title: Understanding the role of proteolytic digestion on discovery and targeted proteomic measurements using liquid chromatography tandem mass spectrometry and design of experiments.
abstract: Workflows in bottom-up proteomics have traditionally implemented the use of proteolysis during sample preparation; enzymatic digestion is most commonly performed using trypsin. This results in the hydrolysis of peptide bonds forming tryptic peptides, which can then be subjected to LC-MS/MS analysis. While the structure, specificity, and kinetics of trypsin are well characterized, a lack of consensus and understanding has remained regarding fundamental parameters critical to obtaining optimal data from a proteomics experiment. These include the type of trypsin used, pH during digestion, incubation temperature as well as enzyme-to-substrate ratio. Through the use of design of experiments (DOE), we optimized these parameters, resulting in deeper proteome coverage and a greater dynamic range of measurement. The knowledge gained from optimization of a discovery-based proteomics experiment was applied to targeted LC-MS/MS experiments using protein cleavage-isotope dilution mass spectrometry for absolute quantification. We demonstrated the importance of these digest parameters with respect to our limit of detection as well as our ability to acquire more accurate quantitative measurements. Additionally, we were able to quantitatively account for peptide decay observed in previous studies, caused by nonspecific activity of trypsin. The tryptic digest optimization described here has eliminated this previously observed peptide decay as well as provided a greater understanding and standardization for a common but critical sample treatment used across the field of proteomics.
authors.affiliation: W.M. Keck Fourier Transform Mass Spectrometry Laboratory, Department of Chemistry, North Carolina State University , Raleigh, North Carolina, 27695, United States.
authors.forename: Philip L
authors.initials: PL
authors.lastname: Loziuk
authors.forename: Jack
authors.initials: J
authors.lastname: Wang
authors.forename: Quanzi
authors.initials: Q
authors.lastname: Li
authors.forename: Ronald R
authors.initials: RR
authors.lastname: Sederoff
authors.forename: Vincent L
authors.initials: VL
authors.lastname: Chiang
authors.forename: David C
authors.initials: DC
authors.lastname: Muddiman
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002853:Chromatography, Liquid / Q000592:standards; D005163:Factor Analysis, Statistical; D006863:Hydrogen-Ion Concentration; D007553:Isotope Labeling; D058977:Molecular Sequence Annotation; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification*; D010940:Plant Proteins / Q000737:chemistry / Q000302:isolation & purification*; D032107:Populus / Q000737:chemistry*; D055550:Protein Stability; D059748:Proteolysis; D040901:Proteomics / Q000379:methods / Q000592:standards*; D012107:Research Design; D013552:Swine; D053719:Tandem Mass Spectrometry / Q000592:standards; D014357:Trypsin / Q000737:chemistry*; D052584:Xylem / Q000737:chemistry*
chemical_list: D010446:Peptide Fragments; D010940:Plant Proteins; D014357:Trypsin