title: Combination of online enzyme digestion with stable isotope labeling for high-throughput quantitative proteome analysis.
abstract: Various enzyme reactors and online enzyme digestion strategies have been developed in recent years. These reactors greatly enhanced the detection sensitivity and proteome coverage in qualitative proteomics. However, these devices have higher rates of miscleavage in protein digestion. Therefore, we investigated the effect of online enzyme digestion on the quantification accuracy of quantitative proteomics using chemical or metabolic isotope labeling approaches. The incomplete digestion would introduce some unexpected variations in comparative quantification when the samples are digested and then chemically isotope labeled in different aliquots. Even when identical protein aliquots are processed on these devices using post-digestion chemical isotope labeling and the CVs of the ratios controlled to less than 50% in replicate analyses, about 10% of the quantified proteins have a ratio greater than two-fold, whereas in theory the ratio is 1:1. Interestingly, the incomplete digestion with enzyme reactor is not a problem when metabolic isotope labeling samples were processed because the proteins are isotopically labeled in vivo prior to their simultaneous digestion within the reactor. Our results also demonstrated that both high quantification accuracy and high proteome coverage can be achieved in comparative proteome quantification using online enzyme digestion even when a limited amount of metabolic isotope labeling samples is used (1683 proteins comparatively quantified from 10(5)  Hela cells).
authors.affiliation: CAS Key Lab of Separation Sciences for Analytical Chemistry, National Chromatographic Research and Analysis Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China.
authors.forename: Fangjun
authors.initials: F
authors.lastname: Wang
authors.forename: Xiaoluan
authors.initials: X
authors.lastname: Wei
authors.forename: Hu
authors.initials: H
authors.lastname: Zhou
authors.forename: Jing
authors.initials: J
authors.lastname: Liu
authors.forename: Daniel
authors.initials: D
authors.lastname: Figeys
authors.forename: Hanfa
authors.initials: H
authors.lastname: Zou
mesh_terms: D000818:Animals; D030562:Databases, Protein; D006367:HeLa Cells; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D007553:Isotope Labeling / Q000379:methods*; D008099:Liver / Q000737:chemistry; D051379:Mice; D010446:Peptide Fragments / Q000032:analysis* / Q000378:metabolism; D020543:Proteome / Q000032:analysis*; D040901:Proteomics / Q000379:methods*; D053719:Tandem Mass Spectrometry; D014357:Trypsin / Q000737:chemistry* / Q000378:metabolism
chemical_list: D010446:Peptide Fragments; D020543:Proteome; D014357:Trypsin