Use Of MALDI-TOF MS Research Articles

Rapid and cost-effective identification of Bartonella species using mass spectrometry

Bacteria of the genus Bartonella are emerging zoonotic bacteria recognized in a variety of human diseases. Due to their poor chemical reactivity, these fastidious bacteria are poorly characterized using routine phenotypic laboratory tests. Identification is usually achieved using molecular techniques that are time-consuming, expensive and technically demanding. Recently, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has emerged as a new technique for bacterial species identification. This study evaluated the use of MALDI-TOF MS for rapid genus and species identification of Bartonella species. Reference strains representing 17 recognized Bartonella species were studied. For each species, MS spectra for four colonies were analysed. The consensus spectrum obtained for each species was unique among spectra obtained for 2843 bacteria within the Bruker database, including 109 alphaproteobacteria. Thirty-nine additional blind-coded Bartonella strains were correctly identified at the species level, including 36 with a significant score. Altogether, these data demonstrate that MS is an accurate and reproducible tool for rapid and inexpensive identification of Bartonella species.

Immuno‐MALDI‐TOF MS: New perspectives for clinical applications of mass spectrometry

The discovery of novel biomarkers by means of advanced detection tools based on proteomic analysis technologies necessitates the development of improved diagnostic methods for application in clinical routine. On the basis of three different application examples, this review presents the limitations of conventional routine diagnostic assays and illustrates the advantages of immunoaffinity enrichment combined with MALDI-TOF MS. Applying this approach increases the specificity of the analysis supporting a better diagnostic recognition, sensitivity, and differentiation of certain diseases. The use of MALDI-TOF MS as detection method facilitates the identification of modified peptides and proteins providing additional information. Further, employing respective internal standard peptides allows for relative and absolute quantitation which is mandatory in the clinical context. Although MALDI-TOF MS is not yet established for clinical routine diagnostics this technology has a high potential for improvement of clinical diagnostics and monitoring therapeutic efficacy.

Consistency between chemotyping of single filaments of Planktothrix rubescens (cyanobacteria) by MALDI‐TOF and the peptide patterns of strains determined by HPLC‐MS

The high metabolic diversity of potentially toxigenic cyanobacteria has been recently demonstrated for several bloom-forming genera. To understand the dynamics of cyanobacterial blooms and fluctuations in toxin content, techniques are required that allow the distinction of clones in field samples on a subspecific taxonomic level. We evaluated the possibility to analyze single filaments of a common toxigenic taxon, Planktothrix rubescens, by comparing peptide profiles obtained from HPLC/MS analyses of bulk biomass to mass spectra of single filaments for 11 clonal strains. For all strains a significant relationship of chromatogram peak area to mass spectrum peak height was found for individual peptides. Individual peptide's response in mass spectrometry compared to HPLC, however, differed, likely attributable to the presence of particular moieties with high proton affinities in the molecules. Thirty-four individual peptides detected in HPLC fractions of all strains could also be detected in the corresponding single filaments. In conclusion, MALDI-TOF MS analysis of single filaments is shown to be an efficient tool for the study of chemotype diversity and dynamics in field studies and laboratory experiments. The good correspondence between peak area and peak height in chromatograms and mass spectra, respectively, is promising with respect to the use of MALDI-TOF MS as a quantitative analytical tool for natural products studies and cyanotoxin research.

Genotyping fetal paternally inherited SNPs by MALDI‐TOF MS using cell‐free fetal DNA in maternal plasma: Influence of size fractionation

