Research Profile: A new approach to shotgun proteomics

Feb 1, 2004 - Although they concede that tandem MS is the gold standard for identifying pep- tides, scientists at the Research Triangle. Institute (RT...
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A new approach to shotgun proteomics Although they concede that tandem MS is the gold standard for identifying peptides, scientists at the Research Triangle Institute (RTI) are challenging the dogma that it is the only way to identify peptides and proteins in shotgun proteomics experiments. In the January 15 issue of Analytical Chemistry (pp 267– 275), Jim Stephenson, Jr., and Benjamin Cargile of RTI’s Mass Spectrometry Research Group laid the theoretical groundwork for a potentially faster way to identify peptides in complex samples that combines separation by isoelectric focusing (IEF) and determination of accurate mass by MS. Most shotgun proteomics experiments involve the trypsin digestion of an organism’s proteome. The peptides generated, including peptides that are unique to specific proteins, are generally separated by multidimensional reversedphase LC. Each LC peak is analyzed by MS, and each of the resulting peaks is the starting material for a second MS analysis to identify the amino acid sequence of the peptide. Stephenson and others argue, however, that so much machine time is required in the MS/MS approach that it is highly improbable in realistic experimental scenarios. Stephenson and Cargile propose an alternative way to identify peptides that combines an “accurate mass tag” strategy with isoelectric point (pI) data. In this approach, a peptide’s mass is measured to a high degree of accuracy and compared to the theoretically “unique” tryptic proteome of an organism. Although accurate mass tags are extremely powerful, compounds other than unique tryptic peptides could have an isotopic mass identical to that of a peptide, which would confound the analysis, says Stephenson. In addition, two or more peptides with different amino acid sequences could have identical masses. However, two or more peptides with identical elemental compositions would most likely have different isoelectric 50 A

points, says Stephenson. He says that researchers can use the isoelectric point of the peptide in question rather than rely on a second stage of MS to identify the amino acid sequence. Stephenson and Cargile assessed the IEF/MS approach in small-scale laboratory evaluations and then conducted computer modeling experiments with the theoJim Stephenson searches for a faster way to identify peptides. retical proteomes of missed [trypsin] cleavages, nonspecific E. coli and the rat Rattus norvegicus. cleavages, nonpeptide material, and postThey originally tested their theory with translational modifications that I see in slab gels (10  7 cm). Current narrowcomplex mixture digests in my lab, I range immobilized pH gradient strips doubt this technique will have much can be used to discriminate by as little chance of working on anything but very as 0.001 pI unit, which improves the simple organisms with small genomes.” accuracy of peptide identification. Stephenson and Cargile are currently Initially, Stephenson feared that pepoptimizing their IEF and accurate mass tide bands would rapidly diffuse in IEF experiments to determine how closely strips because of their low molecular laboratory results duplicate their predicweights, but laboratory experiments retions. “There is a big difference between vealed that peptides fractionate well on doing the calculations and looking at the the strips in 8 M urea. The researchers real data, because you never know what did not use ionic detergents or stains in you will find there,” Stephenson says. the IEF, thereby avoiding two common Mike Kinter, associate professor of cell sources of nonpeptide interference durbiology at the Cleveland Clinic and coauing MS analysis of peptides. thor with Sherman of Protein Sequencing “The idea has merit and certainly and Identification Using Tandem Mass should be explored. It would indeed be useful to eliminate the MS/MS time from Spectrometry, believes that tandem MS is the only way to obtain unequivocal most of our runs,” says Nicholas E. Sheramino acid sequences of peptides. “Pepman, a professor at the University of Virtide amino acid sequence is the most ginia and director of the W. M. Keck Biofundamental type of data that you can medical Mass Spectrometry Laboratory. get,” says Kinter. “Even in the world of However, algorithms are needed that can proteomics, we are seeing a swing away successfully predict the isoelectric points of the theoretically “unique” tryptic pro- from the mass-mapping experiment, to some degree, back to the tandem MS exteome of an organism. Sherman is not periment in part because the instruments convinced that any algorithm can successare continuing to get better, and scienfully predict those isoelectric points. “With any great-sounding technique, tists want to measure that fundamentally important parameter—peptide amino I say show me how it can be used to acid sequence.” a solve problems in real samples,” says —Rex Graham Sherman. “Based on the amount of

A N A LY T I C A L C H E M I S T R Y / F E B R U A R Y 1 , 2 0 0 4

JIMMY W. CRAWFORD, RTI

RESEARCH PROFILE