Integrated and Ultrasensitive Gel Protein Identification

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Abstract
An integrated gel protein identification technology is developed and demonstrated for the effective (∼90% recovery), rapid (less than 5 min), and sensitive identification (as low as 1 ng gel protein loading) of gel-resolved proteins using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). This integrated technology involves on-line combination of electronic protein transfer with nanoscale proteolytic digestion in a capillary platform, enabling electrokinetic-based protein extraction and stacking, real-time proteolytic cleavage of extracted proteins, and direct deposition of protein digests onto MALDI targets. By revisiting the yeast two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) in similar isoelectric point and molecular mass ranges as studied by Gygi and co-workers (Gygi, S. P.; Corthals, G. L.; Zhang, Y.; Rochon, Y.; Aebersold, R. Proc. Natl. Acad. Sci. U.S.A.2000, 97, 9390−9395), we are additionally able to identify a large number of low abundance proteins with codon adaptation index (CAI) values of <0.2 and increase the proteome coverage to nearly 50%. The CAI value distribution for identified yeast proteins now more closely approximates that predicted for the entire yeast proteome. We further note that the current single-capillary methodology can be easily expanded to a multiplexed capillary platform as a ultrahigh throughput and greatly effective tool for linking 2-D PAGE with MS, particularly for the analysis of low-abundance proteins.