Using Capillary Electrophoresis−Selective Tandem Mass Spectrometry To Identify Pathogens in Clinical Samples

Abstract

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.

Capillary electrophoresis‐electrospray‐mass spectrometry in peptide analysis and peptidomics

“…Other groups have directed their attention to the identification of microorganisms by means of peptide analysis of the proteins derived from them [53,73], demonstrating that CE-MS can also be used to identify different microorganisms present in different matrices even in low amount. Besides, Erny and Cifuentes [103] developed a CE-MS mapping approach to obtain 2-D maps of protein digests that allowed easy and simultaneous visual inspection of large dataset as well as to observe immediately relevant differences in closely related samples.…”

“…the electroosmotic flow, these neutral coatings are usually employed in CE-MS using a sheath flow interface, since, as already mentioned above, this interface is less dependant on the EOF. For instance, linear polyacrylamide has been used as coating for peptide analysis by CE-MS [67], as well as poly(vinyl alcohol) (PVA) [37,57,73,85] demonstrating its suitability to prevent peptides adsorption during the analysis of these molecules. In fact, Hu et al [73] showed that it was possible to identify using PVA-coated capillaries and CE-MS/ MS, common pathogen microorganisms in clinical diag- nosis by means of the selective identification of some of their specific peptides selected as biomarkers.…”

“…For instance, linear polyacrylamide has been used as coating for peptide analysis by CE-MS [67], as well as poly(vinyl alcohol) (PVA) [37,57,73,85] demonstrating its suitability to prevent peptides adsorption during the analysis of these molecules. In fact, Hu et al [73] showed that it was possible to identify using PVA-coated capillaries and CE-MS/ MS, common pathogen microorganisms in clinical diag- nosis by means of the selective identification of some of their specific peptides selected as biomarkers. The authors developed a method that included culturing followed by proteins digestion and CE-MS/MS analysis in about 8 h. This procedure allowed the identification of different pathogens in several samples [73].…”

Detection of microbial food contaminants and their products by capillary electromigration techniques

“…Also, the application of this approach to food analysis presents some difficulties due to inherent variability of cellular composition of bacteria under different environmental conditions. To overcome the problem of poor identification induced by limited mass accuracy in the determination of relative molecular mass of proteins when using low resolution MS analyzers, Hu et al [78,79] applied CE-MS/MS to selected peptide ions to obtain partial sequences of protein biomarkers. The coupling CE-MS/MS can provide a highly selective and sensitive analytical method for characterizing the analytes present in complex mixtures.…”

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

“…Proteins that have been extracted from microbial samples can be digested by enzymes; the sequence information of the peptides, obtained by MS/MS, can then be utilized to identify the proteins and deduce the microorganism's source through a proteome database search [23][24][25][26][27][28][29][30]. Recently, the authors' group reported approaches that use the selective proteotypic-peptide analysis (SPA) of protein digests that have been separated through CE to accurately identify bacterial mixtures [31][32][33]. Proteotypic peptides refer to those experimentally observable peptides that identify specific proteins [34].…”