Integrated and ultrasensitive gel protein identification

被引:13
作者
Cooper, JW
Lee, CS [1 ]
机构
[1] Calibrant Biosyst, Rockville, MD 20855 USA
[2] Univ Maryland, Dept Chem & Biochem, College Pk, MD 20742 USA
关键词
D O I
10.1021/ac035318z
中图分类号
O65 [分析化学];
学科分类号
070302 ; 081704 ;
摘要
An integrated gel protein identification technology is developed and demonstrated for the effective (similar to90% 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.
引用
收藏
页码:2196 / 2202
页数:7
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