Quantification of Vitamin B₁₂-Related Proteins in Marine Microbial Systems Using Selected Reaction Monitoring Mass Spectrometry

Mass spectrometry-based proteomic approaches to studying microbial systems enable assessment of taxonomically resolved functional capacity. A subset of these proteomic approaches are absolutely quantitative, enabling comparisons of protein expression patterns between different studies and across environments. This chapter outlines a method for applying quantitative assays in marine microbial communities, using proteins involved in vitamin B₁₂ (cobalamin) utilization and production as specific examples. This approach involves identifying important protein targets, determining taxonomic resolution of the required assays, identifying suitable peptides, developing and optimizing liquid chromatography-selected reaction monitoring mass spectrometry assays (LC-SRM-MS), and processing the resulting data. Implementing the method outlined here results in measurements (fmol diagnostic peptide per μg of total bulk protein) that, in this case, define the nutritional status of microbial community members with respect to vitamin B₁₂, and are comparable across and between marine microbial systems.