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PMID: 20873769 Published · ppublish English Journal Article Research Support, N.I.H., Extramural Research Support, Non-U.S. Gov't

Online nanoflow RP-RP-MS reveals dynamics of multicomponent Ku complex in response to DNA damage.

Journal of proteome research ·Vol. 9 ·No. 12 ·2010-12-03 ·Pages 6242-55

Zhou F, Cardoza JD, Ficarro SB, Adelmant GO, Lazaro JB, Marto JA

Abstract

Tandem affinity purification (TAP) coupled with mass spectrometry has become the technique of choice for characterization of multicomponent protein complexes. While current TAP protocols routinely provide high yield and specificity for proteins expressed under physiologically relevant conditions, analytical figures of merit required for efficient and in-depth LC-MS analysis remain unresolved. Here we implement a multidimensional chromatography platform, based on two stages of reversed-phase (RP) separation operated at high and low pH, respectively. We compare performance metrics for RP-RP and SCX-RP for the analysis of complex peptide mixtures derived from cell lysate, as well as protein complexes purified via TAP. Our data reveal that RP-RP fractionation outperforms SCX-RP primarily due to increased peak capacity in the first dimension separation. We integrate this system with miniaturized LC assemblies to achieve true online fractionation at low (≤5 nL/min) effluent flow rates. Stable isotope labeling is used to monitor the dynamics of the multicomponent Ku protein complex in response to DNA damage induced by γ radiation.

MeSH Terms
Antigens, Nuclear/metabolism Blotting, Western Chromatography, Liquid/methods Chromosomal Proteins, Non-Histone/analysis,metabolism Cluster Analysis DEAD-box RNA Helicases/analysis,metabolism DNA Damage DNA Repair DNA-Binding Proteins/metabolism Guanine Nucleotide Exchange Factors/analysis,metabolism HeLa Cells Heterogeneous-Nuclear Ribonucleoprotein Group C/analysis,metabolism Humans Kinetics Ku Autoantigen Mass Spectrometry/methods Nanotechnology/methods Protein Binding Proteins/analysis,classification,metabolism Proteomics/methods
Chemicals
Antigens, Nuclear Chromosomal Proteins, Non-Histone DNA-Binding Proteins Guanine Nucleotide Exchange Factors HNRNPC protein, human Heterogeneous-Nuclear Ribonucleoprotein Group C Proteins RCC2 protein, human DDX21 protein, human Xrcc6 protein, human DEAD-box RNA Helicases Ku Autoantigen
Authors & Affiliations
6 authors, click to expand affiliations / ORCID
Zhou Feng
Department of Cancer Biology and Blais Proteomics Center, Dana-Farber Cancer Institute, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusettes, United States.
Cardoza Job D
Ficarro Scott B
Adelmant Guillaume O
Lazaro Jean-Bernard
Marto Jarrod A
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Article Info
Journal
Journal of proteome research
Abbr.
J Proteome Res
ISSN
1535-3907
DOI
10.1021/pr1004696
Published
2010-12-03
Epub
2010-00-27
Pages
6242-55
Language
English
Region
United States
NLM ID
101128775
PMCID
PMC2997187
Subset
IM
Grants
NHGRI NIH HHS · P50HG004233 · United States
NHGRI NIH HHS · P50 HG004233 · United States
NINDS NIH HHS · P01 NS047572 · United States
NINDS NIH HHS · P01NS047572 · United States
NHGRI NIH HHS · P50 HG004233-04 · United States
NINDS NIH HHS · P01 NS047572-06 · United States
External Links
PubMed source DOI: 10.1021/pr1004696 PMC Full Text View journal details
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