Abstract
Pseudomonas chlororaphis phage 201 phi 2-1 produces a large structurally complex virion, including the products of 89 phage genes. Many of these proteins are modified by proteolysis during virion maturation. To delineate the proteolytic maturation process, 46 slices from an SDS-polyacrylamide gel were subjected to tryptic digestion and then HPLC-electrospray ionization-tandem mass spectrometry analysis. The scale of the experiment allowed high sequence coverage and detection of mass spectra assigned to peptides with one end produced by trypsin and the other end derived from a maturation cleavage (semitryptic peptides). Nineteen cleavage sites were detected in this way. From these sites, a cleavage motif was defined and used to predict the remaining cleavages required to explain the gel mobility of the processed polypeptide species. Profiling the gel with spectrum counts for specific polypeptide regions was found to be helpful in deducing the patterns of proteolysis. A total of 29 cleaved polypeptides derived from 19 gene products were thus detected in the mature 201 phi 2-1 virion. When combined with bioinformatics analyses, these results revealed the presence of head protein-encoding gene modules. Most of the propeptides that were removed from the virion after processing were acidic, whereas the mature domain remaining in the virion was nearly charge-neutral. For four of these processed virion proteins, the portions remaining in the mature virion were mutually homologous. Spectrum counts were found to overestimate the relative quantity of minor polypeptide species in the virion. The resulting sensitivity for minor species made it possible to observe a small amount of general proteolysis that also affected the virions.
Highlights
The well established processing pattern in phage 2012-1 is available to predict the mature polypeptides formed in the other KZrelated phages
We thank the UTHSCSA Bioinformatics Center for assistance with computational aspects of the project
Summary
The phage 2012-1 virion preparations were generated as described [13], including use of step and buoyant density centrifugations in CsCl. The tandem MS results obtained from the digest of each gel slice were searched separately using Mascot, and the data files were either evaluated individually or combined into data sets for processing by Scaffold (“MudPIT”). For more detailed SC gel profiling, a Perl script was written to process the spectrum report written by Scaffold and produce a formatted spectrum count data file featuring the profile of peptides belonging to specific subregions of the gene product. A hidden Markov model (HMM) corresponding to the logo alignment was prepared with a local implementation of the Sequence Analysis and Modeling System [21, 22] using prior information to set the probability of novel residues occurring in the motif as specified in the associated w0.5 model building script. Perl scripts were written to read Scaffold spectrum reports and Sequence Analysis and Modeling System output files and to graph peptide coverage and cleavage motifs within GBrowse as sequence features. Results were generally consistent with our previous study [13] with only one protein previously considered to be abundant, gp146, falling markedly below this threshold in the current study
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