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BACKGROUND: Recent studies show that the haptoglobin phenotype in individuals with diabetes mellitus is an important factor for predicting the risk of myocardial infarction, cardiovascular death, and stroke. Current methods for haptoglobin phenotyping include PCR and gel electrophoresis. A need exists for a reliable method for high-throughput clinical applications. Mass spectrometry (MS) can in principle provide fast phenotyping because haptoglobin α 1 and α 2, which define the phenotype, have different molecular masses. Because of the complexity of the serum matrix, an efficient and fast enrichment technique is necessary for an MS-based assay. METHODS: MALDI plates were functionalized by ambient ion landing of electrosprayed antihaptoglobin antibody. The array was deposited on standard indium tin oxide slides. Fast immunoaffinity enrichment was performed in situ on the plate, which was further analyzed by MALDI-TOF MS. The haptoglobin phenotype was determined from the spectra by embedded software script. RESULTS: The MALDI mass spectra showed ion signals of haptoglobin α subunits at m/z 9192 and at m/z 15 945. A cohort of 116 sera was analyzed and the reliability of the method was confirmed by analyzing the identical samples by Western blot. One hundred percent overlap of results between the direct immunoaffinity desorption/ionization MS and Western Blot analysis was found. CONCLUSIONS: MALDI plates modified by antihaptoglobin antibody using ambient ion landing achieve low nonspecific interactions and efficient MALDI ionization and are usable for quick haptoglobin phenotyping.
- MeSH
- chromatografie afinitní MeSH
- fenotyp MeSH
- haptoglobiny analýza imunologie MeSH
- lidé MeSH
- povrchové vlastnosti MeSH
- protilátky imunologie MeSH
- software MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice metody MeSH
- western blotting MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Staphylococcus aureus is a major causative agent of infections associated with hospital environments, where antibiotic-resistant strains have emerged as a significant threat. Phage therapy could offer a safe and effective alternative to antibiotics. Phage preparations should comply with quality and safety requirements; therefore, it is important to develop efficient production control technologies. This study was conducted to develop and evaluate a rapid and reliable method for identifying staphylococcal bacteriophages, based on detecting their specific proteins using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) profiling that is among the suggested methods for meeting the regulations of pharmaceutical authorities. Five different phage purification techniques were tested in combination with two MALDI-TOF MS matrices. Phages, either purified by CsCl density gradient centrifugation or as resuspended phage pellets, yielded mass spectra with the highest information value if ferulic acid was used as the MALDI matrix. Phage tail and capsid proteins yielded the strongest signals whereas the culture conditions had no effect on mass spectral quality. Thirty-seven phages from Myoviridae, Siphoviridae or Podoviridae families were analysed, including 23 siphophages belonging to the International Typing Set for human strains of S. aureus, as well as phages in preparations produced by Microgen, Bohemia Pharmaceuticals and MB Pharma. The data obtained demonstrate that MALDI-TOF MS can be used to effectively distinguish between Staphylococcus-specific bacteriophages.
- MeSH
- biologické přípravky izolace a purifikace MeSH
- chemická frakcionace metody MeSH
- lidé MeSH
- replikace viru MeSH
- shluková analýza MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice * metody MeSH
- stafylokokové bakteriofágy klasifikace metabolismus MeSH
- Staphylococcus aureus virologie MeSH
- virové proteiny analýza chemie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Matrix-assisted laser desorption/ionisation (MALDI) of small molecules is challenging and in most cases impossible due to interferences from matrix ions precluding analysis of molecules <300-500 Da. A common matrix such as ferulic acid belongs to an important class of compounds associated with antioxidant activity. If the shared phenolic structure is related to the propensity as an active MALDI matrix then it follows that direct laser desorption/ionisation should be possible for polyphenols. Indeed matrix-less laser desorption/ionisation mass spectrometry is achieved whereby the analyte functions as a matrix and was used to monitor low molecular weight compounds in wine samples. Sensitivity ranging from 0.12-87 pmol/spot was achieved for eight phenolic acids (4-coumaric, 4-hydroxybenzoic, caffeic, ferulic, gallic, protocatechuic, syringic, vanillic) and 0.02 pmol/spot for trans-resveratrol. Additionally, 4-coumaric, 4-hydroxybenzoic, caffeic, ferulic, gallic, syringic, vanillic acids and trans-resveratrol were identified in wine samples using accurate mass measurements consistent with reported profiles based on liquid chromatography (LC)/MS. Minimal sample pre-treatment make the technique potentially appropriate for fingerprinting, screening and quality control of wine samples. Copyright (c) 2009 John Wiley & Sons, Ltd.
In the case of a release of highly pathogenic bacteria (HPB), there is an urgent need for rapid, accurate, and reliable diagnostics. MALDI-TOF mass spectrometry is a rapid, accurate, and relatively inexpensive technique that is becoming increasingly important in microbiological diagnostics to complement classical microbiology, PCR, and genotyping of HPB. In the present study, the results of a joint exercise with 11 partner institutions from nine European countries are presented. In this exercise, 10 distinct microbial samples, among them five HPB, Bacillus anthracis, Brucella canis, Burkholderia mallei, Burkholderia pseudomallei, and Yersinia pestis, were characterized under blinded conditions. Microbial strains were inactivated by high-dose gamma irradiation before shipment. Preparatory investigations ensured that this type of inactivation induced only subtle spectral changes with negligible influence on the quality of the diagnosis. Furthermore, pilot tests on nonpathogenic strains were systematically conducted to ensure the suitability of sample preparation and to optimize and standardize the workflow for microbial identification. The analysis of the microbial mass spectra was carried out by the individual laboratories on the basis of spectral libraries available on site. All mass spectra were also tested against an in-house HPB library at the Robert Koch Institute (RKI). The averaged identification accuracy was 77% in the first case and improved to >93% when the spectral diagnoses were obtained on the basis of the RKI library. The compilation of complete and comprehensive databases with spectra from a broad strain collection is therefore considered of paramount importance for accurate microbial identification.
