Proteome of the large Pseudomonas myovirus 201 phi 2-1: delineation of proteolytically processed virion proteins.
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.
Department of Biochemistry, The University of Texas Health Science Center, San Antonio, Texas 78229-3900, USA.
Julie A
JA
Thomas
Susan T
ST
Weintraub
Kevin
K
Hakala
Philip
P
Serwer
Stephen C
SC
Hardies
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001435:Bacteriophages / Q000235:genetics / Q000378:metabolism*; D036022:Capsid Proteins / Q000737:chemistry / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D020543:Proteome / Q000032:analysis*; D011549:Pseudomonas / Q000821:virology*; D015203:Reproducibility of Results; D014357:Trypsin / Q000378:metabolism; D014764:Viral Proteins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D014771:Virion / Q000378:metabolism*
D036022:Capsid Proteins; D010455:Peptides; D020543:Proteome; D014764:Viral Proteins; D014357:Trypsin