Identification of the -1 translational frameshift sites using a liquid chromatography-tandem mass spectrometric approach.
Translational frameshifting, a ubiquitous mechanism used to produce alternative proteins for different biological purposes, appears in a variety of genes in probably all organisms. In the past, the combinational use of sophisticated expression vectors, specific endopeptidases, and Edman degradation has been the main approach for identification of the translational frameshift sites. Although Edman degradation is highly reliable, it is also time-consuming and costly. In this article, we report a new liquid chromatography-tandem mass spectrometric (LC-MS/MS) approach for identifying the -1 translational frameshift sites. The approach consists of three steps: (i) LC-MS/MS analysis of the protein digests, (ii) primary data analysis using the known mRNA sequence, and (iii) advanced data analysis using a new database containing distinct mRNA sequences with single insertion at particular positions. We first validated our approach by analyzing the previously documented slippery sequence, A4G, from IS3. With this approach, we further determined whether the TTTTTTG (T6G) sequence of IS1372 from Streptomyces lividans had the -1 translational frameshifting potential. The identified amino acid sequence of the transframe peptide indicated that the -1 frameshifting occurred at the T6G motif, as predicted previously. The results on IS3 (A4G) and IS1372 (T6G) suggested that this approach is effective for the translational frameshifting studies.
Tsay
Yeou-Guang
YG
EverNew Biotech Inc., Taipei, Taiwan.
Chen
Chang-Chieh
CC
Hu
Shiau-Ting
ST
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D002853:Chromatography, Liquid; D018965:Frameshifting, Ribosomal; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D010449:Peptide Mapping; D040901:Proteomics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D018629:Spectrometry, Mass, Secondary Ion; D014357:Trypsin
D001426:Bacterial Proteins; C102920:IS1372 protein, Streptomyces lividans; D010446:Peptide Fragments; D012333:RNA, Messenger; D014357:Trypsin