Luis Javier Gonzlez 1 Lila Castellanos-Serra 1 Volker Badock 2 MaylÌn DÌaz 1 Alejandro Moro 1 Silvio Perea 1 Alicia Santos 1 Dalila Paz-Lago 1 Albrecht Otto 2 Eva-Christina Mçller 2 Susanne Kostka 2 Brigitte Wittmann-Liebold 2, * Gabriel PadrÕn 1 1 CenterforGeneticEngineering andBiotechnology, Havana,Cuba 2 Max-DelbrçckCenter forMolecularMedicine, Berlin,Germany Identification pf nuclear proteins of small cell lung cancer cell line H82: An improved procedure for the analysis of silver-stained proteins An efficient method for digestion and extraction of proteolytic peptides from silver- stainedproteinswasappliedtothecharacterizationofnuclearproteinsfromthesmall celllungcancerH82ATCCHTB175)celllinepreviouslyseparatedbyhigh-resolution largeformattwo-dimensionalgelelectrophoresis.From68spots,evenlydistributedon thegelareaandrepresentingawiderangeofspotintensities,6392%)weresuccess- fully identified by matrix-assisted laser desorption/ionization MALDI) or electrospray ionozation-mass spectrometry ESI-MS). In five cases where the identification was notpossible,thepresenceofanintensebackgroundapparentlyduetotheleakageof polymers from the microtubes or other plastics, was detected. Extensive analysis of peptide sequences by ESI MS/MS experiments allowed the identification of post- translational modifications, such as acetylation, phosphorylation, deamidation of asparagine residues and the presence of isoaspartic acid. A new protein variant not reportedinsequencedatabaseswasalsodetected. Keywords: In-gel digestion / Mass spectrometry / Proteomics / Small-cell lung cancer / Two- dimensionalgelelectrophoresis EL5219 1 Introduction Two-dimensional polyacrylamide gel electrophoresis 2-DE) is widely accepted as the most resolving tech- niquefortheanalysisofcomplexmixturesofproteinsin asingleexperiment[1].Differentstainingtechniquesthat use dyes, chemical reactions, radioactive labeling or fluorescent reagents allow the visualization of proteins previously separated by 2-DE [2]. For proteome studies, three attributes of the detection technique are required: a high sensitivity preferable in the order of low nano- grams), the preservation of protein integrity i.e., not introducing chemical modifications) and the compatibil- ity with further procedures for protein identification as massspectrometryMS)followedbydatabasesearch. In analytical gels, protein detection by silver staining [3] is widely used due to its high sensitivity. In the original procedures, an enhancement of sensitivity was accom- plishedbyincludinganearlystepofproteinreactionwith glutaraldehyde. This procedure has been modified sev- eralgroups±attheexpenseofasomewhatreducedsen- sitivity±inordertorenderitcompatiblewithproteiniden- tification by MS [4±6]. There are divergent reports in the literature concerning the efficiency of the identification byMSofsilver-stainedproteinspreviouslyseparatedby 2-DE [7±9]. For preparative purposes, many groups remain reluctant to the use of modified silver-staining, arguing a lower success rate in identification after in-gel digestion. A rapid view to the posters in the 2002 Siena meeting From Genome to Proteome, Siena, Sept. 2±6, 2002)showedapreferenceforcolloidalCoomassieblue andotherCoomassievariantsasstainingmethodforpre- parativepurposes.Acarefulevaluationofpeptidelosses during in-gel digestion and all post-digestion steps has beenaccomplishedwithradioisotopicallylabeledrecom- binant proteins [10]. It was demonstrated that even a minimalhandlingresultedinadsorptiveloosesonplastics tips and tubes) of about 15%, while it increased up to 50% or higher if samples were partially or completely dried to remove organic solvents by vacuum evapora- tion. Inthispaperwedescribesomemodificationsofthepost- digestion steps adapted to the conventional protocols currently in use for in-gel digestion in order to minimize sample handling, in particular, we introduce a two-step elution of the peptides from the C-18 microcolumns for maximizing their recovery. The efficacy of the proposed method is demonstrated by the successful identification of 92% of the analyzed silver-stained spots from 2-DE Correspondence: Dr. Lila Castellanos-Serra, Division of Physi- cal-Chemistry, Center for Genetic Engineering and Biotechnol- ogy Ave31 e/158y 190, Cubanacn, Playa, P.O. Box 6162, CP, 10600,Havana,Cuba E-mail: lila.castellanos@cigb.edu.cu Fax: +537-336008 Abbreviation: SCLC,small-celllungcancer Electrophoresis 2003, 24, 000±000 1 * Presentaddress:WITAGmbH,Warthestr.21,D-14513Teltow, Germany ã 2003WILEY-VCHVerlagGmbH&Co.KGaA,Weinheim 0173-0835/03/01n±1$17.501.50/0 VCH Auftr.-Nr.27481 1. Papierlauf Seite 1 ±me± {1_VCH}el_03_01/el_5219.3d Electrophoresis Mittwoch, den 20. 11. 02, 08 Uhr 58 Proteomics and 2-DE