For example, a dimer of ten different subunits, to see if one subunit is present at only one copy per dimer. Can Edman degradation of the whole complex solve this, by the ratio of different amino acids released in the first several steps?
That might work, as long as none of the N-termini are blocked.
Another method is to run the protein on SDS-PAGE, stain with Coomassie Bue, and measure the stain intensity of each band, assuming all the proteins bind the same number of Coomassie Blue molecules per amino acid. You have to correct for the different sizes of the subunits, of course.
You can also run the protein on HPLC or UPLC or capillary gel electrophoresis under denaturing conditions and use the UV absorbance of each protein's peak and calculated extinction coefficient to quantify the amount of each.
Actually for the specific example I had in mind, the subunit in question is blocked (N-acetyl methionine) so I guess Edman is out. It is one of the smaller subunits and elutes early from reverse-phase HPLC, so that may be the way to go but we need to determine extinction coefficients, maybe by quantitative amino acid analysis of pure samples or calculating from amino acid content (Goldfarb /Anthis and Clore method).
This is a vertebrate membrane protein complex, so heterologous expression of individual subunits and reconstitution would be a challenge. The subunit in question is one of the smallest and presence of an extra copy would not be detectable by SEC, but I guess the idea is: see how much of the subunit you can add before you start to get a separate monomer peak, which could work if reconstitution could be achieved. All good ideas in general, but for this particular case I think HPLC quantitation is best. Thanks!
I've done this myself for large complexes using MALS. MALS, either coupled with SEC or in batch mode, can give a direct measurement of the mass of the complex.
A good reference for HPLC quantitation based on extinction coefficients predicted from AA sequence, in acetonitrile/water mixtures:
Bas J. H. Kuipers and Harry Gruppen (2007) Prediction of Molar Extinction Coefficients of Proteins and Peptides Using UV Absorption of the Constituent Amino Acids at 214 nm To Enable Quantitative Reverse Phase High-Performance Liquid Chromatography\u2212Mass Spectrometry Analysis
https://pubs.acs.org/doi/10.1021/jf070337l
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