Lactobacillus delbrueckii subspecies characterization by MudPIT analysis
We have characterized three strains belonging to L. delbrueckii subspecies (bulgaricus, B; lactis, L; L.delbrueckii 23, M) highlighting their divergent origin, different galactose metabolic capabilities and we have demonstrated potential probiotic features.
Sample Processing Protocol
Total extract of each Lactobacillus samples (4 biological replicates for each condition) was resuspended in Rapigest 0.2% v/v (Waters Corporation, Milford, MA, USA) and after incubation at 100°C for 15min and quantification, each sample was digested first with trypsin (1:50) at 37°C o/n and then a second aliquot of trypsin (1:100) was added to samples. Finally, after acidification with TFA 0.5% (v/v) and precipitation of Rapigest, samples were desalted by PepClean C-18 spin column (Pierce Biotechnology, Rockford, IL, USA). MudPIT analyse were conducted with a linear ion trap mass spectrometer LTQ (Thermo Fisher Scientific, Waltham, MA, USA). The separation of peptides was obtained in the first dimension with eight steps of increasing ammonium chloride concentration, and in the second dimension through an acetonitrile gradient.
Data Processing Protocol
Experimental mass spectra were correlated to tryptic peptide sequences by comparing with theoretical mass spectra, obtained by in silico digestion of a protein database downloaded from the NCBI website (www.ncbi.nlm.nih.gov/Ftp/index.htlm) containing Lactobacillus delbrueckii subsp. bulgaricus and Lactobacillus delbrueckii subsp. lactis sequences. Data processing was performed using the 3.3.1. Bioworks version, based on SEQUEST algorithm (University of Washington, licensed to Thermo Finnigan Corp., San Josè, CA, USA), and the following parameters: Xcorr scores greater than 1.5 for singly charged peptide ions and 2.0 and 2.5 for doubly and triply charged ions, respectively, the peptide probability ≤0.001 and the protein consensus score value ≥10. These filters guaranteed that the resulting proteins have a probability value p≤0.001.
Zanni E, Schifano E, Motta S, Sciubba F, Palleschi C, Mauri P, Perozzi G, Uccelletti D, Devirgiliis C, Miccheli A. Combination of Metabolomic and Proteomic Analysis Revealed Different Features among Lactobacillus delbrueckii Subspecies bulgaricus and lactis Strains While In Vivo Testing in the Model Organism Caenorhabditis elegans Highlighted Probiotic Properties. Front Microbiol. 2017 Jun 28;8:1206. eCollection 2017 PubMed: 28702021