title: A one-step preparation method of monolithic enzyme reactor for highly efficient sample preparation coupled to mass spectrometry-based proteomics studies.
abstract: Mass spectrometry (MS) coupled to sample preparation and separation techniques has become a primary tool for proteomics studies. However, due to sample complexity, it is often challenging to achieve fast and efficient sample preparation prior to MS analysis. In recent decades, monolithic materials have been developed not only as chromatographic media, but also as efficient solid supports for immobilizing multiple types of affinity reagents. Herein, the N-acryloxysuccinimide-co-acrylamide-co-N,N'-methylenebisacrylamide (NAS-AAm-Bis) monolith was fabricated within silanized 200 μm i.d. fused-silica capillaries and was used as an immobilized enzyme reactor (IMER). The column was conjugated with trypsin/Lys-C and Lys-N enzymes to allow enzymatic digestions to occur while protein mixture was loaded onto the IMER column followed by MS-based proteomics analysis. Similar MS signal and protein sequence coverage were observed using protein standard bovine serum albumin (BSA) compared to in-solution digestion. Furthermore, mouse serum, yeast, and human cell lysate samples were also subjected to enzymatic digestion by both IMER (in seconds to minutes) and conventional in solution digestion (overnight) for comparison in large-scale proteomics studies. Comparable protein identification results obtained by the two methods highlighted the potential of employing NAS-based IMER column for fast and highly efficient sample preparation for MS analysis in proteomics studies.
authors: School of Pharmacy, University of Wisconsin, 777 Highland Avenue, Madison, WI 53705, United States.
authors: Shan
authors: S
authors: Jiang
authors: School of Pharmacy, University of Wisconsin, 777 Highland Avenue, Madison, WI 53705, United States.
authors: Zichuan
authors: Z
authors: Zhang
authors: School of Pharmacy, University of Wisconsin, 777 Highland Avenue, Madison, WI 53705, United States; Department of Chemistry, University of Wisconsin, 1101 University Avenue, Madison, WI 53706, United States. Electronic address: lingjun.li@wisc.edu.
authors: Lingjun
authors: L
authors: Li
mesh_terms: D000180:Acrylic Resins / Q000737:chemistry; D000818:Animals; D019149:Bioreactors; D002417:Cattle; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004800:Enzymes, Immobilized; D006801:Humans; D051379:Mice; D011506:Proteins / Q000032:analysis*; D040901:Proteomics; D044967:Serum; D012710:Serum Albumin, Bovine / Q000032:analysis; D012822:Silicon Dioxide; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014357:Trypsin / Q000737:chemistry*; D015003:Yeasts / Q000737:chemistry
chemical_list: D000180:Acrylic Resins; D004800:Enzymes, Immobilized; D011506:Proteins; D012710:Serum Albumin, Bovine; D012822:Silicon Dioxide; D014357:Trypsin
keywords: Database search; Digestion; ESI-orbitrap-MS; IMER; MALDI-MS; Monolithic column; Protein identification; Proteomics