title: Fast Enzymatic Processing of Proteins for MS Detection with a Flow-through Microreactor.
abstract: The vast majority of mass spectrometry (MS)-based protein analysis methods involve an enzymatic digestion step prior to detection, typically with trypsin. This step is necessary for the generation of small molecular weight peptides, generally with MW < 3,000-4,000 Da, that fall within the effective scan range of mass spectrometry instrumentation. Conventional protocols involve O/N enzymatic digestion at 37 ºC. Recent advances have led to the development of a variety of strategies, typically involving the use of a microreactor with immobilized enzymes or of a range of complementary physical processes that reduce the time necessary for proteolytic digestion to a few minutes (e.g., microwave or high-pressure). In this work, we describe a simple and cost-effective approach that can be implemented in any laboratory for achieving fast enzymatic digestion of a protein. The protein (or protein mixture) is adsorbed on C18-bonded reversed-phase high performance liquid chromatography (HPLC) silica particles preloaded in a capillary column, and trypsin in aqueous buffer is infused over the particles for a short period of time. To enable on-line MS detection, the tryptic peptides are eluted with a solvent system with increased organic content directly in the MS ion source. This approach avoids the use of high-priced immobilized enzyme particles and does not necessitate any aid for completing the process. Protein digestion and complete sample analysis can be accomplished in less than ~3 min and ~30 min, respectively.
authors.affiliation: Biological Sciences, Virginia Tech; malazar@vt.edu.
authors.forename: Iulia M
authors.initials: IM
authors.lastname: Lazar
authors.affiliation: Biological Sciences, Virginia Tech.
authors.forename: Jingren
authors.initials: J
authors.lastname: Deng
authors.affiliation: Biological Sciences, Virginia Tech.
authors.forename: Nicole
authors.initials: N
authors.lastname: Smith
mesh_terms: D004800:Enzymes, Immobilized / Q000737:chemistry*; D008970:Molecular Weight; D010455:Peptides / Q000032:analysis*; D011506:Proteins / Q000737:chemistry; D012822:Silicon Dioxide / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014357:Trypsin / Q000737:chemistry
chemical_list: D004800:Enzymes, Immobilized; D010455:Peptides; D011506:Proteins; D012822:Silicon Dioxide; D014357:Trypsin