Automated protein identification using atmospheric-pressure matrix-assisted laser desorption/ionization.
Atmospheric-pressure matrix-assisted laser desorption/ionization (AP-MALDI) ion trap mass spectrometry (ITMS) has been evaluated for automated protein identification. By using signal averaging and long ion-injection times, protein identification limits in the 50-fmol range are achieved for standard protein digests. Data acquisition requires 7.5 min or less per sample and the MS/MS spectra files are automatically processed using the SEQUEST database searching algorithm. AP-MALDI-ITMS was compared with the widely used methods of microLC/MS/MS (ion trap) and automated MALDI-TOF peptide mass mapping. Sample throughput is 10-fold greater using AP-MALDI compared with microcapillary liquid chromatography/tandem mass spectrometry (microLC/MS/MS). The protein sequence coverage obtained from AP-MALDI-MS/MS spectra matched by SEQUEST is lower compared with microLC/MS/MS and MALDI-TOF mass mapping. However, by using the AP-MALDI full-scan peptide mass fingerprint spectrum, sequence coverage is increased. AP-MALDI-ITMS was applied for the analysis of Coomassie blue stained gels and was found to be a useful platform for rapid protein identification.
Mehl
John T
JT
Molecular Profiling, Merck Research Laboratories, Rahway, NJ 07065, USA. john.mehl@merck.com
Cummings
John J
JJ
Rohde
Ellen
E
Yates
Nathan N
NN
D000818:Animals; D001322:Autoanalysis; D002374:Catalase / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D011506:Proteins / Q000737:chemistry*; D051381:Rats; D017207:Rats, Sprague-Dawley; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D011506:Proteins; D002374:Catalase