Filip Duša

Timely identification of highly pathogenic bacteria is crucial for efficient mitigation of the connected harmful health effects. Matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) of intact cells enables fast identification of the microorganisms based on their mass spectrometry protein fingerprint profiles. However, the MALDI‐TOF MS examination must be preceded by a time‐demanding cultivation of the native bacteria to isolate representative cell samples to obtain indicative fingerprints. Isoelectric focusing (IEF) is capable of separating bacterial cells according to their isoelectric point while effectively removing other non‐focusing compounds from sample matrix. In this work, we present a divergent‐flow IEF chip (DF‐IEF chip) fractionation as an alternative way for sample clean‐up and concentration of bacterial cells to prepare samples usable for following MALDI‐TOF MS analysis without the need of time‐demanding cultivation. By means of DF‐IEF chip method, we processed four species of highly pathogenic bacteria (Bacillus anthracis, Brucella abortus, Burkholderia mallei, and Yersinia pestis) inactivated with H₂O₂ vapors or by heat treatment at 62.5°C for 24 h. The DF‐IEF chip method continually separated and concentrated the inactivated bacterial cells for subsequent detection using MALDI‐TOF MS. The content of the inactivated bacteria in the DF‐IEF chip fractions was evaluated with the MS analysis, where inactivated Y. pestis was found to be the most efficiently focusing species. Sensitivity analysis showed limits as low as 2 × 10⁵ colony forming units per mL for inactivated B. anthracis.

Introduction: Cyanobacterium Arthrospira platensis (AP) (Nordstedt) Gomont contains high content of phycobiliproteins (PBP), which are an important source for food industry. Methods effectively extracting proteins contained in AP cells are demanded to provide a supply of the material. Water‐based extraction methods are advisable due to the high solubility of the PBP. Objectives: Extraction techniques such as ultrasound assisted extraction (UAE) and pressurized water extraction (PWE) are popular due to their environmental friendliness, better extraction efficiency, and faster extraction process. In this paper, efficiency of the two methods is compared. Materials and Methods: PWE along with UAE is utilized for release of PBP from the AP cells. The extraction parameters including time, temperature, pressure, and ultrasound intensity are tested to obtain the most efficient setup. The methods were evaluated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE), and the replicates of PWE extracts were further analyzed by capillary isoelectric focusing with laser‐induced fluorescence (cIEF‐LIF). Results: The developed PWE method using higher pressure treatment at lower temperature was significantly faster than UAE methods, and the SDS‐PAGE results showed a high content of phycobiliproteins in the extracts. cIEF‐LIF analysis showed that the sequential PWE of individual samples was repeatable, and the mild extraction provided a fluorescent profile similar to the commercially available C‐phycocyanin standard. Conclusion: Pressurized water extraction was shown to be an efficient, rapid, and well‐automated extraction method for AP proteins in general, including bioactive phycobiliproteins. Obtained results encourage the use of PWE in small‐scale analytical applications for primary extraction of proteins.

In this work, we compare labeling by two negatively charged fluorescent labels, 8‐aminopyrene‐1,3,6‐trisulfonic acid (APTS) and 8‐(2‐hydrazino‐2‐oxoethoxy)pyrene‐1,3,6‐trisulfonic acid (Cascade Blue hydrazide [CBH]). Effectiveness of the labeling chemistries were investigated by 4‐hydroxybenzaldehyde and maltoheptaose followed by LC/UV‐MS and CE/LIF analysis, respectively. The reaction yield of APTS labeling was determined to be only ∼10%. This is due to reduction of almost 90% of the analyte by sodium cyanoborohydride to alcohol, which cannot be further labeled via reductive amination. However, the CBH labeling provides ∼90% reaction yield based on the LC/UV‐MS measurements. The significantly higher labeling yield was also confirmed by CE/LIF measurements. Finally, the more effective hydrazone formation technique of CBH was characterized and applied for N‐linked glycan analysis by CE/LIF.