Identification of bacteria in juice/lettuce using magnetic nanoparticles and selected reaction monitoring mass spectrometry.

Ensuring food safety requires a rapid and reliable method for detecting food-borne pathogens. Mass spectrometry has been demonstrated as a powerful tool to classify pure bacterial species. However, matrix interference from food backgrounds may lead to false results because of the suppression of microbial signals. It is useful to develop a method for bacterial enrichment and marker identification in food samples. Magnetic zirconia nanoparticles were used to concentrate spiked microorganisms from apple juice/lettuce under specific conditions (pH 4.5). Bacterial identification was achieved using nanoLC-MS. Selected reaction monitoring of bacteria-related peptides was applied for the first time to identify bacteria including Staphylococcus aureus and Escherichia coli. This study presents an accurate means for bacterial identification in food matrixes using MS. The analysis time is less than 90 min and the minimum concentration of E. coli detected was 5 × 103 CFU/mL. The interaction between bacteria and the magnetic nanoparticles was electrostatic and nonspecific, in contrast to immunoassays which require specific antibodies. The targeted peptide analysis focuses on the bacterial markers, thus significantly simplifying the analysis and leading to an accurate identification of bacteria.

Functionalized magnetic iron oxide (Fe3O4) nanoparticles for capturing gram-positive and gram-negative bacteria.

The development of nanotechnology in biology and medicine has raised the need for conjugation of nanoparticles (NPs) to biomolecules. In this study, magnetic and functionalized magnetic iron oxide nanoparticles were synthesized and used as affinity probes to capture Gram-positive/negative bacteria. The morphology and properties of the magnetic NPs were examined by transmission electron microscopy, Fourier transform infrared spectroscopy, and zeta potential measurements. Furthermore, this study investigated the interaction between functionalized magnetic nanoparticles and Gram positive/negative bacteria. The positively and negatively charged magnetic nanoparticles include functionalities of Fe3O4, SiO2, TiO2, ZrO2, poly ethyleneimine (PEI) and poly acrylic acid. Their capture efficiencies for bacteria were investigated based on factors such as zeta potential, concentration and pH value. PEI particles carry a positive charge over a range of pH values from 3 to 10, and the particles were found to be an excellent candidate for capturing bacteria over such pH range. Since the binding force is mainly electrostatic, the architecture and orientation of the functional groups on the NP surface are not critical. Finally the captured bacteria were analyzed using matrix-assisted laser desorption/ionization mass spectrometry. The minimum detection limit was 10(4) CFU/mL and the analysis time was reduced to be less than 1 hour. In addition, the detection limit could be reduced to an extremely low concentration of 50 CFU/mL when captured bacteria were cultivated.

Evaluating the potential nonthermal microwave effects of microwave-assisted proteolytic reactions.

Microwave-assisted proteolytic digestion methods have evolved into a highly effective approach and serve as an alternative to conventional overnight digestion. This approach typically exploits the unique microwave properties to facilitate the digestion of proteins into their peptides within minutes. Conventional digestion is carried out at 37°C while microwave-assisted digestion requires much higher and sometimes inconsistent temperatures. Thus, this study aims to investigate whether the faster reaction rate is due to the microwave quantum effect or the thermal effect. Quantitative mass spectrometry was used to conduct kinetic analysis of tryptic digestion for several proteins by microwave and conventional heating. The percentages of digestion products relative to internal standards showed no significant difference between microwave and conventional heating conditions at the same digestion temperature. The optimum temperature for tryptic digestion was determined to be 50°C. Furthermore, this study compares the digestion completeness indicators of several proteins under microwave and conventional heating. Again, the values obtained from microwave and conventional heating were similar given identical temperatures. The overall results prove that a nonthermal effect does not exist in microwave-assisted tryptic digestion. Therefore, conventional heating at high temperatures (50°C) can be also used to accelerate digestion reactions.

Use of polyethylenimine-modified magnetic nanoparticles for highly specific enrichment of phosphopeptides for mass spectrometric analysis.

Phosphopeptides have been isolated and concentrated by use of polyethyleneimine (PEI)-modified magnetic nanoparticles as an extremely specific affinity probe. The particles specifically captured phosphopeptides from a tryptic digest of a protein mixture that contained 0.07% (mole/mole) phosphoproteins, which is the highest specificity obtained to date. The time required for enrichment of the phosphopeptides was 1 min only. PEI-modified magnetic nanoparticles carry positive charges over a wide range of pH-between 3 and 11. This feature means the particles are effectively dispersed in solution during phosphopeptide capture. Mass spectrometric analysis revealed the very high efficiency of enrichment of phosphopeptides that contain both single and multiply-phosphorylated sites. The detection limit in the analysis of phosphopeptides obtained from both bovine α-casein and ß-casein by matrix-assisted laser desorption/ionization mass spectrometry was 5 fmol. This approach was also used to enrich the phosphopeptides in a protein digest obtained from non-fat milk.

Identifying bacterial species using CE-MS and SEQUEST with an empirical scoring function.

CE-MS/MS analysis of proteolytic digests of bacterial cell extracts was combined with SEQUEST searching and a new scoring system to identify bacteria species in bacterial mixtures. Searches of MS/MS spectra against protein databases enabled the identification of bacterial species by the matching of the proteins associated with the corresponding species. An empirical scoring function was obtained by evaluating the SEQUEST search results of 38 samples that contained single bacterial species. The scoring by the empirical function helped move up the positive identification results from their original positions in the ranking based on Xcorr values alone. Therefore, the identification of bacteria in the samples that contained bacterial mixtures was improved. Bacterial species in 20 bacterial mixtures, including one real sample, were correctly identified by database searches and the new scoring function.

Using capillary electrophoresis-selective tandem mass spectrometry to identify pathogens in clinical samples.

Analysis of microbial mixtures in complex systems, such as clinical samples, using mass spectrometry can be challenging because the specimens may contain mixtures of several pathogens or both pathogens and nonpathogens. We have successfully applied capillary electrophoresis-selective MS/MS of unique peptide marker ions to the identification of common pathogens in clinical diagnosis. We searched the CE-MS/MS spectra acquired from the proteolytic digests of pure bacterial cell extracts against protein databases. The identified peptides that matched a protein associated with a particular pathogen were selected as marker ions to identify that bacterium in clinical specimens. Thirty-four clinical specimens, obtained from pus, wound, sputum, and urine samples, were analyzed using both biochemical and selective MS/MS methods. The bacteria in these clinical samples were cultivated directly, without prior isolation of a pure colony, before performing the selective MS/MS analyses. The bacteria analyzed included both Gram-positive and -negative strains. The match with respect to the pathogens identified was good between the biochemical and the selective MS/MS methods; the matching rate was 91%. The rate was as high as 97% when not considering two specimens for which the bacteria were not grown successfully. Two of the specimens that we identified using the biochemical method as containing two bacterial species were confirmed also through selective tandem MS analysis.