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

  • Erin M. BertrandEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1849)


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.

Key words

Targeted metaproteomics Mass spectrometry Microbial systems Selected reaction monitoring 


  1. 1.
    Vogel C, Marcotte EM (2012) Insights into the regulation of protein abundance from proteomic and transcriptomic analyses. Nat Rev Genet 13:227–232CrossRefGoogle Scholar
  2. 2.
    Mesuere B, Devreese B, Debyser G et al (2012) Unipept: tryptic peptide-based biodiversity analysis of metaproteome samples. J Proteome Res 11:5773–5780CrossRefGoogle Scholar
  3. 3.
    Saito MA, Bertrand EM, Dutkiewicz S et al (2011) Iron conservation by reduction of metalloenzyme inventories in the marine diazotroph Crocosphaera watsonii. Proc Natl Acad Sci U S A 108:2184–2189CrossRefGoogle Scholar
  4. 4.
    Bertrand EM, Moran DM, McIlvin MR et al (2013) Methionine synthase interreplacement in diatom cultures and communities: implications for the persistence of B12 use by eukaryotic phytoplankton. Limnol Oceanogr 58:1431–1450CrossRefGoogle Scholar
  5. 5.
    Bertrand EM, Saito MA, Jeon YJ et al (2011) Vitamin B12 biosynthesis gene diversity in the Ross Sea: the identification of a new group of putative polar B12 biosynthesizers. Environ Microbiol 13:1285–1298CrossRefGoogle Scholar
  6. 6.
    Picotti P, Bodenmiller B, Aebersold R (2013) Proteomics meets the scientific method. Nat Methods 10:24–27CrossRefGoogle Scholar
  7. 7.
    Lange V, Malmström JA, Didion J et al (2008) Targeted quantitative analysis of Streptococcus pyogenes virulence factors by multiple reaction monitoring. Mol Cell Proteomics 7:1489–1500CrossRefGoogle Scholar
  8. 8.
    Picotti P, Aebersold R (2012) Selected reaction monitoring–based proteomics: workflows, potential, pitfalls and future directions. Nat Methods 9:555–566CrossRefGoogle Scholar
  9. 9.
    Gerber SA, Rush J, Stemman O et al (2003) Absolute quantification of proteins and phosphoproteins from cell lysates by tandem MS. Proc Natl Acad Sci U S A 100:6940–6945CrossRefGoogle Scholar
  10. 10.
    MacLean B, Tomazela DM, Shulman N et al (2010) Skyline: an open source document editor for creating and analyzing targeted proteomics experiments. Bioinformatics 26:966–968CrossRefGoogle Scholar
  11. 11.
    Mesuere B, Debyser G, Aerts M et al (2014) The Unipept metaproteomics analysis pipeline. Proteomics 15:1437–1442CrossRefGoogle Scholar
  12. 12.
    Mesuere B, Willems T, Jeugt F, Van d et al (2016) Unipept web services for metaproteomics analysis. Bioinformatics 32:1746–1748CrossRefGoogle Scholar
  13. 13.
    Saito MA, Dorsk A, Post AF et al (2015) Needles in the blue sea: sub-species specificity in targeted protein biomarker analyses within the vast oceanic microbial metaproteome. Proteomics 15:3521–3531CrossRefGoogle Scholar
  14. 14.
    Eyers CE, Lawless C, Wedge DC et al (2011) CONSeQuence: prediction of reference peptides for absolute quantitative proteomics using consensus machine learning approaches. Mol Cell Proteomics 10:M110.003384CrossRefGoogle Scholar
  15. 15.
    Mallick P, Schirle M, Chen SS et al (2007) Computational prediction of proteotypic peptides for quantitative proteomics. Nat Biotechnol 25:125–131CrossRefGoogle Scholar
  16. 16.
    Keeling PJ, Burki F, Wilcox HM et al (2014) The Marine Microbial Eukaryote transcriptome Sequencing Project (MMETSP): illuminating the functional diversity of eukaryotic life in the oceans through transcriptome sequencing. PLoS Biol 12:e1001889CrossRefGoogle Scholar
  17. 17.
    Bertrand EM, Allen AE, Dupont CL et al (2012) Influence of cobalamin scarcity on diatom molecular physiology and identification of a cobalamin acquisition protein. Proc Natl Acad Sci U S A 109:E1762–E1771CrossRefGoogle Scholar
  18. 18.
    Pruitt KD, Tatusova T, Maglott DR (2005) NCBI Reference Sequence (RefSeq): a curated non-redundant sequence database of genomes, transcripts and proteins. Nucleic Acids Res 33:D501–D504CrossRefGoogle Scholar
  19. 19.
    Bertrand EM, McCrow JP, Moustafa A et al (2015) Phytoplankton-bacterial interactions mediate micronutrient colimitation at the coastal Antarctic sea ice edge. Proc Natl Acad Sci U S A 112:9938–9943CrossRefGoogle Scholar
  20. 20.
    Saito MA, McIlvin MR, Moran DM et al (2014) Multiple nutrient stresses at intersecting Pacific Ocean biomes detected by protein biomarkers. Science 345:1173–1177CrossRefGoogle Scholar
  21. 21.
    Wiśniewski JR, Gaugaz FZ (2015) Fast and sensitive total protein and peptide assays for proteomic analysis. Anal Chem 87:4110–4116CrossRefGoogle Scholar
  22. 22.
    Eze JMO, Dumbroff EB (1982) A comparison of the Bradford and Lowry methods for the analysis of protein in chlorophyllous tissue. Can J Bot 60:1046–1049CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of BiologyDalhousie UniversityHalifaxCanada

Personalised recommendations