title: Capillary isoelectric focusing of probiotic bacteria from cow's milk in tapered fused silica capillary with off-line matrix-assisted laser desorption/ionization time-of-flight mass spectrometry identification.
abstract: In this study, combination of capillary isoelectric focusing (CIEF) in tapered fused silica (FS) capillary with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is presented as an efficient approach for unambiguous identification of probiotic bacteria in real sample. For this purpose, bacteria within genus Lactobacillus were selected as model bioanalytes and cow's milk was selected as a biological sample. CIEF analysis of both the cultivated bacteria and the bacteria in the milk was optimized and isoelectric points characterizing the examined bacteria were subsequently determined independently of the bacterial sample origin. The use of tapered FS capillary significantly enhanced the separation capacity and efficiency of the CIEF analyses performed. In addition, the cell number injected into the tapered FS capillary was quantified and an excellent linearity of the calibration curves was achieved which enabled quantitative analysis of the bacteria by CIEF with UV detection. The minimum detectable number of bacterial cells was 2×10(6) mL(-1). Finally, cow's milk spiked with the selected bacterium was analyzed by CIEF in tapered FS capillary, the focused and detected bacterial cells were collected from the capillary, deposited onto the cultivation medium, and identified using MALDI-TOF MS afterward. Our results have revealed that the proposed procedure can be advantageously used for unambiguous identification of probiotic bacteria in a real sample.
authors.affiliation: Institute of Analytical Chemistry of the ASCR, vvi, Brno, Czech Republic. horka@iach.cz
authors.forename: Marie
authors.initials: M
authors.lastname: Horká
authors.forename: Pavel
authors.initials: P
authors.lastname: Karásek
authors.forename: Jiří
authors.initials: J
authors.lastname: Salplachta
authors.forename: Filip
authors.initials: F
authors.lastname: Růžička
authors.forename: Marie
authors.initials: M
authors.lastname: Vykydalová
authors.forename: Anna
authors.initials: A
authors.lastname: Kubesová
authors.forename: Vladimír
authors.initials: V
authors.lastname: Dráb
authors.forename: Michal
authors.initials: M
authors.lastname: Roth
authors.forename: Karel
authors.initials: K
authors.lastname: Slais
mesh_terms: D000818:Animals; D002138:Calibration; D002417:Cattle; D019075:Electrophoresis, Capillary / Q000379:methods; D005504:Food Analysis / Q000379:methods*; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing / Q000295:instrumentation / Q000379:methods*; D007778:Lactobacillus / Q000302:isolation & purification*; D008892:Milk / Q000382:microbiology*; D061352:Pasteurization; D019936:Probiotics / Q000032:analysis*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
keywords: Capillary isoelectric focusing; Lactic acid bacteria; Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; Milk; Tapered fused silica capillary