Intact (whole) cell matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF MS) is an established method for biotyping in clinical microbiology as well as for revealing phenotypic shifts in cultured eukaryotic cells. Intact cell MALDI-TOF MS has recently been introduced as a quality control tool for long-term cultures of pluripotent stem cells. Despite the potential this method holds for revealing minute changes in cells, there is still a need for improving the ionization efficiency or peak reproducibility. Here we report for the first time that supplementation by fine particles of black phosphorus to the standard MALDI matrices, such as sinapinic and α-cyano-4-hydroxycinnamic acids enhance intensities of mass spectra of particular amino acids and peptides, presumably by interactions with aromatic groups within the molecules. In addition, the particles of black phosphorus induce the formation of small and regularly dispersed crystals of sinapinic acid and α-cyano-4-hydroxycinnamic acid with the analyte on a steel MALDI target plate. Patterns of mass spectra recorded from intact cells using black phosphorus-enriched matrix were more reproducible and contained peaks of higher intensities when compared to matrix without black phosphorus supplementation. In summary, enrichment of common organic matrices by black phosphorus can improve discrimination data analysis by enhancing peak intensity and reproducibility of mass spectra acquired from intact cells.
- MeSH
- Amino Acids analysis chemistry MeSH
- Cell Culture Techniques methods MeSH
- Cell Line MeSH
- Phosphorus chemistry MeSH
- Humans MeSH
- Human Embryonic Stem Cells MeSH
- Peptides analysis chemistry MeSH
- Reproducibility of Results MeSH
- Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization methods standards MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
RATIONALE: Explorative statistical analysis of mass spectrometry data is still a time-consuming step. We analyzed critical factors for application of principal component analysis (PCA) in mass spectrometry and focused on two whole spectrum based normalization techniques and their application in the analysis of registered peak data and, in comparison, in full spectrum data analysis. We used this technique to identify different metabolic patterns in the bacterial culture of Cronobacter sakazakii, an important foodborne pathogen. METHODS: Two software utilities, the ms-alone, a python-based utility for mass spectrometry data preprocessing and peak extraction, and the multiMS-toolbox, an R software tool for advanced peak registration and detailed explorative statistical analysis, were implemented. The bacterial culture of Cronobacter sakazakii was cultivated on Enterobacter sakazakii Isolation Agar, Blood Agar Base and Tryptone Soya Agar for 24 h and 48 h and applied by the smear method on an Autoflex speed MALDI-TOF mass spectrometer. RESULTS: For three tested cultivation media only two different metabolic patterns of Cronobacter sakazakii were identified using PCA applied on data normalized by two different normalization techniques. Results from matched peak data and subsequent detailed full spectrum analysis identified only two different metabolic patterns - a cultivation on Enterobacter sakazakii Isolation Agar showed significant differences to the cultivation on the other two tested media. The metabolic patterns for all tested cultivation media also proved the dependence on cultivation time. CONCLUSIONS: Both whole spectrum based normalization techniques together with the full spectrum PCA allow identification of important discriminative factors in experiments with several variable condition factors avoiding any problems with improper identification of peaks or emphasis on bellow threshold peak data. The amounts of processed data remain still manageable. Both implemented software utilities are available free of charge from http://uprt.vscht.cz/ms.
