Multiplexed proteomic reactor for the processing of proteomic samples.
We report the development of a 96-well plate proteomic reactor for gel-free processing of minute amounts of complex proteomic samples. The device performs multiplexed trapping, enrichment, and biochemical processing of proteins, resulting in concentrated peptide solutions ready for mass spectrometric analysis. Individual wells on the reactor can process up to 2 microg of protein. We also report the coupling of the plate proteomic reactor with protein fractionation using size-exclusion chromatography for large-scale identification of proteins. To illustrate the potential of this approach, we separated 400 microg of MCF7 cell lysate using size-exclusion chromatography and processed 35 protein fractions on the reactor plate. Using stringent criteria when searching the data, a total of 875 unique proteins were identified. More relaxed searching conditions associated with a 1% false positive rate led to the identification of 2683 unique proteins, meaning that one protein was identified per 3-10 ng of total protein lysate loaded on the reactor plate.
Hou
Weimin
W
The Ottawa Institute of Systems Biology, University of Ottawa, 451 Smyth Road, Ottawa, Ontario, K1H 8M5 Canada.
Ethier
Martin
M
Smith
Jeffrey C
JC
Sheng
Yinglun
Y
Figeys
Daniel
D
D014408:Biomarkers, Tumor / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D019149:Bioreactors; D001943:Breast Neoplasms / Q000473:pathology; D045744:Cell Line, Tumor; D002850:Chromatography, Gel / Q000379:methods; D006801:Humans; D011506:Proteins / Q000032:analysis* / Q000737:chemistry; D040901:Proteomics / Q000295:instrumentation / Q000379:methods*
D014408:Biomarkers, Tumor; D011506:Proteins