The determination of fetal point mutations from fetal cell-free DNA (cf-DNA) in maternal plasma is technically challenging due to the preponderance of maternal sequences. It has recently been shown that fetal cf-DNA sequences are smaller than maternal ones and that the selection of small cf-DNA fragments by size fractionation by agarose gel electrophoresis leads to the enrichment of fetal cf-DNA sequences, thereby permitting the detection of otherwise masked fetal point mutations. In a separate development, the use of MALDI-TOF MS has also been shown to facilitate the detection of fetal point mutations from cf-DNA in maternal plasma. In this study, a combination of these approaches was examined. cf-DNA was extracted from 18 maternal plasma samples, 10 taken at term and 8 obtained early in the second trimester. A total of 41 SNP loci were examined in size-fractionated and total cf-DNA using either a conventional homogeneous MassEXTEND (hME) assay or a nucleotide-specific single allele base extension reaction (SABER) assay. The analysis of total cf-DNA indicated that size fractionation considerably enhanced the sensitivity of the standard hME assay, especially for samples taken early in pregnancy. Size fractionation also rendered the signals obtained by the SABER assay more precise.

In Vitro and In Situ Tracking of Choline-Phospholipid Biogenesis by MALDI TOF-MS

The quantitative monitoring of newly synthesized species of phosphatidylcholines (PCs) and sphingomyelins (SMs) has been achieved in cultured human lens epithelial cells, both in situ and in vitro, with the use of MALDI TOF-MS. As the cells were cultured with deuterated choline-d(9), new peaks that differed from the hydrogenated species by 9.06 Da appeared in the mass spectra. The initial rates of appearance of all deuterated species of PCs were comparable and 4 times higher than those for SMs. After 12 h, those rates began to decrease for PCs but not for deuterated SMs, whose relative contents continued to increase throughout the 72 h of the experiment. The differences in initial rates are consistent with the reported initial generation of PCs, their subsequent degradation, and transfer of their headgroup, phosphorylcholine, to SMs. To further test the ability of MALDI TOF-MS to quantify changes in phospholipid (PL) metabolic pathways, myriocin, an inhibitor of SM synthesis, was added to the cells. In vitro and in situ results revealed a decrease in SMs and an unexpected increase in some PCs. With the use of other deuterated precursors and in combination with postsource decay or tandem MS/MS, this approach could allow the simultaneous tracking of the biosynthesis of multiple PL classes while providing details on their acyl chains.

Processing of Polymeric Dosage Forms for Advanced Drug Delivery: From Melt-Extrudates to Nanoparticles

Pharmaceutical technology has been directed towards solving the challenges brought by changes in drug discovery characterized by the advent, on the one hand, of sparingly water-soluble drugs exhibiting low oral bioavailability and, on the other, by the so-called biopharmaceuticals, namely peptide and protein drugs as well as nucleic acid materials. The article reviews some of the results obtained in the laboratory in connection with oral and parenteral drug delivery. Hot-melt extrudates for oral administration appear to be very efficient for the sustained release of highly water-soluble drugs, whereas pH-sensitive nanoparticles have proved very beneficial for increasing the bioavailability of protease inhibitors. Special attention is also paid to the scaling up aspect of the nanoparticle preparation methods. As for the parenteral route, biodegradable implants and nanoparticles are examined for their potential to extend drug action. The important issue of premature degradation of peptide or protein drugs during manufacturing of particles is also addressed using tetracosactide and insulin as model drugs. In this respect, emphasis is put on the use of MALDI-TOF MS for the direct assessment of the primary structure of proteins such as insulin in nanoparticles and simultaneous quantification. Finally, various means for preventing rapid clearance of colloidal carriers from the bloodstream by the reticuloendothelial system are described, in particular the utilization of pegylated biodegradable polymers. The reduction of nanoparticle phagocytosis opens perspectives for improved drug targeting of organs.

MALDI-TOF mass spectrometric detection of multiplex single base extended primers. A study of 17 y-chromosome single-nucleotide polymorphisms.

