Lesson from the Stoichiometry Determination of the Cohesin Complex: A Short Protease Mediated Elution Increases the Recovery from Cross-Linked Antibody-Conjugated Beads

Abstract

Affinity purification of proteins using antibodies coupled to beads and subsequent mass spectrometric analysis has become a standard technique for the identification of protein complexes. With the recent transfer of the isotope dilution mass spectrometry principle (IDMS) to the field of proteomics, quantitative analyses—such as the stoichiometry determination of protein complexes—have become achievable. Traditionally proteins were eluted from antibody-conjugated beads using glycine at low pH or using diluted acids such as HCl, TFA, or FA, but elution was often found to be incomplete. Using the cohesin complex and the anaphase promoting complex/cyclosome (APC/C) as examples, we show that a short 15−60 min predigestion with a protease such as LysC (modified on-bead digest termed protease elution) increases the elution efficiency 2- to 3-fold compared to standard acid elution protocols. While longer incubation periods—as performed in standard on-bead digestion—led to partial proteolysis of the cross-linked antibodies, no or only insignificant cleavage was observed after 15−60 min protease mediated elution. Using the protease elution method, we successfully determined the stoichiometry of the cohesin complex by absolute quantification of the four core subunits using LC-SRM analysis and 19 reference peptides generated with the EtEP strategy. Protease elution was 3-fold more efficient compared to HCl elution, but measurements using both elution techniques are in agreement with a 1:1:1:1 stoichiometry. Furthermore, using isoform specific reference peptides, we determined the exact STAG1:STAG2 stoichiometry within the population of cohesin complexes. In summary, we show that the protease elution protocol increases the recovery from affinity beads and is compatible with quantitative measurements such as the stoichiometry determination of protein complexes.

Abstract

A modified on-bead digestion termed “protease elution” increased the recovery of cohesin and APC/C complexes 2- to 3-fold from affinity-beads compared to standard acid elution protocols with only little sample contamination from antibody-peptides in contrast to standard on-bead digestion. Absolute quantification of cohesin revealed a 1:1:1:1 stoichiometry of the four core subunits, and isoform specific quantification revealed a STAG1:STAG2 ratio of 1:12 to 1:15 within the population of cohesin complexes.