In-line separation by capillary electrophoresis prior to analysis by top-down mass spectrometry enables sensitive characterization of protein complexes.
Intact protein analysis via top-down mass spectrometry (MS) provides a bird's eye view over the protein complexes and complex protein mixtures with the unique capability of characterizing protein variants, splice isoforms, and combinatorial post-translational modifications (PTMs). Here we applied capillary electrophoresis (CE) through a sheathless CE-electrospray ionization interface coupled to an LTQ Velos Orbitrap Elite mass spectrometer to analyze the Dam1 complex from Saccharomyces cerevisiae. We achieved a 100-fold increase in sensitivity compared to a reversed-phase liquid chromatography coupled MS analysis of recombinant Dam1 complex with a total loading of 2.5 ng (12 amol). N-terminal processing forms of individual subunits of the Dam1 complex were observed as well as their phosphorylation stoichiometry upon Mps1p kinase treatment.
Department of Chemical Physiology, The Scripps Research Institute , 10550 North Torrey Pines Road, La Jolla, California 92037, United States.
Xuemei
X
Han
Yueju
Y
Wang
Aaron
A
Aslanian
Bryan
B
Fonslow
Beth
B
Graczyk
Trisha N
TN
Davis
John R
JR
Yates
D001665:Binding Sites; D018797:Cell Cycle Proteins / Q000032:analysis* / Q000378:metabolism; D019075:Electrophoresis, Capillary / Q000379:methods*; D008869:Microtubule-Associated Proteins / Q000032:analysis* / Q000378:metabolism; D008970:Molecular Weight; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D021122:Protein Subunits / Q000032:analysis / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D015203:Reproducibility of Results; D029701:Saccharomyces cerevisiae Proteins / Q000032:analysis* / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
D018797:Cell Cycle Proteins; C116003:DAM1 protein, S cerevisiae; D008869:Microtubule-Associated Proteins; D021122:Protein Subunits; D029701:Saccharomyces cerevisiae Proteins; D017346:Protein Serine-Threonine Kinases; C093416:MPS1 protein, S cerevisiae
capillary electrophoresis; phosphorylation site mapping; phosphorylation stoichiometry; post-translational modification; protein complexes; top-down mass spectrometry