Quantitative Tagless Copurification: A Method to Validate and Identify Protein-Protein Interactions.
Identifying protein-protein interactions (PPIs) at an acceptable false discovery rate (FDR) is challenging. Previously we identified several hundred PPIs from affinity purification - mass spectrometry (AP-MS) data for the bacteria Escherichia coli and Desulfovibrio vulgaris These two interactomes have lower FDRs than any of the nine interactomes proposed previously for bacteria and are more enriched in PPIs validated by other data than the nine earlier interactomes. To more thoroughly determine the accuracy of ours or other interactomes and to discover further PPIs de novo, here we present a quantitative tagless method that employs iTRAQ MS to measure the copurification of endogenous proteins through orthogonal chromatography steps. 5273 fractions from a four-step fractionation of a D. vulgaris protein extract were assayed, resulting in the detection of 1242 proteins. Protein partners from our D. vulgaris and E. coli AP-MS interactomes copurify as frequently as pairs belonging to three benchmark data sets of well-characterized PPIs. In contrast, the protein pairs from the nine other bacterial interactomes copurify two- to 20-fold less often. We also identify 200 high confidence D. vulgaris PPIs based on tagless copurification and colocalization in the genome. These PPIs are as strongly validated by other data as our AP-MS interactomes and overlap with our AP-MS interactome for D.vulgaris within 3% of expectation, once FDRs and false negative rates are taken into account. Finally, we reanalyzed data from two quantitative tagless screens of human cell extracts. We estimate that the novel PPIs reported in these studies have an FDR of at least 85% and find that less than 7% of the novel PPIs identified in each screen overlap. Our results establish that a quantitative tagless method can be used to validate and identify PPIs, but that such data must be analyzed carefully to minimize the FDR.
From the ‡Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720;
Maxim
M
Shatsky
§Genomics Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720;
Ming
M
Dong
¶OB/GYN Department, University of California San Francisco-Sandler-Moore Mass Spectrometry Core Facility, University of California, San Francisco, California 94143;
Haichuan
H
Liu
‖Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720;
Lee Lisheng
LL
Yang
§Genomics Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720;
Megan
M
Choi
**Earth Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720;
Mary E
ME
Singer
**Earth Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720;
Jil T
JT
Geller
¶OB/GYN Department, University of California San Francisco-Sandler-Moore Mass Spectrometry Core Facility, University of California, San Francisco, California 94143;
Susan J
SJ
Fisher
¶OB/GYN Department, University of California San Francisco-Sandler-Moore Mass Spectrometry Core Facility, University of California, San Francisco, California 94143;
Steven C
SC
Hall
‡‡Department of Civil and Environmental Engineering, University of Tennessee, Knoxville, Tennessee 37996; §§Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831;
Terry C
TC
Hazen
From the ‡Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720; ¶¶Department of Plant and Microbial Biology, University of California, Berkeley, California 94720;
Steven E
SE
Brenner
‖‖Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720.
Gareth
G
Butland
‖Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720;
Jian
J
Jin
¶OB/GYN Department, University of California San Francisco-Sandler-Moore Mass Spectrometry Core Facility, University of California, San Francisco, California 94143;
H Ewa
HE
Witkowska
From the ‡Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720; mdbiggin@lbl.gov jmchandonia@lbl.gov.
John-Marc
JM
Chandonia
§Genomics Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720; mdbiggin@lbl.gov jmchandonia@lbl.gov.
Mark D
MD
Biggin
D001426:Bacterial Proteins / Q000378:metabolism*; D002846:Chromatography, Affinity / Q000379:methods; D016969:Desulfovibrio vulgaris / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D013058:Mass Spectrometry / Q000379:methods; D025941:Protein Interaction Mapping / Q000379:methods; D060066:Protein Interaction Maps; D040901:Proteomics / Q000379:methods*
D001426:Bacterial Proteins