title: Integrated and ultrasensitive gel protein identification.
abstract: An integrated gel protein identification technology is developed and demonstrated for the effective ( approximately 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.
authors.affiliation: Calibrant Biosystems, Rockville, Maryland 20855, USA.
authors.forename: Jonathan W
authors.initials: JW
authors.lastname: Cooper
authors.forename: Cheng S
authors.initials: CS
authors.lastname: Lee
mesh_terms: D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D004800:Enzymes, Immobilized / Q000737:chemistry; D005782:Gels / Q000737:chemistry; D008567:Membranes, Artificial; D036103:Nanotechnology / Q000379:methods; D011506:Proteins / Q000032:analysis* / Q000737:chemistry*; D012680:Sensitivity and Specificity; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000295:instrumentation / Q000379:methods; D013997:Time Factors; D014357:Trypsin / Q000737:chemistry
chemical_list: D004800:Enzymes, Immobilized; D005782:Gels; D008567:Membranes, Artificial; D011506:Proteins; D014357:Trypsin