Efficient Chymotryptic Proteolysis Enhanced by Infrared Radiation for Peptide Mapping Sheng Wang, # Ting Liu, # Luyan Zhang, and Gang Chen* School of Pharmacy and Department of Chemistry, Fudan University, Shanghai 200032, China Received June 28, 2008 Abstract: Infrared (IR) radiation was employed to enhance the efficiency of chymotryptic proteolysis for peptide mapping in this work. Protein solutions containing chy- motrypsin in sealed transparent Eppendorf tubes were allowed to digest under an IR lamp at 37 °C. BSA and cytochrome c (Cyt-c) were digested by IR-assisted chy- motryptic proteolysis to demonstrate the feasibility and performance of the novel digestion approach and the digestion time was significantly reduced to 5 min. The obtained digests were further identified by MALDI-TOF MS with the sequence coverages that were comparable to those obtained by using conventional in-solution digestion. The suitability of IR-assisted chymotryptic pro- teolysis to complex proteins was demonstrated by digest- ing human serum. The present proteolysis strategy is simple and efficient, offering great promise for high- throughput protein identification. Keywords: Infrared • Proteolysis • Mass Spectrometry • Protein • Chymotrypsin Introduction Proteomics is the large-scale study of proteins, particularly their structures and functions. As one of the fastest developing areas in biological research, it has drawn more and more research attention. 1,2 One of its most important tasks is to develop efficient and rapid approaches to identifying various proteins. Protein digestion is a key procedure prior to mass spectrometry (MS) identification. It is of high importance to develop novel methods to achieve a highly efficient proteolysis for MS-based peptide mapping because conventional in- solution digestion is time-consuming. 3,4 A variety of approaches have been developed to enhance the efficiency of proteolysis. Trypsin has been immobilized in the channels of microchips by sol-gel encapsulation, 5,6 covalent linking 7 and multilayer assembly 8,9 approaches to fabricate microfluidic enzymatic reactors for protein digestion. Because a high amount of enzyme could be immobilized in the channels, the digestion time was significantly reduced to less than 5 s compared to 12 h for conventional in-solution digestion. Besides microfluidic chips, trypsin and chymotrypsin have also been immobilized in the inner bores of fused silica capillaries to fabricate flow-through bioreactors for proteoly- sis. 10-12 In addition, a variety of trypsin-immobilized magnetic particles have been dispersed in protein solutions to carry out proteolysis with the aid of heat 13 or microwaves 14,15 and the digestion time was less than 5 min. Conventional in-solution digestion has been commonly used in proteolysis. However, the autolysis of protease would gener- ate interfering fragments. A low weight ratio (typically 1:20 to 1:100) between protease and protein was usually employed and resulted in long digestion time (typically 12 h at 37 °C). 4 It is a challenging task to enhance the digestion efficiency of con- ventional in-solution protein digestion. Recently, microwaves were employed to enhance the efficiency of conventional in- solution proteolysis significantly. 16,17 The typical digestion time of microwave-assisted proteolysis was in the range of 5-20 min. Moreover, it was reported that ultrasonic waves could also accelerate the conventional in-solution digestion of proteins and the digestion time was reduced to 1 min. 18 As an important form of electromagnetic wave, infrared (IR) ray has wavelengths between about 750 nm and 1 mm and has found a wide range of applications. 19 In a previous report, we have employed IR radiation as an energy source to promote tryptic proteolysis for the first time and the digestion could complete within 5-10 min. 20 R-Chymotrypsin is another commonly used protease that selectively catalyzes the hydrolysis of peptide bonds on the C-terminal side of tyrosine, phenylalanine, tryptophan, and leucine. The typical time of in-solution chymotryptic proteolysis is in the range of 12-24 h. 21,22 It is of high interest to demonstrate the possibility of employing IR radiation as an energy source to enhance the efficiency of in-solution chy- motryptic proteolysis. In this work, IR radiation was employed to enhance the digestion efficiency of in-solution chymotryptic proteolysis. Protein solutions containing chymotrypsin in sealed transpar- ent Eppendorf tubes were allowed to be exposed to IR radiation to perform high efficient proteolysis. The novel IR-assisted chymotryptic proteolysis approach has been coupled with MALDI-TOF MS for the digestion and peptide mapping of BSA and cytochrome c (Cyt-c). The digestion time was significantly reduced to 5 min compared to 12 h for conventional in-solution digestion. The operation procedure, feasibility, and perfor- mance of IR-assisted chymotryptic proteolysis are reported in the following sections. Experimental Section 1. Reagent and Solutions. Acetonitrile (ACN) and am- monium bicarbonate (NH 4 HCO 3 ) were both purchased from * To whom correspondence should be addressed. E-mail: gangchen@ fudan.edu.cn. Fax: +86-21-64187117. # These author contributed equally to this work. 10.1021/pr800476s CCC: $40.75  2008 American Chemical Society Journal of Proteome Research 2008, 7, 5049–5054 5049 Published on Web 10/18/2008