Montserrat Carrascal 1 Sonia Carujo 2 Oriol Bachs 2 Joaquin Abian 1 1 Structural and Biological Mass Spectrometry Unit, Department of Medical Bioanalysis, IIBB-CSIC, IDIBAPS, Barcelona, Spain 2 Department de Biologia Cellular i Anatomia Patològica, Facultat de Medicina, IDIBAPS, Universitat de Barcelona, Barcelona, Spain Identification of p21 Cip1 binding proteins by gel electrophoresis and capillary liquid chromatography microelectrospray tandem mass spectrometry Proteins bound to a glutathione-S-transferase-p21 Cip1 affinity column were separated by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and identified using tandem mass spectrometry. Capillary liquid chromatography coupled to microelectrospray tandem mass spectrometry (capLC-mESI MS/MS) in an ion trap allowed identification of the proteins present in the gel bands. Of eleven bands ana- lyzed, fifty-three proteins were identified. More than one hundred tryptic peptides were detected on-line, automatically fragmented and used for protein characterization in databases. Samples were also analyzed by off-line nanospray and matrix-assisted laser desorption/ionization mass spectrometry. CapLC-mESI MS/MS was the most efficient technique for the analysis of these protein mixtures. Keywords: p21 Cip1 / Protein-protein interactions / Mass spectrometry / Protein characterization / Capillary liquid chromatography microelectrospray tandem mass spectrometry PRO 0179 1 Introduction To understand biological processes it is essential to iden- tify and characterize interacting proteins and protein complexes. Proteins involved in the same processes can be characterized after their isolation in conditions pro- moting specific interactions. For example, protein com- plexes can be isolated by copurification using common biochemical procedures [1]. Immunoprecipitation meth- ods and affinity chromatography also allow the selective isolation of interacting proteins and protein complexes [2–4]. Currently, protein mixtures are efficiently characterized following a general strategy that consists of protein iso- lation by 2-DE and subsequent identification using mass spectrometry. This procedure has been applied in studies such as those on the differential protein expression in tumoral cells and tissues [5], changes in protein profile after treatment with drugs [6, 7], alterations in phosphory- lation patterns [8], characterization of isoforms [9] or the description of tissue specific or cellular-compartment specific proteic profiles [10–12]. Given its high separation efficiency, 2-DE is an appropriate technique for the sep- aration of the thousands of proteins present in a bio- logical fluid, cell or tissue. In the study of protein interactions, the previous selection of the protein set by the methods described above greatly reduces the complexity of the sample. In this case, 1-D SDS-PAGE could replace 2-DE, thereby providing a rapid and more convenient separation method [1, 13, 14]. For example, Mann and co-workers [2] characterized the Nup85p complex using a procedure that involved immu- no affinity purification of the complex, chemical cross- linking of the protein components, 1-D SDS-PAGE separ- ation and MS identification. Protein characterization is mostly carried out by pep- tide mass mapping (PMM) using matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) or by peptide sequence analysis by nanospray tandem mass spectrometry (nESI-MS/MS). Several studies have reported that MALDI-TOF MS PMM has some limitations in protein characterization when there is more than one protein in the gel band or when only a few proteolytic peptides are detected [15]. The presence of protein mixtures in 1-D gel bands is probably common even when selective prefractionation pro- cedures are used. In most cases, this hinders the auto- matic characterization of individual proteins and, when a match is obtained, it usually corresponds to a dominant protein in a mixture of low abundance proteins that remain undetected. More specific information about samples requires manual treatment of the peptide map to eliminate the first group of peptides identified from the Correspondence: Dr. Joaquin Abian, Structural and Biological Mass Spectrometry Unit, Department of Medical Bioanalysis, IIBB-CSIC, IDIBAPS, Roselló 161 7 a Planta, E-08036-Barcelona, Spain E-mail: jambam@iibb.csic.es Fax: 134-93-363-83-01 Abbreviations: CapLC, capillary liquid chromatography; CHCA, a-cyano-4-hydroxycinnamic acid; FSC, fused silica capillary; PMM, peptide mass mapping Proteomics 2002, 2, 455–468 455 ª WILEY-VCH Verlag GmbH, 69451 Weinheim, 2002 1615-9853/02/0404–455 $17.50+.50/0