- MeSH
- Bacteria chemie klasifikace MeSH
- bakteriologické techniky metody MeSH
- mezinárodní spolupráce MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice metody MeSH
- testování odbornosti laboratoří * MeSH
- Publikační typ
- časopisecké články MeSH
- multicentrická studie MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Evropa MeSH
This study presents a timely, reliable, and sensitive method for identification of pathogenic bacteria in clinical samples based on a combination of capillary electrophoresis with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. In this respect, a part of a single-piece fused silica capillary was etched with supercritical water with the aim of using it for static or dynamic cell-surface adhesion from tens of microliter sample volumes. The conditions for this procedure were optimized. Adhered cells of Staphylococcus aureus (methicillin-susceptible or methicillin-resistant) and of Pseudomonas aeruginosa were desorbed and preconcentrated from the rough part of the capillary surface using transient isotachophoretic stacking from a high conductivity model matrix. The charged cells were swep and separated again in micellar electrokinetic chromatography using a nonionogenic surfactant. Static adhesion of the cells onto the roughened part of the capillary is certainly volumetric limited. Dynamic adhesion allows the concentration of bacteria from 100 μL volumes of physiological saline solution, bovine serum, or human blood with the limits of detection at 1.8 × 102, 1.7 × 103, and 1.0 × 103 cells mL-1, respectively. The limits of detection were the same for all three examined bacterial strains. The recovery of the method was about 83% and it was independent of the sample matrix. A combination of capillary electrophoresis with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry required at least 4 × 103 cells mL-1 to obtain reliable results. The calibration plots were linear (R2 = 0.99) and the relative standard deviations of the peak area were at most 2.2%. The adhered bacteria, either individual or in a mixture, were online analyzed by micellar electrokinetic chromatography and then collected from the capillary and off-line analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry without interfering matrix components.
- MeSH
- Bacteria izolace a purifikace MeSH
- bakteriální adheze MeSH
- bakteriologické techniky MeSH
- elektroforéza kapilární metody MeSH
- koncentrace vodíkových iontů MeSH
- micely MeSH
- oxid křemičitý chemie MeSH
- Pseudomonas aeruginosa izolace a purifikace MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice metody MeSH
- Staphylococcus aureus izolace a purifikace MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Cellulose-based preparative isoelectric focusing was used for preseparation and concentration of uropathogens Staphylococcus aureus, Escherichia coli, Enterococcus faecalis, Staphylococcus epidermidis, Candida albicans, and Candida parapsilosis in a urine sample containing a high concentration of human serum albumin. For the visibility of the colorless microbial zones in the separation medium, the microbial cells were labeled with red nonionogenic tenside (1-[[4-(phenylazo)phenyl]azo]-2-hydroxy-3-naphthoic acid polyethylene glycol ester, PAPAN). A very short incubation time, about 2 min, was sufficient for the adsorption of 0.001% (w/v) PAPAN onto the cell surface at the optimized conditions. As low as 103 cells of E. coli (pI 4.6) resuspended in 100 μL of urine sample and spiked with 0.1 mg mL-1 of human serum albumin (pI 4.8) were successfully preseparated and concentrated using this method. Because the pI values of the labeled microorganisms remained unchanged, the focused red zones of microbial cells were collected from the separation media and further analyzed by either capillary isoelectric focusing or matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The viability of the cells extracted from the collected zones was also confirmed. The proposed method provides reliable, relatively fast, and cost-effective identification of uropathogens in urine specimens with a high level of albumin.
- MeSH
- Bacteria klasifikace izolace a purifikace MeSH
- barvení a značení metody MeSH
- houby klasifikace izolace a purifikace MeSH
- infekce močového ústrojí mikrobiologie MeSH
- isoelektrická fokusace MeSH
- lidé MeSH
- lidský sérový albumin analýza MeSH
- povrchově aktivní látky chemie MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
In our laboratory, we have been studying the reductive processes that occur during matrix-assisted laser desorption/ionization (MALDI) experiments. Recently, we have finished an analysis of the DHB matrix effect on the azo group in cyclic peptides. However, deep understanding of disulfide bond behaviour during a mass spectrometry (MS) experiment is much more important in proteomics as its reduction can cause serious errors in protein spectra interpretation. Therefore, we have focused on intra- and intermolecular disulfide bonds as well as disulfide bonds connecting cysteine and 2-thio-5-nitrobenzoic acid (TNB, Ellman's reagent modification) in model peptides during MALDI MS measurements. While the reduction was not observed for intra- and intermolecular cysteine-cysteine disulfide bonds, the disulfide connection between cysteine and TNB was always affected. It was proved that TNB and Ellman's reagent can act as a matrix itself. The results obtained enabled us to propose a reaction mechanism model which is able to describe the phenomena observed during the desorption/ionization process of disulfide-containing molecules.
- MeSH
- cystein chemie MeSH
- disulfidy chemie MeSH
- ionty chemie MeSH
- kyselina dithionitrobenzoová chemie MeSH
- molekulární sekvence - údaje MeSH
- nitrobenzoany chemie MeSH
- peptidy chemie MeSH
- sekvence aminokyselin MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice metody MeSH
- sulfhydrylové sloučeniny chemie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
