PMC

A) Absolute quantification with isotopes; alternative sources of isotopically labeled peptides are indicated. In all cases, the absolute concentration of the standards must be determined prior to use in mass spectrometry. B) General principle behind the use of isotopic labels in quantitative proteomics. In the precursor (MS1), the mass to charge ratios (m/z) of all co-eluting peptides are monitored, and their intensity recorded. Since isotopically labeled peptides have different m/z, they are distinguished from each other in the MS1 scan: Relative differences in abundance are proportional to their intensities. Identification (here of the light, green, species) is performed in the MS/MS (or MS2) spectrum. C) Quantification based on spectral counting. Different unique peptides from the same protein may be sequenced; spectral counts refers to the sum of all spectra mapped to a given protein.

A) Graphical representation of a direct protein-protein interaction. The two circles (referred to as “nodes”) represent each of the proteins engaged in an interaction, and the line linking them (the “edge”) represents the interaction. B) Interaction network (or “hairball”) representing ~ 500 interactions amongst ~100 proteins (generated by Cytoscape []). C) Protein complexes in a cell. Here, the green protein is found in three different biochemically defined complexes (direct interactions are depicted by contact between the nodes). Not shown here is the relative abundance of these three complexes. D) Unweighted graphical representation (spoke expansion) of the interactions established by the green protein after affinity-purification coupled to mass spectrometry. The organization in different complexes is lost (from this single AP-MS analysis) and direct and indirect interactions are represented in the same manner, as they are indistinguishable in the mass spectrometer. E) Iterative AP-MS helps to resolve complex organization surrounding a central bait. After identification of each of the interaction partners for the green protein, these can be in turned cloned, and analyzed by mass spectrometry. This recapitulates the complex organization shown in (C), though it does not indicates direct or direct interactors. F) Adding binary data to AP-MS data is beneficial to reconstitute the assembly of individual complexes. The dashed lines represent demonstrated (thickest lines) or predicted (thinner lines) direct interactions (the likelihood of a direct interaction is proportional to edge thickness). G) Complexes are not always present in the cell in the same abundances; here, complex 1 is more abundant than complex 2, itself more abundant than complex 3. Most of the green protein will reside in complex 1. H) Quantitative mass spectrometry data provides the relative abundance of each of the interactors for the green protein. This information is shown here as the thickness of the edges.