Protein Complexes

Mapping

The most efficient way to profile the content of a given protein sample, in this case a protein complex (usually from a pull-down), is to employ an SDS-PAGE separation in the first dimension and then excise the lane into segments (number of segments is dependent on sample complexity); each segment is then in-gel digested with trypsin and analyzed by nano LC/MS/MS. Each segment is searched against a concatenated forward and reverse database and the search results assembled into a non-redundant list from the entire lane using Scaffold. This is now an established method in the protein mass spectrometry community and is commonly referred to as GeLC/MS. The use of SDS-PAGE gels allows high resolution protein separations and a non-biased sampling of protein classes (hydrophobic, acidic, etc); it is also tolerant to buffer systems typically used in cell biology removing any need for the sample clean up often required prior to mass spectrometry. Through careful optimization of protocols at each stage of the process (SDS-PAGE, digestion, nano LC, mass spectrometry and database searching), NextGen is able to achieve outstanding levels of sensitivity and coverage

Ref: Labib, K. et al. Nature Cell Biology, 10, 395 (2008)

Ref: Labib, K. et al. Nature Cell Biology, 8, 358 (2006)

An example is shown below for an immunoprecipitation of a mouse protein in a cell line. The gel was stained with coomassie and excised in to 40 segments. Data were searched against the IPI mouse database and matched to 456 proteins with at least two unique peptides per protein. The protein identification statistics are shown in the adjacent table, note the absence of any reverse hits.

Quantitation

Whilst qualitatively profiling the protein content of a complex may be sufficient for many studies, it is often required that the relative levels of complex members be monitored across several experiments, for example when looking at separate pull downs of each member of a complex. We are able to efficiently determine relative levels by following the same GeLC/MS workflow for each sample and employing spectral counting to perform quantitation.

Here, the number of peptides matching to a protein and, importantly, the number of times those peptides are observed (spectral count) allows inference of protein abundance between samples. The use of peptide internal standards allows us to measure absolute levels of each protein and therefore infer stoichiometric information about members of a complex.