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Liver tissue
Soluble fraction
Membrane fraction In-solution digestion for express analysis
In-gel digestion for in-depth analysis
Nano-LC/MS/MS
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Isoelectric point (pl )
Bioinformatics analysis
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Lots of IDs. Small samples of liver tissue are subjected to in-depth (left) or express (right) proteomics analysis by nano-LC/MS/MS.
ers showed that sub-parts-per-million mass accuracy could be obtained with the LTQ–Orbitrap (Mol. Cell. Proteomics 2005, 4, 2010−2021). A background ion of ambient air generated by ESI is used as a reference compound or a “lock mass” to calibrate m/z values in separate MS runs. Ordinarily, stringent search criteria require two peptides from the same protein to be identified in a single run. However, the calibration allows the transfer of single-peptide protein identifications between separate MS runs, increasing the number of proteins identified per run by ~30%. To determine the false-positive rate, the MS data were searched against a reversed database. The high mass ac-
2918 Journal of Proteome Research • Vol. 6, No. 8, 2007
MATTHIAS MANN
In the drug development pipeline, liver toxicity can be an irreversible clog. Liver toxicological screening is an important step in the process leading to drug approval by the U.S. Food and Drug Administration. However, even drugs that appeared to be nontoxic in animal studies and clinical trials have been withdrawn from the market after causing liver damage in patients. For comprehensive toxicological screening, a high-throughput method that can detect drug-induced changes in liver biopsies is needed. In this issue of JPR (pp 2963−2972), Matthias Mann and co-workers at the Max Planck Institute of Biochemistry (Germany), the Beijing Institute of Genomics, and Proxeon Biosystems A/S (Denmark) describe the development of a rapid proteomics method for the analysis of mouse liver by MS. The researchers used a simplified fractionation procedure with highaccuracy MS to generate the largest mouse liver proteome to date (2210 unique proteins) with fractions of the time and starting material required previously. Mann says, “Normally, MS proteomic studies use elaborate fractionation procedures. This means that no matter how sensitive the mass spectrometer, you need a lot of starting material.” Mann and co-workers homogenized 20 mg of mouse liver tissue (1/10 of the previously required amount) and then fractionated the homogenate into soluble and membrane protein fractions. Each fraction was run in one lane of an SDS-PAGE gel, and each lane of the Coomassie-stained gel was cut into 10 slices. Proteins were digested, extracted, and analyzed by LC/MS/MS. This simple fractionation procedure bypasses traditional labor-intensive fractionation steps as well as the isolation of individual protein spots from 2DE gels. “We think the starting material can be pared down even further, but we’re already getting into the range of tissue that can be obtained from
needle biopsies,” says Mann. Advanced MS techniques were used to obtain broad proteome coverage from the small amount of fractionated material. The peptide fractions were analyzed by LC/MS/MS with a hybrid linear quadrupole ion trap (LTQ)−Orbitrap mass spectrometer. In a previous study, Mann and co-work-
(M log ol. 10 we igh t)
Express proteomics of the mouse liver
curacy of the LTQ–Orbitrap allowed stringent identification criteria that eliminated all false positives. A total of 2210 unique proteins were identified. Importantly, 47% were annotated as membrane proteins, which typically are underrepresented in proteomics data sets. The identified proteins had a broad range of functions, including roles in metabolism, cell organization, and biogenesis. As expected, protein domains found in drug-metabolizing enzymes and uptake/efflux transporters were overrepresented in liver compared with the whole mouse proteome. Comparison of the proteome coverage of the new method with that of previous 2DE-based procedures revealed that the simplified fractionation procedure, combined with LTQ–Orbitrap MS, identified proteins over a broader range of physicochemical properties. With the goal of further improving clinical applicability, the researchers determined the number of proteins that could be identified in a single LC/MS/ MS run without extra fractionation or electrophoresis steps. Surprisingly, in a pilot experiment, 783 unique proteins were identified in a single 2-hour run from 10 mg of mouse liver tissue. Transferring protein identifications among nine single runs increased the number of identified proteins to an average of 1269 per run. For toxicological screening, future researchers could construct a database containing MS data from 50−100 runs, and subsequent runs could be compared against this database after calibration to the lock mass to allow extensive proteome coverage of a sample with a single LC/MS/MS run. According to Mann, “For clinical applications, you want an analysis technique that is simple and fast. Although multiple fractionation steps will theoretically increase the proteome coverage, I think the work is better spent refining MS to make it more sensitive.” Mann predicts that the new method will be useful for high-throughput toxicological screening of small amounts of liver tissue and body fluids. —Laura Cassiday
© 2007 American Chemical Society
