Capillary electrophoresis/electrospray ionization high mass accuracy time-of-flight mass spectrometry for protein identification using peptide mapping.
Capillary electrophoresis/electrospray ionization (CE/ESI) high mass accuracy time-of-flight mass spectrometry was used for the first time to characterize small proteins using peptide mapping. To identify small proteins, the intact proteins were first analyzed to obtain their average molecular weights with errors less than 1 Da. On-line capillary electrophoresis mass spectrometry of the tryptic digests of these small proteins was then performed to obtain the accurate molecular weights of the peptides with accuracies of approximately 10 ppm. Next, this information was used for the identification of the proteins using a protein database. It was found that high mass accuracy is an effective tool in reducing the list of most-likely proteins generated by the database. In addition, on-line collision-induced dissociation of the completely or partially resolved capillary electrophoresis peaks of the protein digests was used to unambiguously identify the sequences of these peptides. Each CE/ESI-MS analysis used only 5 nL of sample containing approximately 120 fmol of each peptide in protein digests. The results indicate that the combination of capillary electrophoresis and high resolution, high mass accuracy time-of-flight mass spectrometry is a viable option for the identification of small proteins using peptide mapping.
Department of Chemistry and Biochemistry, University of Texas at Austin 78712, USA.
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Cao
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Moini
D002138:Calibration; D003574:Cytochrome c Group / Q000737:chemistry; D019075:Electrophoresis, Capillary; D006868:Hydrolysis; D013058:Mass Spectrometry; D008970:Molecular Weight; D009211:Myoglobin / Q000737:chemistry; D010449:Peptide Mapping / Q000379:methods*; D011506:Proteins / Q000737:chemistry*; D014357:Trypsin
D003574:Cytochrome c Group; D009211:Myoglobin; D011506:Proteins; D014357:Trypsin