Published in the Journal of Dairy Science in April 2014:
Quantitative profiling of bacteriocins present in dairy-free probiotic preparations of Lactobacillus acidophilus by nanoliquid chromatography-tandem mass spectrometry
Renu Nandakumar, PhD and Kesh Talapatra PhD, PhD, MBA
Bacteriocins are a heterogeneous group of ribosomally synthesized peptides or proteins with antimicrobialactivity, produced predominantly by lactic acid bacteria, with potential applications as biopreservativesand probiotics. We describe here a novel strategy based on a bottom-up, shotgun proteomic approach usingnanoliquid chromatography-tandem mass spectrometry (nanoLC-MS/MS) with multiple fragmentationtechniques for the quantitative profiling of bacteriocins present in the probiotic preparations of Lactobacillusacidophilus. A direct LC-MS/MS analysis with alternate collision-induced dissociation, high-energy collisiondissociation, and electron-transfer dissociation fragmentation following a filter-assisted size-exclusionsample prefractionation has resulted in the identification of peptides belonging to 37 bacteriocins or relatedproteins. Peptides from lactacin F, helveticin J, lysin, avicin A, acidocin M, curvaticin FS47, and carocin Dwere predominant. The process of freeze drying under vacuum was observed to affect both the diversity andabundance of bacteriocins. Data acquisition using alternating complementary peptide fragmentationmodes, especially electron-transfer dissociation, has significantly enhanced the peptide sequence coverageand number of bacteriocin peptides identified. Multienzyme proteolytic digestion was observed to increasethe sample complexity and dynamic range, lowering the chances of detection of low-abundant bacteriocin peptides by LC-MS/MS. An analytical platform integrating size exclusion prefractionation, nanoLC-MS/MSanalysis with multiple fragmentation techniques, and data-dependent decision tree-driven bioinformatic dataanalysis is novel in bacteriocin research and suitable for the comprehensive bioanalysis of diverse, low-abundant bacteriocins in complex samples.