A/Prof Akifumi Hosoda

Event Date: 
Tuesday, June 27, 2017 - 18:00 - 18:15
Mejio University (Nagoya, Japan)

Proteotyping of methanogenic archaea by the ribosomal protein mass spectrum


Microbial identification methods that analyze whole cells or cell lysates by MALDI-TOF MS fingerprinting have been developed to determine microbial species in the clinical research field. However, these whole cell fingerprinting methods, which can determine bacteria at the species level, have difficulty in classifying bacteria at the strain level. Therefore, we developed a bioinformatics-based approach called the S10-GERMS method (S10-spc-alpha operon gene encoding ribosomal protein mass spectrum), to identify bacteria at the strain level by using ribosomal subunit proteins (R-proteins) as biomarkers. In this study, we analyzed whether anaerobic methanogenic archaea {two strains of Methanococcus maripaludis (S2 & C6) and three strains of Methanosarcina barkeri (Wiesmoor, 227 & MS) etc.} could be classified at strain level by the S10-GERMS method.
A molecular weight database for R-proteins of genome sequenced strains was constructed by DNA sequences and calculation of molecular weight. Analyzed methanogens were cultured and resuspended in ethanol (70%). Cells were mixed with 1/5 vol % of matrix solution {Sinapinic acid (15 mg/mL) in 50% acetonitrile with 1% trifluoroacetate}. Then, mass spectra of cells were observed with positive linear mode.
In the case of two strains of M. maripaludis that are closely affiliated with 16S rRNA gene sequences (99% similarity), four-sevenths of R-proteins chosen as biomarkers differed in their mass spectra (both theoretical and observed), indicating that strain S2 and C6 are classified as different strains according to this method. M. barkeri could also be identified using R-protein. These results implied that S10-GERMS method provides an identification platform for environmental samples.