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 with two-dimensional topographic map analysis of in-gel tryptic digest products.
Protein spots from two-dimensional (2-D) gel electrophoresis of a human erythroleukemia cell line have been identified by analysis of the in-gel tryptic digests using capillary high performance liquid chromatography (HPLC) separation with on-line detection using electrospray ionization mass spectrometry (ESI-MS). This is performed using an electrospray/ion trap storage/reflectron time-of-flight mass spectrometer system (ESI-IT-reTOFMS). A 2-D topographic mapping display developed to process the on-line data acquired with this TOF system has been used to obtain mass identification of each peptide, even though the capillary HPLC only provides limited separation capability of the tryptic peptide mixtures studied herein. Using this method, a substantial fraction of the protein sequence can be covered and identified using 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, and the tryptic peptide map into the database a unique match for identification of the protein generally results. It is also demonstrated that a much improved coverage of the protein sequence is obtained by this method relative to matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS).
Chen
Y
Y
Department of Chemistry, School of Medicine, The University of Michigan, Ann Arbor, MI 48109-1055, USA.
Jin
X
X
Misek
D
D
Hinderer
R
R
Hanash
S M
SM
Lubman
D M
DM
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D016208:Databases, Factual; D015180:Electrophoresis, Gel, Two-Dimensional; D006801:Humans; D007526:Isoelectric Point; D004915:Leukemia, Erythroblastic, Acute / Q000378:metabolism*; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000032:analysis* / Q000737:chemistry; D010446:Peptide Fragments / Q000032:analysis / Q000737:chemistry; D010449:Peptide Mapping; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014357:Trypsin / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D009363:Neoplasm Proteins; D010446:Peptide Fragments; D014357:Trypsin