Efficient in-gel proteolysis accelerated by infrared radiation for protein identification.
In this report, infrared (IR) radiation was employed to enhance the efficiency of in-gel proteolysis for MS-based protein identification. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the target protein bands excised from polyacrylamide gel were cut into small pieces that were further treated in trypsin solution. Subsequently, the wet gel pieces sealed in transparent Eppendorf tubes were exposed to an IR lamp to perform IR-assisted in-gel digestion. To demonstrate the feasibility and performance of the novel digestion approach, it was employed to digest BSA and cytochrome c (Cyt-c) in polyacrylamide gels after SDS-PAGE separations. The results indicated that IR radiation substantially enhanced the efficiency of in-gel proteolysis and the digestion time was significantly reduced to 5 min compared to 16 h for conventional in-gel digestion. The obtained digests were further identified by MALDI-TOF MS with improved sequence coverages. The suitability of IR-assisted in-gel proteolysis to real protein samples was demonstrated by digesting and identifying human serum albumin in gel separated from human serum by SDS-PAGE. The present proteolysis strategy is simple and efficient, offering great promise for the high-throughput protein identification in proteomics research.
School of Pharmacy and Department of Chemistry, Fudan University, Shanghai 200032, China.
Huimin
H
Bao
Ting
T
Liu
Xian
X
Chen
Gang
G
Chen
D000180:Acrylic Resins / Q000737:chemistry; D000818:Animals; D002417:Cattle; D045304:Cytochromes c / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007259:Infrared Rays; D013058:Mass Spectrometry / Q000379:methods; D010455:Peptides / Q000737:chemistry; D011506:Proteins / Q000032:analysis*; D040901:Proteomics / Q000379:methods*; D012709:Serum Albumin / Q000737:chemistry*; D012710:Serum Albumin, Bovine / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D013997:Time Factors; D000099166:Polyacrylamides
D000180:Acrylic Resins; D045304:Cytochromes c; D010455:Peptides; D011506:Proteins; D012709:Serum Albumin; D012710:Serum Albumin, Bovine; D000099166:Polyacrylamides