Online nanoflow RP-RP-MS reveals dynamics of multicomponent Ku complex in response to DNA damage.
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.
Zhou
Feng
F
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
JD
Ficarro
Scott B
SB
Adelmant
Guillaume O
GO
Lazaro
Jean-Bernard
JB
Marto
Jarrod A
JA
D034961:Antigens, Nuclear / Q000378:metabolism; D015153:Blotting, Western; D002853:Chromatography, Liquid / Q000379:methods*; D002868:Chromosomal Proteins, Non-Histone / Q000032:analysis / Q000378:metabolism; D016000:Cluster Analysis; D053487:DEAD-box RNA Helicases / Q000032:analysis / Q000378:metabolism; D004249:DNA Damage; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000378:metabolism; D020662:Guanine Nucleotide Exchange Factors / Q000032:analysis / Q000378:metabolism; D006367:HeLa Cells; D034482:Heterogeneous-Nuclear Ribonucleoprotein Group C / Q000032:analysis / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D000072200:Ku Autoantigen; D013058:Mass Spectrometry / Q000379:methods*; D036103:Nanotechnology / Q000379:methods; D011485:Protein Binding; D011506:Proteins / Q000032:analysis / Q000145:classification / Q000378:metabolism; D040901:Proteomics / Q000379:methods
D034961:Antigens, Nuclear; D002868:Chromosomal Proteins, Non-Histone; D004268:DNA-Binding Proteins; D020662:Guanine Nucleotide Exchange Factors; C503635:HNRNPC protein, human; D034482:Heterogeneous-Nuclear Ribonucleoprotein Group C; D011506:Proteins; C477650:RCC2 protein, human; C501726:DDX21 protein, human; C000606509:Xrcc6 protein, human; D053487:DEAD-box RNA Helicases; D000072200:Ku Autoantigen