PMID- 24144163
OWN - NLM
STAT- MEDLINE
DCOM- 20140827
LR  - 20131209
IS  - 1535-3907 (Electronic)
IS  - 1535-3893 (Linking)
VI  - 12
IP  - 12
DP  - 2013 Dec 6
TI  - Understanding the role of proteolytic digestion on discovery and targeted 
      proteomic measurements using liquid chromatography tandem mass spectrometry and 
      design of experiments.
PG  - 5820-9
LID - 10.1021/pr4008442 [doi]
AB  - 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.
FAU - Loziuk, Philip L
AU  - Loziuk PL
AD  - W.M. Keck Fourier Transform Mass Spectrometry Laboratory, Department of 
      Chemistry, North Carolina State University , Raleigh, North Carolina, 27695, 
      United States.
FAU - Wang, Jack
AU  - Wang J
FAU - Li, Quanzi
AU  - Li Q
FAU - Sederoff, Ronald R
AU  - Sederoff RR
FAU - Chiang, Vincent L
AU  - Chiang VL
FAU - Muddiman, David C
AU  - Muddiman DC
LA  - eng
PT  - Journal Article
PT  - Research Support, U.S. Gov't, Non-P.H.S.
DEP - 20131101
PL  - United States
TA  - J Proteome Res
JT  - Journal of proteome research
JID - 101128775
RN  - 0 (Peptide Fragments)
RN  - 0 (Plant Proteins)
RN  - EC 3.4.21.4 (Trypsin)
SB  - IM
MH  - Amino Acid Sequence
MH  - Animals
MH  - Cattle
MH  - Chromatography, Liquid/standards
MH  - Factor Analysis, Statistical
MH  - Hydrogen-Ion Concentration
MH  - Isotope Labeling
MH  - Molecular Sequence Annotation
MH  - Molecular Sequence Data
MH  - Peptide Fragments/chemistry/*isolation & purification
MH  - Plant Proteins/chemistry/*isolation & purification
MH  - Populus/*chemistry
MH  - Protein Stability
MH  - Proteolysis
MH  - Proteomics/methods/*standards
MH  - Research Design
MH  - Swine
MH  - Tandem Mass Spectrometry/standards
MH  - Trypsin/*chemistry
MH  - Xylem/*chemistry
EDAT- 2013/10/23 06:00
MHDA- 2014/08/29 06:00
CRDT- 2013/10/23 06:00
PHST- 2013/10/23 06:00 [entrez]
PHST- 2013/10/23 06:00 [pubmed]
PHST- 2014/08/29 06:00 [medline]
AID - 10.1021/pr4008442 [doi]
PST - ppublish
SO  - J Proteome Res. 2013 Dec 6;12(12):5820-9. doi: 10.1021/pr4008442. Epub 2013 Nov 
      1.