Identification of proteins from two-dimensional gel electrophoresis of human erythroleukemia cells using capillary high performance liquid chromatography/electrospray-ion trap-reflectron time-of-flight mass spectrometry withtwo-dimensional topographic map analysis of in-gel tryptic digest products

Protein spots from two-dimensional (2-D) gel electrophoresis of a human ery throleukemia cell line have been identified by analysis of the in-gel trypt ic digests using capillary high performance liquid chromatography (HPLC) se paration with on-line detection using electrospray ionization mass spectrom etry (ESI-MS), This is performed using an electrospray/ion trap storage/ref lection time-of-flight mass spectrometer system (ESI-IT-reTOFMS). A 2-D top ographic mapping display developed to process the on-line data acquired wit h this TOF system has been used to obtain mass identification of each pepti de, even though the capillary HPLC only provides limited separation capabil ity of the tryptic peptide mixtures studied herein. Using this method, a su bstantial fraction of the protein sequence can be covered and identified us ing the tryptic map. It is demonstrated that by entering the cell species, the approximate MW and pi range as determined by 2-D gel electrophoresis, a nd the tryptic peptide map into the database a unique match for identificat ion of the protein generally results. It is also demonstrated that a much i mproved coverage of the protein sequence is obtained by this method relativ e to matrix-assisted laser desorption/ionization mass spectrometry (MALDI-M S). Copyright (C) 1999 John Wiley & Sons, Ltd.