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Atmospheric pressure matrix-assisted laser desorption/ionization mass spectrometry (AP-MALDI MS) was applied to develop a proteomics-based method to detect and identify Neisseria species. Heat-inactivated clinical isolate cell suspensions of Neisseria gonorrhoeae and strains belonging to five serogroups (A, B, C, W135, and Y) of Neisseria meningitidis were subjected to on-probe protein/peptide extraction and tryptic digestion followed by AP-MALDI tandem MS (MS/MS)-based proteomic analysis. Amino acid sequences derived from three protonated peptides with m/z values of 1743.8, 1894.8, and 1946.8 were identified by AP-MALDI MS/MS and MASCOT proteome database search analysis as belonging to neisserial acyl carrier protein, neisserial-conserved hypothetical protein, and neisserial putative DNA binding protein, respectively. These three peptide masses can thus be potential biomarkers for neisserial species identification by AP-MALDI MS.
Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has been identified as a powerful technique to develop methods for rapid identification of microorganisms.1–3 Atmospheric pressure MALDI (AP-MALDI) is a new ionization technique which offers both decoupled ion production and specificity. The AP-MALDI source easily interfaces with most commercially available ion trap MS and quadrupole/time-of-flight hybrids, providing researchers with analytical versatility.4 In classical AP-MALDI, the ions created by laser irradiation are extracted into an atmospheric pressure inlet of a mass spectrometer with the aid of a static electric field. In this AP-MALDI configuration, positioning of the laser beam directly on axis with an entrance capillary provides the best sensitivity. Recent development of AP-MALDI source with a pulsed dynamic focusing attribute that entrains and focuses the ions into the mass spectrometer has significantly enhanced the sensitivity of AP-MALDI MS. In the present study, an AP-MALDI ion source (www.apmaldi.com; Mass Tech, Inc., Columbia, MD) interfaced with a commercial ion trap mass spectrometer (Thermo Finnigan, San Jose, CA) was used to develop a rapid identification protocol for Neisseria species. Among the Neisseria species, Neisseria gonorrhoeae and Neisseria meningitidis are most commonly associated with human infections. Based on the chemical structures of N. meningitidis capsular polysaccharide coats and the serological properties, 13 serogroups have been identified, of which 5 serogroups (A, B, C, W135, and Y) are mainly responsible for most of the diseases. Despite the advancements in antibiotic treatments, both of these pathogens cause a significant worldwide health problem. Due to the sudden onset and rapid progression of the deadly meningococcal disease, caused mainly by N. meningitidis infections,5 and due to serious clinical consequences in N. gonorrhoeae infections,6 methods for rapid and accurate identification of both these pathogens in clinical specimens are constantly pursued. To screen for or confirm an infection, current gonorrhea diagnostics involve testing a bodily fluid or urine sample for the presence of N. gonorrhoeae. Eight commercially available N.gonorrhoeae diagnostic kits based on either biochemical or immunological tests have recently been evaluated.7 The immunological kits were shown to be more sensitive and specific than the biochemical kits for N.gonorrhoeae identification. It has been verified that some of the biochemical methods misidentified 30–35% of the tested pathogenic strains. New tests were then developed, where cerebrospinal fluid samples were subjected to automated polymerase chain reaction (PCR) analysis for neisserial diagnostics.8–13 PCR-based identification methods introduced for N. meningitidis strains detect the target genes siaD (in serogroups B, C, W135, and Y) and mynA (in serogroup A). However, these tests often have a number of limitations and challenges, such as susceptibility to inhibition by substances present in patient samples, cost effectiveness, laborious and time-consuming bacterial culturing, nucleic acid extraction, and purification steps prior to PCR amplification and detection.14
A targeted proteomics approach to the rapid identification of Bacillus sp. in bacterial cell mixtures by MALDI MS was recently developed.15 AP-MALDI in combination with an ion trap mass spectrometer has been shown to provide high-quality MS/MS data and high sensitivity. AP-MALDI MS is also a relatively simple and rapid analytical method. Reported here is a similar AP-MALDI MS/MS-based proteomics assay method adapted for the rapid identification of pathogenic Neisseria. The aim of this study was to detect heat-inactivated clinical isolate neisserial cell suspensions by AP-MALDI MS and to determine the limits of detection for such cell suspensions.16 Here we report the results of our rapid (< 20 min) and sensitive method for identifying unique Neisseria-specific biomarker peptides to detect the presence of 103–104 cells/sample by on-probe protein/peptide extraction and in situ tryptic digestion followed by AP-MALDI-MS/MS.
