chemical ionization
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Peptide samples derived from enzymatic in-gel digestion of proteins resolved by gel electrophoresis often contain high amount of salts originating from reaction and separation buffers. Different methods are used for desalting prior to matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS), e.g. reversed-phase pipette tip purification, on-target washing, adding co-matrices, etc. As a suitable matrix for MALDI MS of peptides, alpha-cyano-4-hydroxycinnamic acid (CHCA) is frequently used. Crystalline CHCA shows the ability to bind peptides on its surface and because it is almost insoluble in acidic water solutions, the on-target washing of peptide samples can significantly improve MALDI MS signals. Although the common on-target washing represents a simple, cheap and fast procedure, only a small portion of the available peptide solution is efficiently used for the subsequent MS analysis. The present approach is a combination of the on-target washing principle carried out in a narrow-end pipette tip (e.g. GELoader tip) and preconcentration of peptides from acidified solution by passing it through small CHCA crystals captured inside the tip on a glass microfiber frit. The results of MALDI MS analysis using CHCA-tip peptide preconcentration are comparable with the use of homemade POROS R2 pipette tip microcolumns. Advantages and limitations of this approach are discussed. Copyright 2009 John Wiley & Sons, Ltd.
- MeSH
- chemická frakcionace metody MeSH
- kyseliny kumarové chemie MeSH
- peptidy chemie izolace a purifikace MeSH
- soli chemie izolace a purifikace MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice metody MeSH
- tandemová hmotnostní spektrometrie metody MeSH
- Publikační typ
- práce podpořená grantem MeSH
Silver-ion high-performance liquid chromatography (HPLC) coupled to atmospheric pressure chemical ionization mass spectrometry (APCI-MS) is used for the regioisomeric analysis of triacylglycerols (TGs). Standard mixtures of TG regioisomers are prepared by the randomization reaction from 8 mono-acid TG standards (tripalmitin, tristearin, triarachidin, triolein, trielaidin, trilinolein, trilinolenin and tri-gamma-linolenin). In total, 32 different regioisomeric doublets and 11 triplets are synthesized, separated by silver-ion HPLC using three serial coupled chromatographic columns giving a total length of 75cm. The retention of TGs increases strongly with the double bond (DB) number and slightly for regioisomers having more DBs in sn-1/3 positions. DB positional isomers (linolenic vs. γ-linolenic acids) are also separated and their reverse retention order in two different mobile phases is demonstrated. APCI mass spectra of all separated regioisomers are measured on five different mass spectrometers: single quadrupole LC/MSD (Agilent Technologies), triple quadrupole API 3000 (AB SCIEX), ion trap Esquire 3000 (Bruker Daltonics), quadrupole time-of-flight micrOTOF-Q (Bruker Daltonics) and LTQ Orbitrap XL (Thermo Fisher Scientific). The effect of different types of mass analyzer on the ratio of [M+H-R(i)COOH](+) fragment ions in APCI mass spectra is lower compared to the effect of the number of DBs, their position on the acyl chain and the regiospecific distribution of acyl chains on the glycerol skeleton. Presented data on [M+H-R(i)COOH](+) ratios measured on five different mass analyzers can be used for the direct regioisomeric determination in natural and biological samples.
- MeSH
- hmotnostní spektrometrie s elektrosprejovou ionizací metody MeSH
- hmotnostní spektrometrie metody MeSH
- isomerie MeSH
- molekulová hmotnost MeSH
- triglyceridy chemie MeSH
- vysokoúčinná kapalinová chromatografie metody MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- srovnávací studie MeSH
Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry is used for the determination of molecular weights of different chemical compounds. We describe here the use of MALDI-TOF mass spectrometry to detect a carbapenem antibiotic, meropenem, and its degradation products. Buffered meropenem solution (0.1 mM Tris-HCl, pH 6.8) was mixed with an overnight culture of bacteria. After 3-h incubation, the reaction mixture was centrifuged, and the supernatant was analyzed by MALDI-TOF mass spectrometry. The presence or absence of peaks representing meropenem and its sodium salts was crucial. The average turnaround time of this test, considering the use of overnight culture, is 4 h. We validated this method for the detection of resistance to carbapenems in Enterobacteriaceae and Pseudomonas aeruginosa mediated by carbapenemase production. A total of 124 strains, including 30 carbapenemase-producing strains, were used in the study. The sensitivity of this method is 96.67%, with a specificity of 97.87%. Our results demonstrate the ability of this method to routinely detect carbapenemases in Enterobacteriaceae and Pseudomonas spp. in laboratories. This assay is comparable with a labor-intensive imipenem-hydrolyzing spectrophotometric assay that is a reference method for the detection of carbapenemase. As demonstrated here, MALDI-TOF mass spectrometry may be used in microbiological laboratories not only for microbial identification but also for other applications, such as studies of mechanisms of antibiotic resistance.
