Determination of protein stoichiometry within protein complexes using absolute quantification and multiple reaction monitoring.
Many cellular processes are driven by protein complexes. Although the identification of protein components in such complexes has become almost a routine matter, accurate determination of their stoichiometry within a protein complex is still a challenge. We have established a method to determine the stoichiometries of protein complexes using absolute quantification (AQUA) with the help of synthetic standard peptides in combination with multiple reaction monitoring (MRM). Our approach is exemplified by the analysis of the human spliceosomal hPrp19/CDC5L complex, which consists of seven individual proteins and plays a crucial role in the assembly of the fully catalytically active spliceosome during pre-mRNA splicing. We evaluated several conditions for complete hydrolysis of the protein complex and found that the denaturing conditions under which hydrolysis is performed are absolutely crucial for accurately determining protein stoichiometries within this complex. In addition, we tested the suitability of different AQUA peptides and further compared different MS techniques to read out the relative signal intensities that were then used in absolute quantification. Our analyses revealed that dependent on the denaturing conditions different stoichiometries within the complex were obtained. The most consistent results were obtained by enzymatic hydrolysis in the presence of acetonitrile in combination with MRM.
Bioanalytical Mass Spectrometry Group, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Göttingen, Germany.
Carla
C
Schmidt
Christof
C
Lenz
Michael
M
Grote
Reinhard
R
Lührmann
Henning
H
Urlaub
D000595:Amino Acid Sequence; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000302:isolation & purification; D045643:DNA Repair Enzymes / Q000737:chemistry / Q000302:isolation & purification; D006801:Humans; D006868:Hydrolysis; D046912:Multiprotein Complexes / Q000737:chemistry*; D009687:Nuclear Proteins / Q000737:chemistry / Q000302:isolation & purification; D000072260:RNA Splicing Factors; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000302:isolation & purification; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D017381:Spliceosomes / Q000737:chemistry
C508357:CDC5L protein, human; D018797:Cell Cycle Proteins; D046912:Multiprotein Complexes; D009687:Nuclear Proteins; D000072260:RNA Splicing Factors; D016601:RNA-Binding Proteins; D045643:DNA Repair Enzymes; C417041:PRPF19 protein, human