Experimental determination of translational starts using peptide mass mapping and tandem mass spectrometry within the proteome of Mycobacterium tuberculosis.
Identification of protein translation start sites is largely a bioinformatics exercise, with relatively few confirmed by N-terminal sequencing. Translation start site determination is critical for defining both the protein sequence and the upstream DNA which may contain regulatory motifs. It is demonstrated here that translation start sites can be determined during routine protein identification, using MALDI-MS and MS/MS data to select the correct N-terminal sequence from a list of alternatives generated in silico. Applying the method to 13 proteins from Mycobacterium tuberculosis, 11 predicted translational start sites were confirmed, and two reassigned. The authors suggest that these data (be they confirmation or reassignments) are important for the annotation of both this genome and those of organisms with related genes. It was also shown that N-acetylation, reported to be rare in prokaryotes, was present in three of the 13 proteins (23 %), suggesting that in the mycobacteria this modification may be common, and an important regulator of protein function, although more proteins need to be analysed. This method can be performed with little or no additional experimental work during proteomics investigations.
Rison
Stuart C G
SCG
The Royal Veterinary College, Royal College Street, London NW1 0TU, UK.
Mattow
Jens
J
Max-Planck-Institute for Infection Biology, Core Facility Protein Analysis, Campus Charité Mitte, Schumannstr. 21/22, D-10117 Berlin, Germany.
Jungblut
Peter R
PR
Max-Planck-Institute for Infection Biology, Core Facility Protein Analysis, Campus Charité Mitte, Schumannstr. 21/22, D-10117 Berlin, Germany.
Stoker
Neil G
NG
The Royal Veterinary College, Royal College Street, London NW1 0TU, UK.
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D001483:Base Sequence; D018387:Codon, Initiator; D006801:Humans; D008969:Molecular Sequence Data; D009169:Mycobacterium tuberculosis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010442:Peptide Chain Initiation, Translational / Q000235:genetics*; D010449:Peptide Mapping / Q000379:methods*; D010455:Peptides / Q000737:chemistry; D020543:Proteome; D053719:Tandem Mass Spectrometry / Q000379:methods*
D001426:Bacterial Proteins; D018387:Codon, Initiator; D010455:Peptides; D020543:Proteome