Protein analysis by mass spectrometry and sequence database searching: A proteomic approach to identify human lymphoblastoid cell line proteins

Lymphoblastoid cell lines correspond to in vitro EBV-immortalized lymphocyt e B-cells. These cells display a suitable model for experiments dealing wit h changes in protein expression occurring upon B-cell differentiation, afte r drug treatment, or after inhibition of some transcription factors. For al l these reasons we have undertaken an effort aimed at developing a hematopo ietic cell line protein two-dimensional electrophoresis (2-DE) database, co ntaining B-lymphoblastoid 2-DE maps. In this work, matrix-assisted laser de sorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) peptide mass fingerprinting analysis was adopted for protein identification. The p eptide mass fingerprinting identification and the sequence coverage obtaine d on colloidal Coomassie blue (CBB) stained gel was close to that obtained using zinc-imidazole staining. Everything considered, CBB being more confor table for subsequent spot manipulations, CBB staining was chosen for identi fication of a larger number of polypeptides. The results suggest that retic ulation of the gel can interfere preventing the uptake of the enzyme during the in-gel digestion step. Consequently, low molecular mass proteins appea r more difficult to identify by mass fingerprinting. Finally, the informati on provided in this study allows the construction of a new annoted referenc e map of human lymphoblastoid cell proteins. Among the identified proteins 60% were not yet positioned on 2-DE maps in three of the most important wel l-documented databases. The annoted map will be accessible via Internet on the LBPP server at URL:http://www-smbh.univ-paris13.fr/lbtp/index.htm.