- MeSH
- antibakteriální látky analýza MeSH
- bakteriální proteiny metabolismus MeSH
- beta-laktamasy metabolismus MeSH
- Enterobacteriaceae enzymologie MeSH
- lidé MeSH
- Pseudomonas aeruginosa enzymologie MeSH
- senzitivita a specificita MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice metody MeSH
- thienamyciny analýza MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
- práce podpořená grantem MeSH
The newly developed interface-free capillary electrophoresis-nanospray/mass spectrometry system (CE-nESI/MS) was applied for rapid analysis of the cardioprotective drug dexrazoxane and its hydrolysed form ADR-925 in deproteinized blood plasma samples. The aim of this study was to test the simplest possible CE-nESI/MS instrumentation for analyses of real samples. This interface-free system, utilizing single piece of a narrow bore capillary as both the electrophoretic separation column and the nanospray emitter, was operated at a flow rate of 30nL/min. Excellent electrophoretic separation and sensitive nanospray ionization was achieved with the use of only one high voltage power supply. In addition, hydrophobic external coating was developed and tested for additional stability of the nanospray ionization. To our knowledge this is the first study devoted to the analysis of dexrazoxane and ADR-925 by capillary electrophoresis-mass spectrometry.
RATIONALE: Hyphenation of atmospheric pressure chemical ionization (APCI) mass spectrometry with capillary and micro high-performance liquid chromatography (HPLC) is attractive for many applications, but reliable ion sources dedicated to these conditions are still missing. There are a number of aspects to consider when designing such an ion source, including the susceptibility of the ionization processes to ambient conditions. Here we discuss the importance of ion source housing for APCI at low flow rates. METHODS: Selected compounds dissolved in various solvents were used to study ionization reactions at 10 μL/min flow rate. APCI spectra were generated using the Ion Max-S source (Thermo Fisher Scientific) operated with or without the ion source housing. RESULTS: The APCI spectra of most compounds measured in the open and enclosed ion sources were markedly different. The differences were explained by water and oxygen molecules that entered the plasma region of the open ion source. Water tended to suppress charge transfer processes while oxygen diminished electron capture reactions and prevented the formation of acetonitrile-related radical cations useful for localizing double bonds in lipids. The effects associated with the ion source housing were significantly less important for compounds that are easy to protonate or deprotonate. CONCLUSIONS: The use of ion source housing prevented alternative ionization channels leading to unwanted or unexpected ions. Compared with the conventional flow rate mode (1 mL/min), the effects of ambient air components were significantly higher at 10 μL/min, emphasizing the need for ion source housing in APCI sources dedicated to low flow rates.
- Publikační typ
- časopisecké články MeSH
This contribution is the second part of the project on strategies used in the selection of electrolyte systems for anionic ITP with ESI-mass spectrometric detection. It presents ITP as a powerful tool for selective stacking of anionic analytes, performed in a nonconventional way in moving-boundary systems where two co-anions are present in both the leading and terminating zones. The theoretical background is given to substantiate the conditions for the existence and migration of ITP boundaries in moving-boundary systems and stacking of analytes at these boundaries. The practical aspects of the theory are shown in form of stacking-window diagrams that bring immediate information about which analytes are stacked in a given system. The presented theory and strategy are illustrated and verified on the example of analysis of a model mixture of salicylic acid, ibuprofen and diclofenac, and comparison of regular and free-acid ITP with moving-boundary ITP systems formed by formic and propionic acids and ammonium as counterion.
The use of trypsin for protein digestion is hampered by its autolysis and low thermostability. Chemical modifications have been employed to stabilize the enzyme. Modified trypsin (e.g. methylated) usually enables performing digestions at elevated temperatures, but it still produces autolytic peptides. In this work, unmodified bovine trypsin was subjected to a microscale affinity chromatography on Arginine Sepharose (ASE) or Benzamidine Sepharose (BSE), which utilized the principle of active-site ligand binding. Trypsin was retained on the sorbents in ammonium bicarbonate as a binding buffer. After washings to remove unbound impurities, the enzyme was eluted by arginine as a free ligand (from ASE) or by diluted hydrochloric acid (from BSE). MALDI-TOF mass spectrometry confirmed removal of large molecular fragments as well as autolytic and other background peptides. Consequently, the purified trypsin was tested for its performance in procedures of in-gel digestion of protein standards and selected urinary proteins from real samples. It has been shown that the affinity purification of trypsin decreases significantly the number of unmatched peptides in peptide mass fingerprints. The presence of arginine in the digestion buffer was found to reduce intensity of autolytic peptides. As a result, the described purification procedure is applicable in a common proteomic routine.
- MeSH
- chromatografie afinitní MeSH
- elektroforéza v polyakrylamidovém gelu MeSH
- mikrochemie metody MeSH
- peptidové mapování metody MeSH
- proteinové hydrolyzáty chemie MeSH
- proteinurie moč MeSH
- skot MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice metody MeSH
- trypsin izolace a purifikace MeSH
- zvířata MeSH
- Check Tag
- skot MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
