Miniscule Fingerprints for Big Bad Bugs

Clostridium botulinum: The name might not strike fear in every reader’s heart, but maybe it should. This lethal pathogen causes botulism, a form of food poisoning characterized by abdominal cramps, difficulty swallowing and speaking, and paralysis¹, and it has potential for use as a bioterror weapon. Highly lethal microbes like this make blindingly apparent the urgent need for clear-cut, efficient identification methods. Over the past few years, proteomic strategies using mass spectrometry have found new biomarkers with very promising health applications. Current techniques have not come without complications and shortcomings. The ‘shotgun proteomics‘ strategy involves first digesting microbial proteins into peptides, separating them with liquid chromatography, and sequencing with highly sensitive and selective mass spectrometry. Researchers then use database search algorithms such as SEQUEST or Mascot to identify proteins correlated with specific microbes.² Unfortunately, these analysis methods often show false positives: each algorithm uses a different scoring system and selects a different number of peptides. Depending on the threshold for error entered by the user may turn up completely different biomarkers. To deal with this, in 2010, one group of researchers from the UK Health Protection Agency (HPA) developed a ‘pipeline’ protocol to prevent false positives. Their strategy used a BLAST search of the Mass Spec data to narrow down candidate biomarkers against a control and then rigorously matched several algorithms against each other to maximize the chance of correct identification. They successfully used this technique to select eight C. botulinum biomarkers with high confidence.³ Hopefully, those kinds of advances in proteomic analysis techniques and improved algorithms will continue to advance microbe identification. Specificity and quality of mass spectrometry equipment continues to remain a focal point for researchers trying to identify new biomarkers for microbial strains. In the above C. botulinum demonstration, Al-Shahib et al. found an LTQ Orbitrap mass spectrometer in conjunction with a Dionex HPLC system worked well for them. In 2012, University of California researchers used the LTQ Orbitrap mass spectrometer to identify biomarkers for radiation risk in human skin bacteria. The production of porphyrins in these bacteria change in a manner that correlates with human cancer risk, so characterizing these strains of bacteria in radiation-exposed patients may help with pre-symptomatic risk diagnosis.⁴ Whatever the mass spectrometer used, with continuing improvement of proteomic techniques we hope to see many more such discoveries in the future. References

1. A.D.A.M. Medical Encyclopedia. Pubmed Health Botulism Symptoms. National Institute of Health, 15 2012. Web. 16 Dec 2012
2. Nesvizhskii, Alexey I. (2007) Protein Identification by Tandem Mass Spectrometry and Sequence Database Searching.
3. Al-Shahib, Ali, et al. (2010) Coherent pipeline for biomarker discovery using mass spectrometry and bioinformatics. BMC Bioinformatics. 11, (p. 437) Web. 16 Dec. 2012
4. Wang, Y. (2012) The Response of Human Skin Commensal Bacteria as a Reflection of UV Radiation: UV-B Decreases Porphyrin Production. PLoS One. 7(10):e47798

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