- MeSH
- Principal Component Analysis MeSH
- Bacteriological Techniques MeSH
- Time Factors MeSH
- Cronobacter sakazakii growth & development metabolism MeSH
- Culture Media MeSH
- Software MeSH
- Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization methods standards statistics & numerical data MeSH
- Publication type
- Journal Article MeSH
BACKGROUND AND AIMS: Haemophilus influenzae new strain acquisition has been demonstrated to increase the relative risk of acute exacerbation fourfold in contrast to colonisation or chronic infection by the same strain in chronic obstructive pulmonary disease (COPD). Unfortunately, molecular typing techniques are not suitable for routine use due to cost, labour-intensity and need for special expertise. We tested two techniques potentially useful for routine typing, namely the newly available MALDI-TOF MS and the modified McRAPD compared to MLST as the gold standard. METHODS: In 10 patients (10.8%) suffering from COPD or cystic fibrosis, H. influenzae isolates were recovered repeatedly at different timepoints from the same patient during the study period. This allowed for thirteen pairwise comparisons of typing results in isolates recovered consecutively from the same patient to test the ability of the techniques to uncover new strain acquisition. RESULTS: MLST detected 9 cases of new strain acquisition among the 13 pairwise comparisons. However, MALDI-TOF MS reported all 13 pairs as different and thus new. In contrast, McRAPD was able to differentiate all the new strain acquisitions from pre-existing ones, both by visual inspection of melting profiles and by Relative Significant Difference values. CONCLUSIONS: Unlike MALDI-TOF MS, McRAPD appears to be a suitable candidate for routine discrimination of new strain acquisitions because of its accuracy and, rapid, easy and economic performance.
- MeSH
- Pulmonary Disease, Chronic Obstructive diagnosis MeSH
- Cystic Fibrosis diagnosis MeSH
- Diagnosis, Differential MeSH
- Haemophilus influenzae isolation & purification MeSH
- Haemophilus Infections diagnosis MeSH
- Humans MeSH
- Sensitivity and Specificity MeSH
- Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization methods standards MeSH
- Bacterial Typing Techniques methods standards MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Evaluation Study MeSH
A new method, reversed phase liquid chromatography with off-line surface-assisted laser desorption/ionization mass spectrometry (RPLC-SALDI MS) for the determination of brassicasterol (BR), cholesterol (CH), stigmasterol (ST), campesterol (CA) and β-sitosterol (SI) in oil samples has been developed. The sample preparation consisted of alkaline saponification followed by extraction of the unsaponificable fraction with diethyl ether. The recovery of the sterols ranged from 91 to 95% with RSD less than 4%. Separation of the five major sterols on a C18 column using methanol-water gradient was achieved in about 10min. An on-line UV detector was employed for the initial sterol detection prior to effluent deposition using a laboratory-built spotter with 1:73 splitter. Off-line SALDI MS was then applied for mass determination/identification and quantification of the separated sterols. Ionization of the nonpolar analytes was achieved by silver ion cationization with silver nanoparticles used as the SALDI matrix providing limits of detection 12, 6 and 11fmol for CH, ST and SI, respectively. Because of the incorporated splitter, the effective limits of detection of the RPLC-SALDI MS analysis were 4, 3 and 4pmol (or 0.08, 0.06 and 0.08μg/mL) for CH, ST and SI, respectively. For quantification, 6-ketocholestanol (KE) was used as the internal standard. The method has been applied for the identification and quantification of sterols in olive, linseed and sunflower oil samples. The described off-line coupling of RPLC to SALDI MS represents an alternative to GC-MS for analysis of nonpolar compounds.
- MeSH
- Cholestadienols chemistry isolation & purification MeSH
- Cholesterol analogs & derivatives chemistry isolation & purification MeSH
- Chromatography, Reverse-Phase methods standards MeSH
- Phytosterols chemistry isolation & purification MeSH
- Ketocholesterols chemistry isolation & purification MeSH
- Linseed Oil analysis chemistry MeSH
- Plant Oils analysis chemistry MeSH
- Reference Standards MeSH
- Sitosterols chemistry isolation & purification MeSH
- Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization methods standards MeSH
- Stigmasterol chemistry isolation & purification MeSH
- Silver chemistry MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
V tomto sdělení prezentujeme naše dosavadní výsledky práce s hmotnostním spektrometrem SELDI TOF, které se týkají přípravy a možnosti analýzy jednotlivých druhů biologického materiálu obecně studovaných v onkologickém preklinickém výzkumu (sérum, plazma, buněčné frakce, nativní tkáň).
We present our pilot results on application of SELDI TOF mass spectrometry in cancer research. We discuss applicability of this technology on various clinical material commonly studied in cancer patients such as serum, plasma, cell fractions, and native tissues.
