Experimental evaluation of protein identification by an LC/MALDI/on-target digestion approach.
Tryptic digestion of proteins continues to be a workhorse of proteomics. Traditional tryptic digestion requires several hours to generate an adequate protein digest. A number of enhanced accelerated digestion protocols have been developed in recent years. Nonetheless, a need still exists for new digestion strategies that meet the demands of proteomics for high-throughput and rapid detection and identification of proteins. We performed an evaluation of direct tryptic digestion of proteins on a MALDI target plate and the potential for integrating RP HPLC separation of protein with on-target tryptic digestion in order to achieve a rapid and effective identification of proteins in complex biological samples. To this end, we used a Tempo HPLC/MALDI target plate deposition hybrid instrument (ABI). The technique was evaluated using a number of soluble and membrane proteins and an MRC5 cell lysate. We demonstrated that direct deposition of proteins on a MALDI target plate after reverse-phase HPLC separation and subsequent tryptic digestion of the proteins on the target followed by MALDI TOF/TOF analysis provided substantial data (intact protein mass, peptide mass and peptide fragment mass) that allowed a rapid and unambiguous identification of proteins. The rapid protein separation and direct deposition of fractions on a MALDI target plate provided by the RP HPLC combined with off-line interfacing with the MALDI MS is a unique platform for rapid protein identification with improved sequence coverage. This simple and robust approach significantly reduces the sample handling and potential loss in large-scale proteomics experiments. This approach allows combination of peptide mass fingerprinting (PMF), MS/MS peptide fragment fingerprinting (PPF) and whole protein MS for both protein identification and structural analysis of proteins.
Getie-Kebtie
Melkamu
M
Tumor Vaccines and Biotechnology Branch, Division of Cellular and Gene Therapies, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD 20892, USA.
Franke
Peter
P
Aksamit
Robert
R
Alterman
Michail A
MA
D002460:Cell Line; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D011506:Proteins / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D014357:Trypsin / Q000737:chemistry
D011506:Proteins; D014357:Trypsin