Infrared-assisted proteolysis using trypsin-immobilized silica microspheres for peptide mapping.
A novel proteolysis approach was developed by using infrared (IR) radiation and trypsin-immobilized silica microspheres. Protein solutions containing trypsin-immobilized microspheres in sealed transparent Eppendorf tubes were allowed to digest under an IR lamp at 37 degrees C. The feasibility and performance of the present proteolysis approach were demonstrated by the digestion of BSA and cytochrome c (Cyt-c) and the digestion time was significantly reduced to 5 min. The obtained digests were identified by MALDI-TOF-MS with the sequence coverages of 54% (BSA) and 83% (Cyt-c) that were better than those obtained by conventional in-solution tryptic digestion. The suitability of the new digestion approach to complex proteins was demonstrated by digesting human serum. The present proteolysis strategy is simple and efficient and will find a wide range of application in protein identification.
Bao
Huimin
H
School of Pharmacy and Department of Chemistry, Fudan University, Shanghai, China.
Lui
Ting
T
Zhang
Luyan
L
Chen
Gang
G
D045304:Cytochromes c / Q000737:chemistry; D004800:Enzymes, Immobilized / Q000378:metabolism*; D006801:Humans; D007259:Infrared Rays; D008863:Microspheres; D010449:Peptide Mapping / Q000379:methods*; D011506:Proteins / Q000737:chemistry; D012710:Serum Albumin, Bovine / Q000737:chemistry; D012822:Silicon Dioxide / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D017550:Spectroscopy, Fourier Transform Infrared; D014357:Trypsin / Q000378:metabolism*
D004800:Enzymes, Immobilized; D011506:Proteins; D012710:Serum Albumin, Bovine; D012822:Silicon Dioxide; D045304:Cytochromes c; D014357:Trypsin