALDI-MS. Anal Chem 74, 4410–4416.CrossRefGoogle Scholar
  3. López-Ferrer, D., Petritis, K., Hixson, K.K., Heibeck, T.H., Moore, R.J., Belov, M.E., Camp, D.G., 2nd, and Smith, R.D. (2008). Application of pressurized solvents for ultrafast trypsin hydrolysis in proteomics: proteomics on the fly. J Proteome Res 7(8), 3276–3281.CrossRefGoogle Scholar
  4. Warscheid, B., and Fenselau, C. (2004). A targeted proteomics approach to the rapid identification of bacterial cell mixtures by matrix-assisted laser desorption/ionization mass spectrometry. Proteomics 4, 2877–2892.CrossRefGoogle Scholar

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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