N. meningitidis clinical isolates used in this study were F8238 (serogroup A), H44/76 (serogroup B), BB1850 (serogroup B), 2120 (serogroup C), S1975 (serogroup Y), and S4383 (serogroup W135). N. gonorrhoeae strains were GC192, GC MS11, and GC186 which were generously provided by Dr. Margaret Bash from the clinical isolate collection of the Center for Biologics Evaluation and Research (Food and Drug Administration, Bethesda, MD). Freezer stocks of N.gonorrhoeae strains were grown on gonococcal base agar plates (15 g polypeptone peptone, 4 g potassium phosphate dibase, 1 g potassium phosphate monobase, 5 g NaCl, and 15 g Bacto Agar per liter) supplemented with 0.4% glucose and 0.68 mM Fe(NO3)3. N. meningitidis strains were grown on brain heart infusion agar plates (37 g brain heart infusion, 15 g Bacto Agar supplemented with 0.1% fetal bovine serum) at 37°C in a CO2 incubator. Overnight plate cultures were suspended in water. One portion was heat inactivated at 60°C for 45 min and the other portion was used for colony counting after making suitable serial dilutions.
One microliter of heat-inactivated aqueous culture suspensions (103–104 cells/μL) were placed on C18 coated17,18 MALDI target plate at 50°C. Proteins from bacterial cell suspension were selectively extracted (~ 2 min) on the probe with 1 μL of 50% NH4OH (basic protein extraction method). Extracted proteins were digested in situ (~ 1 min) on the probe via addition of 1 μL of trypsin immobilized on beads (Pierce, Rockford, IL) followed immediately by 1 μL of acetonitrile. Sample spots were allowed to dry and subsequently washed with 3 μL water. Generated peptides were subjected to AP-MALDI MS analysis after the addition of 1 μL of matrix (10 mg of α-cyano-4-hydroxycinnamic acid in 1 mL of 70% acetonitrile containing 0.1% trifluoroacetic acid). This whole sample preparation process typically takes less than 5 min. To estimate the minimal cell concentration required for the limit of detection, we performed a series of dilutions of the gonococcal strain GCMS 11 stock suspension followed by AP-MALDI-MS-based detection.
AP-MALDI mass spectra were recorded on an LCQ-Deca XP ion trap mass spectrometer (Thermo Finnigan) equipped with an AP-MALDI ion source featuring pulsed dynamic focusing (MassTech, Columbia, MD) and an all solid state laser (355 nm) operating at a laser firing rate of 10 Hz. Laser shots were rastered manually across the sample. Spectra were accumulated in 1-min intervals and averaged. Recorded tandem MS data were submitted to Mascot (Matrix Science, London, UK)19,20 peptide search against the NCBInr public proteome database with taxonomy restricted to bacteria. Peptide and MS/MS fragment ion mass tolerances were set to ±1.0 and ±1.5 Da, respectively, with an allowance of one missed cleavage by trypsin.
