research articles Holzmann et al. Figure 4. Absolute quantiﬁcation of isolated cohesin subunits by LC-SRM analysis on a 5500 QTRAP. (a) Comparing 15, 30, and 60 min LysC protease elution. Data are mean ( SD of four measured peptides in case of SMC1, SMC3 and RAD21 and of three measured peptides in case of STAG1 and STAG2, respectively. Measurements of cohesin peptides were performed in duplicate. (b) Calculation of complex stoichiometry relative to the bait (RAD21) protein. (c) Comparing 30 min LysC predigestion and HCl elution. Data are mean ( SD of four measured peptides in case of SMC1, SMC3 and RAD21 and of three measured peptides in case of STAG1 and STAG2, respectively. Measurements of cohesin peptides were performed in duplicate. (d) Calculation of complex stoichiometry relative to the bait (RAD21) protein. First, to perform absolute quantiﬁcation according to the IDMS principle we generated 19 internal reference peptides (Table S1, Supporting Information) using our previously de- scribed EtEP method (Figure S6, Supporting Information, and ref 31). The sequences of selected reference peptides including an N-terminal K/R were blasted against the nonredundant (nr) database and all, but two peptides (VEDELK from SCC1 and EDLLR from STAG1), were found to be proteotypic. LC-MS/ MS analysis of afﬁnity puriﬁed cohesin identiﬁed no other peptides from proteins that share these two peptides, thus allowing inclusion of VEDELK and EDLLR for quantiﬁcation. Our EtEP method allows the cost-efﬁcient generation of an equimolar mixture of reference peptides of high accuracy by using an “equalizer peptide” to circumvent amino acid analysis and by using the mTRAQ reagent to introduce the isotopic label (Figure S6, Supporting Information). As peptides are only surrogates of the protein to be quantiﬁed, we selected 4 peptides per cohesin subunit to increase the accuracy of quantiﬁcation. In case of STAG, 3 peptides are speciﬁc for isoform 1, 3 are speciﬁc for isoform 2 and one peptide is speciﬁc for both isoforms. We then set up a scheduled SRM method monitoring 3 transitions each for light (sample) and heavy (reference) peptide (Table S1, Figure S9, Supporting Information). Transitions were optimized for intensity by direct infusion of peptides and ramping of collision energy. To allow accurate quantiﬁcation by calculating light/heavy ratios we determined the linear range of the reference peptides. This is of particular importance for peptides that will not be close to a light/heavy ratio of 1 and are of low abundance. For our set of peptides this scenario was expected for STAG1 speciﬁc peptides as STAG1 was shown to be the less abundant of the two STAG isoforms. For all reference peptides MS detector signal response was linear in the range from 1.56 to 100 fmol and most important, all STAG1 speciﬁc peptides showed a linear response from 390 amol to 100 fmol (Figure S7, Supporting Information). Time course experiments of the protease elution analyzed on the monolithic column and by MS suggested that a 15 min predigestion might be sufﬁcient to release proteins from the afﬁnity beads (Figure 3 and Table S4, Supporting Information). Here, using absolute quantiﬁcation we validated this ﬁnding and determined the complex stoichiometry to test whether different LysC predigestion periods inﬂuence the analysis. 786 Journal of Proteome Research Vol. 10, No. 2, 2011 Figure 4a and b and Figure S8 (Supporting Information) show the result of the SRM analysis where we spiked 25 fmol of each reference peptide to the digested complex eluted by 15 min, 30 and 60 min LysC predigestion, respectively. Apparently LysC predigestion for as short as 15 min is sufﬁcient to release the complex from the antibody, although longer predigestion led to a minor increase in abundance of measured cohesin subunits. Most importantly, measurements and subsequent stoichiometry calculation of all 3 time points are in agreement with a 1:1:1:1 stoichiometry of the four core cohesin subunits. We also performed absolute quantiﬁcation from preparations that used trypsin to elute the protein complex from the antibody beads, followed by a single 16 h trypsin digestion overnight (Figures S12-S13, Supporting Information). However, strong differences in quantitative results from peptides derived from the same protein, as well as a high number of missed cleavage sites identiﬁed in LC-MS/MS experiments indicated in-complete digestion, a situation not compatible with absolute quantiﬁcation. This result again highlights the importance of assessing the completeness of sample digestion and the measurement of at least two to three peptides per protein. Results using trypsin elution followed by overnight trypsin digestion are presented in the Supporting Information. Fur- thermore, because a change in stoichiometry determination after digestion under denaturing conditions was reported,16 we also performed LysC elution and subsequent LysC-trypsin digestion in the presence of 2 M urea (30 min LysC elution). Measurements and calculation of stoichiometry were again in agreement with a 1:1:1:1 stoichiometry (data not shown). To test whether the LysC elution protocol introduces a bias in the quantitative composition of complex subunits compared to a standard acid elution we again determined the stoichi- ometry of cohesin isolated from soluble extract of HeLa cells. As a reference elution protocol we used HCl elution, which was the most efﬁcient mTRAQ compatible elution protocol in the initial labelfree experiments. Quantiﬁcation using the estab- lished SRM method revealed again a 1:1:1:1 stoichiometry of the four cohesin subunits independently of the elution method used (Figure 4c and d). Furthermore, the experiment conﬁrmed the signiﬁcantly better elution efﬁciency of the LysC elution (30 min) compared to acid elution. For quantiﬁcation of STAG1 the higher recovery had practical relevance. We set a minimum transition intensity of 500 cps over background in our SRM