Identification of trichloroethanol visualized proteins from two-dimensional polyacrylamide gels by mass spectrometry.
Proteins visualized by 2,2,2-trichloroethanol (TCE) on two-dimensional electrophoresis gels are efficiently identified by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) and MS/MS. In a previous study, a method was developed that placed TCE in the polyacrylamide gel so that protein bands can be visualized without staining in less than 5 min. A visible fluorophore is generated by reaction of TCE with tryptophan that allows for protein visualization. In this study, MALDI-TOF MS and LC-MS/MS are used to identify randomly selected Escherichia coli proteins. The identification of TCE visualized proteins is compared to the identification of Coomassie brilliant blue (CBB) stained proteins from two-dimensional gel electrophoresis of E. coli proteins. This study demonstrated that TCE visualized proteins are compatible with protein identification by MALDI-TOF peptide mass fingerprinting. For 10 randomly selected spots, TCE visualization lead to statistically significant identification of 5 proteins and CBB visualization lead to identification of 6 proteins. TCE visualized proteins are also shown to be well suited for protein identification using LC-MS/MS. In 16 spots selected for MS/MS analysis, TCE samples lead to the identification of 79 peptides; while CBB samples lead to the identification of 65 peptides. TCE samples also supported the identification of more proteins. The low stoichiometry of labeling of tryptophan residues does not require inclusion of this modification for database searches. In addition to being a rapid visualization technique compatible with MS, TCE visualization utilizes rapid washing conditions for sample preparation of proteins spots excised from polyacrylamide gels.
Ladner
Carol L
CL
Department of Biological Sciences, University of Calgary, Calgary, AB, Canada T2N 1N4.
Edwards
Robert A
RA
Schriemer
David C
DC
Turner
Raymond J
RJ
D000595:Amino Acid Sequence; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D029968:Escherichia coli Proteins / Q000032:analysis / Q000737:chemistry; D005023:Ethylene Chlorohydrin / Q000031:analogs & derivatives* / Q000737:chemistry; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D010449:Peptide Mapping / Q000379:methods*; D011506:Proteins / Q000032:analysis* / Q000737:chemistry; D012394:Rosaniline Dyes / Q000737:chemistry; D012680:Sensitivity and Specificity
D029968:Escherichia coli Proteins; D011506:Proteins; D012394:Rosaniline Dyes; D005023:Ethylene Chlorohydrin; C005849:2,2,2-trichloroethanol; C004692:coomassie Brilliant Blue