The serial dilution experiments indicated that the limit of detection for Neisseria spp. is approximately 103 cells per sample. AP-MALDI MS spectra were first collected in m/z range of 500–2000 Da, and then all peptide peaks displaying an intensity higher than 30% (MS intensities were normalized to the highest peak at 100%) were selected for further AP-MALDI MS/MS fragmentation. Mass spectra generated from on-probe peptic digests of the N. meningitidis and N. gonorrhoeae strains and respective MS/MS spectra are presented in Figures 1 and and2.2. The observed and calculated masses along with the derived amino acid sequences of the three neisserial biomarker peptide precursor ions, their mapped protein and organism hits, and their Mascot scores and expectation values are presented in Table 1. The precursor peptide with m/z of 1743.8 was assigned to amino acid sequence found in acyl carrier protein from N. meningitidis Z2491 and another acyl carrier protein from N. gonorrhoeae FA1090. Another observed precursor peptide ion with m/z 1946.9 had the peptide sequence QGDTVTLVGFGTFYVGER. This was mapped to two proteins in the database: (i) a putative DNA binding protein from N. meningitidis Z2491 and (ii) a DbhA protein from N. gonorrhoeae FA 1090. The precursor peptide ion peak with m/z 1894.8 was less intense in most of the strains and was assigned to a peptide sequence found in two conserved hypothetical proteins in the protein database: one from N. meingitidis MC58 and the other from N. gonorrhoeae FA1090. These three protonated peptide precursor ions (1743.8, 1894.8, 1946.9) were present in all of the nine strains (three strains of N. gonorrhoeae and six strains of N. meningitidis) tested. Neisserial acyl carrier protein identified in this work is a small protein (78 amino acid residues) with assigned function of acyl carrier activity in fatty acid and phospholipid biosynthesis. Also, the putative DNA binding protein or DbhA protein present in both the species is a small protein with 89 amino acid residues. The third conserved hypothetical protein was slightly bigger with a 154-amino-acid sequence.
A detailed description of the N. meningitidis (for serogroup B strain MC58 and serogroup A strain Z2491) and N.gonorrhoeae (strain FA1090) genomes is available through The Institute of Genome Research–Microbial Database on the World Wide Web (www.tigr.org). Neisserial proteome database among other prokaryotic and eukaryotic protein information is publicly made available by institutions such as NCBInr. The objective of this work is to develop rapid protocols to generate a considerable small number of tryptic peptides from heat-inactivated whole bacterial cells of Neisseriae that are subjected to AP-MALDI tandem mass spectrometry followed by MASCOT database search to confirm the presence of target species using neisserial species specific biomarker peptides. Three peptide masses (1743.8, 1894.8, 1946.9) observed in the AP-MALDI mass spectra (Figures 1 and and2)2) from the basic extraction protocol are found to be specific biomarker peptides for neisserial species. Although the protocol used in this study did not generate any peptide biomarker that could differentiate N. meningitidis from N.gonorrhoeae, additional research by modifying this protocol to exploit the morphological and biochemical differences between these two species may yield other protein/peptide biomarkers towards achieving that goal. One and two dimensional electrophoretic separations, followed by mass spectrometric analysis and analysis by proteomic tools have been applied to many pathogenic subcellular fractions, cell walls, and outer membrane preparations; mainly for exploring vaccine candidates and to develop Web-accessible proteome databases for microbial research in the post-genomics era.21–27 A bioinformatics comparative analysis of the N. meningitidis and N. lactamica outer membrane proteome has been recently published.28 In this report, we present a rapid biomarker identification protocol which avoids timeframes and consumables inherent to subcellular fractionations and gel electrophoretic separations. AP-MALDI interfaced with mass spectrometer enables us to completely automate the analysis including sample processing, mass spectral data collection, and data interpretation. We are currently working on developing protocols to identify peptide biomarkers to distinguish between the two Neisseria sp., and to apply the developed protocols to cerebrospinal fluid and urine clinical samples from patients. Other possible implications of these methods are to explore the novel common surface protein vaccine candidates for both N. meningitidis and N. gonorrhoeae.
The authors wish to thank Dr. Margaret Bash and Craig. A. Hammack of the Center for Biologics Evaluation and Research, Food and Drug Administration, for their help in providing the Neisseria strains for this study.