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Integrated and Ultrasensitive Gel Protein Identification Jonathan W. Cooper†,‡ and Cheng S. Lee*,‡
Calibrant Biosystems, Rockville, Maryland 20855, and Department of Chemistry and Biochemistry, University of Maryland, College Park, Maryland 20742
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) out of 58 proteins predicted to be present in this range of yeast 2-D PAGE were identified in their studies even when loading the gel with far more protein than optimum for separation.9 In addition to these high-abundance proteins, we were able to identify 11 additional proteins, including 8 low-abundance proteins with CAI values of 70% (see Figure 4B and Table 3) was obtained for the identification of RNA polymerase I-specific transcription initiation factor (YMR270C/RRN9). Furthermore, 10 low-abundance proteins, such as YPL053C, YLL006W, YNL330C, YMR 270C, YGL100W, YGL213C, YMR028W, YDR400W, YBR246W, and YDL024C, were predicted to be present in the selected area of yeast 2-D PAGE because their mRNA expressions have been reported.24 Four of these low-abundance proteins, including YLL006W/MMM1, YNL330C/RPD3, YMR270C/RRN9, YGL213C/SKI8, were identified in this study using the integrated gel protein identification technology (see Figure 4A). CONCLUSION The integrated gel protein identification technology, combining electronic protein transfer with in situ membrane-based proteolytic (24) Velculescu, V. E.; Zhang, L.; Zhou, W.; Vogelstein, J.; Basrai, M. A.; Bassett, D. E., Jr.; Hieter, P.; Vogelstein, B.; Kinzler, K. W. Cell 1997, 88, 243-251.
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digestion, enables rapid and sensitive identification of gel-resolved proteins using MALDI-MS. A key feature of this integrated approach is the elimination of sensitivity limitations imposed by multiple sample handling steps in the current processes for linking 2-D PAGE with MS,5-9 such as gel spot excision, on-gel proteolytic digestion, peptide extraction/concentration, and MALDI target deposition. The sensitivity of the technology is demonstrated by the detection of standard proteins from a gel protein loading as low as 1 ng as well as the identification of low-abundance proteins in complex yeast cell lysates. Besides the simplicity in the procedures and instrument requirements, the translation of singlecapillary setup to a multiplexed capillary format similar to that
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employed in a DNA sequencer will provide ultrahigh throughput and comprehensive analysis of gel-resolved proteins and their isoforms. ACKNOWLEDGMENT Support for this work by the National Cancer Institute under Grant R44 CA94400 is gratefully acknowledged.
Received for review November 7, 2003. Accepted February 10, 2004. AC035318Z
