Proteins have been generated in bulk via bacterially derived methods for years, but limitations in protein size and post-transcriptional modifications have demanded the development and use of other expression systems.
Western blot-based methods have the advantage of simultaneously detecting the protein size and the degree of covalent modification of the target protein.
In this work, we introduce a method for the estimation of the absolute quality of individual protein structure models which is independent of protein size and can be used to both assess isolated chains as well as entire oligomeric assemblies.
Lane M, protein size markers.
Since spectrum counts depend on protein size (larger proteins giving rise to more tryptic peptides), we normalized spectrum counts to protein molecular weights, which we have previously found to be an appropriate method of normalization.
As a result of the Mamavirus genome annotation (see supplementary methods and file 1, Supplementary Material online), 1,023 open reading frames (ORFs) were identified as putative protein-coding genes, with the average predicted protein size of 343 amino acids (aa).
Using a combination of prediction tools (supplementary methods and file 1, Supplementary Material online), 930 open reading frames (ORFs) were identified as putative protein-coding genes, with the mean predicted protein size of 290 amino acids (aa).
The three smallest proteins of HIV-1 (TAT, VPR and VPU) yielded no significant results, which indicates that at protein sizes of less than 100 amino acids our method becomes insensitive.
The relative migration of the detected polypeptides correlated well with the predicted sizes of the untagged and tagged versions of the protein [for size correlations see Materials & Methods].
The Number column indicates the number of clusters identified by each method, the Size column indicates the average number of proteins in each cluster; the Discard% indicates the percentage of proteins not assigned to any cluster.
