Lesson from the Stoichiometry Determination of the Cohesin Complex: A Short Protease Mediated Elution Increases the Recovery from Cross-Linked Antibody-Conjugated Beads Johann Holzmann,*,† Johannes Fuchs,†,‡ Peter Pichler,§ Jan-Michael Peters,† and Karl Mechtler†,‡ Research Institute of Molecular Pathology (IMP), 1030 Vienna, Austria, Institute of Molecular Biotechnology (IMBA), 1030 Vienna, Austria, and CD-Laboratory for Proteome Analysis, 1030 Vienna, Austria Received September 10, 2010 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 analysesssuch as the stoichiometry determination of protein complexesshave 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 periodssas performed in standard on-bead digestionsled 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. Keywords: protease elution • immunoadsorption • stoichiometry • protein complex • absolute quantiﬁcation • EtEP • protein isoforms Introduction Proteins assemble into dynamic macromolecular complexes that regulate fundamental cellular processes such as cell cycle progression and mitosis.1,2 Afﬁnity puriﬁcation coupled to tandem mass spectrometry (AP-MS) has been used extensively to decipher the composition of protein complexes and their networks involved in a variety of biochemical processes in different organisms.2-10 But in contrast to the large number of expression proteomic studies that have generated quantita- tive data, large-scale AP-MS protein interaction studies have led to mostly qualitative data. Only recently, a small number of studies aimed to investigate the dynamics of protein * To whom correspondence should be addressed. Johann Holzmann, Ph.D. Research Institute of Molecular Pathology (IMP), Vienna, Austria Dr. Bohr-Gasse 7 1030 Vienna, Austria. Phone: +43-1-79044-4293. Fax: +43-1- 79044-110. E-mail: johann.holzmann@imp.ac.at. † Research Institute of Molecular Pathology. ‡ Institute of Molecular Biotechnology. § CD-Laboratory for Proteome Analysis. 780 Journal of Proteome Research 2011, 10, 780–789 Published on Web 11/03/2010 complexes within protein interaction networks using afﬁnity puriﬁcation coupled to quantitative MS (AP-QMS) (reviewed in ref 11). One promising application of AP-QMS is the determination of protein complex stoichiometry, which is important informa- tion about the structural organization of a complex.10,12-16 Accurate and precise quantiﬁcation is crucial for stoichiometry determination based on absolute quantiﬁcation of the interact- ing constituents. Stoichiometry determination is particularly susceptible to errors in sample preparation; for example, it has been shown that choice of protein digest conditions can affect the results for the stoichiometry.16 As the hydrolysis conditions have to be optimized for every complex to achieve complete digestion, stoichiometry determination has not been addressed in high-throughput studies so far and has therefore been limited in use to the characterization of individual protein complexes. Immunoadsorption is one of the most widely used methods in biochemistry to purify proteins and their interaction part- 10.1021/pr100927x  2011 American Chemical Society