Extended Data Fig. 7: Inferring protein complexes and subunit stoichiometry from proteome correlation profiling using SEC–MS.

a, Molecular mass (MW) of monomeric proteins (determined from sequence) plotted against the mass determined from the apex of the elution profile for proteins identified by SEC–MS fractions of flower tissue (sFL). Inset shows the molecular mass calibration of the SEC column using a protein calibration standard (mass between 44 and 690 kDa). The distribution of proteins annotated in Araport11 is shown at the top. Many proteins show a much higher apparent molecular mass than would be expected from their sequences (data points above the x = y line). This suggests that these proteins engage in physical protein interactions that are sufficiently stable during SEC separation. b, SEC traces of proteins from five well-characterized protein complexes for flower, leaf and root tissue. Although the resolution of SEC separations is not very high, the complex subunits show very strong co-elution behaviour and the SEC separations of the five complexes are reproducible between tissues. CoA carboxylase n = 4 proteins; CDC48 n = 3 proteins; RubisCO n = 4 proteins; prefoldin n = 6 proteins; SCS n = 3 proteins. c, Intensity-normalized SEC elution profile of proteins for flower tissue. Proteins are ordered based on the SEC fraction in which their intensity peaks and the data are displayed as a heat map (n = 2,485 protein traces). Co-eluting proteins were grouped into ‘trace modules’ (Methods). Proteins in trace modules may represent members of protein complexes and thus serve as candidates for further experimental validation. d, To quantify how well protein complexes can be detected using co-expression analysis from data in the tissue atlas (TA) or by SEC–MS, a summary statistic termed ‘complex index’ was calculated (Methods). The complex index is 1 when all subunits of a complex are identified in the same module and no other proteins are contained in the module. Bar plots show examples for complex indices obtained from the different datasets and are divided into large (>4 subunits) and small (≤4 subunits) protein complexes (according to UniProt). Co-expression alone generates many candidates of interactors, but combining co-expression and SEC–MS analysis is an efficient way to prioritize candidates for follow-up experiments. e, Subunit heterogeneity within the coatomer complex. The coatomer complex consists of seven subunits, five of which (α, β, β′, ε and ζ) can be provided by twelve paralogues of these five genes. Plots show the protein proportions of these paralogues in all 30 tissues (data from tissue atlas). The coatomer complex has a similar composition in most tissues. A notable exception is seed tissues, in which production of subunit ζ-1 dominates over the two other paralogous proteins, suggesting that the coatomer complex in seed tissue also preferentially contains the ζ-1 subunit. Tissues are coloured as in Fig. 1. f, Absolute SEC intensity traces of individual complex subunits for determining subunit stoichiometry. Examples from left to right: the chaperonin complex (flower, 8 proteins, ratio of all subunits: 1:1), the 26S proteasome core and lid (flower, 14+17 proteins, ratio of all subunits: 1:1), the COP9 signalosome (flower, CSN; 8 proteins, ratio of all subunits: 1:1) and the CESA1–CESA3–CESA6 complex (root, 3 proteins, ratio of all subunits: 1:1). CSN3 and CSN5 were detected both as part of the CSN complex and in monomeric form. g, Top, total intensity of protein complex subunits across all tissues for the complexes shown in f (subunit intensities from the tissue atlas). Middle, relative proportion (mean ± s.d.; n = 30 tissues) of subunits across tissues (Methods). For the CESA complex, ratios were calculated for the subunit combinations CESA1–CESA3–CESA6 and CESA4–CESA7–CESA8. The stoichiometries determined from the tissue expression data are generally well-aligned with the expected 1:1 ratio of subunits in these complexes. As noted in f, a substantial amount of CSN5 was detected as a monomer in the SEC analysis, and the tissue expression atlas also shows higher relative expression of this protein compared with all other complex partners. This suggests that the protein is produced in excess over what is required for the COP9 complex (as observed previously¹²³), and may therefore indicate an additional function within the cell.

Source data