Automated protein identification by the combination of MALDI MS and MS/MS spectra from different instruments.
The identification of proteins separated on two-dimensional gels is most commonly performed by trypsin digestion and subsequent matrix-assisted laser desorption ionization (MALDI) with time-of-flight (TOF). Recently, atmospheric pressure (AP) MALDI coupled to an ion trap (IT) has emerged as a convenient method to obtain tandem mass spectra (MS/MS) from samples on MALDI target plates. In the present work, we investigated the feasibility of using the two methodologies in line as a standard method for protein identification. In this setup, the high mass accuracy MALDI-TOF spectra are used to calibrate the peptide precursor masses in the lower mass accuracy AP-MALDI-IT MS/MS spectra. Several software tools were developed to automate the analysis process. Two sets of MALDI samples, consisting of 142 and 421 gel spots, respectively, were analyzed in a highly automated manner. In the first set, the protein identification rate increased from 61% for MALDI-TOF only to 85% for MALDI-TOF combined with AP-MALDI-IT. In the second data set the increase in protein identification rate was from 44% to 58%. AP-MALDI-IT MS/MS spectra were in general less effective than the MALDI-TOF spectra for protein identification, but the combination of the two methods clearly enhanced the confidence in protein identification.
Levander
Fredrik
F
Department of Protein Technology, Electrical Measurements, Lund University, Lund, Sweden.
James
Peter
P
D001331:Automation; D001426:Bacterial Proteins / Q000032:analysis; D005656:Fungal Proteins / Q000032:analysis; D013058:Mass Spectrometry / Q000295:instrumentation / Q000379:methods; D011506:Proteins / Q000032:analysis*; D040901:Proteomics / Q000295:instrumentation* / Q000379:methods; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000295:instrumentation / Q000379:methods*
D001426:Bacterial Proteins; D005656:Fungal Proteins; D011506:Proteins