One of the most promising techniques for typing of multiple single-nucleotide polymorphism (SNP) is detection of single base extension primers (SBE) by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). We present a new MALDI-TOF MS protocol for typing of multiple SNPs in a single reaction. Biotin-labeled ddNTPs were used in the SBE reaction and solid phase-bound monomeric avidin was used as capturing/purification scheme allowing the exclusive release of the SBE products under gentle conditions using 5% triethylamine. We dubbed this method monomeric avidin triethylamine purification. The biotin-labeled ddNTPs contained linkers with different masses ensuring a clear separation of the alleles even for SBE primers with a mass of 10 300 Da. Furthermore, only 25-350 fmol of SBE primers were necessary in order to obtain reproducible MALDI-TOF spectra. Similar signal intensities were obtained in the 5500-10 300 m/z mass range by increasing the concentration of the longer SBE primers in the reaction. To validate the technique, 17 Y-chromosome SNPs were analyzed in 200 males. The precision and accuracy of the mass determination were analyzed by parametric statistic, and the potential use of MALDI-TOF MS for SNP typing is discussed.

Analysis of the products of the anionic oligomerisation of a phosphaalkene using MALDI-TOF mass spectrometry.

The first use of MALDI-TOF MS in the study of the products of RLi (R = Me, Bu) initiated oligomerisation of P[double bond, length as m-dash]C bonds is reported. These studies may be considered as models for analogous polymerisation reactions. The detected linear products with R-P and C-H end-groups are consistent with a chain growth mechanism similar to that known for olefins. Interestingly, the results suggest that backbiting may compete with chain growth.

Tag‐Reporter and Resin Capture—Release Strategy in Oligosaccharide Synthesis

AbstractFor Abstract see ChemInform Abstract in Full Text.

Tag-reporter and resin capture--release strategy in oligosaccharide synthesis.

Low molecular weight poly(ethylene glycol) (LWPEG) has been found to be useful as a "tag" in oligosaccharide synthesis due to its high polarity. Real-time reaction monitoring was achieved by use of MALDI-TOF MS in the glycosylation reactions and color tests in the deprotection of the chloroacetyl group. Further, a cysteine-supported insoluble resin enables the purification of the chloroacetyl-bound compounds on soluble PEG.

One-step purification of recombinant yeast 6-phosphofructo-2-kinase after the identification of contaminants by MALDI-TOF MS.

His-tagged yeast 6-phosphofructo-2-kinase was overexpressed in the yeast strain DFY658 under the control of the Gal1 promoter. Here we describe a simple and fast purification protocol for the recombinant enzyme under native conditions using a HiTrap affinity column loaded with CuSO(4). The use of MALDI-TOF MS after in-gel-digestion enabled us to identify a critical contamination of the end product as yeast alcohol dehydrogenase1 (Adh1p). After identification this contaminant could be efficiently removed by carrying out the washing steps at 25 degrees C instead of at 4 degrees C. To reduce the cellular proteolytic activities a low phosphate concentration in the growth medium was applied. This simple modification of the yeast cell growth conditions increased significantly the yield of the recombinant protein.

MALDI-TOF MS analysis of isoflavones in soy products.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a new technique having a number of advantages for food analysis. This study is the first to demonstrate the use of MALDI-TOF MS to identify isoflavones in soy samples. 2',4',6'-Trihydroxyacetophenone (THAP) and 2,5-dihydroxybenzoic acid (DHB) were both good matrices for isoflavones, but DHB was chosen as the best because it worked well for sample extracts, with good spot-to-spot repeatability. Isoflavones were predominantly ionized in a protonated form with a very small amount of sodium or potassium adduct ions. Fragmentation occurred only through loss of glycosidic residues. Daidzin showed more than twice the response of genistin using MALDI-TOF MS. A simple solid phase extraction of isoflavones from soy samples was developed for MALDI-TOF MS analysis. MALDI-TOF MS can provide an isoflavone profile in 2 min and serves as a powerful tool to identify and study processing changes of isoflavones in soy products.

MALDI-TOF MS analysis of food flavonol glycosides.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a new technique that is having a great impact on food analysis. This study is the first to demonstrate the use of MALDI-TOF MS to identify flavonol glycosides in food samples. 2',4',6'-Trihydroxyacetophenone was chosen as the best matrix because it worked for crude sample extracts and ionized flavonol glycosides in both positive and negative MALDI-TOF MS modes. In the positive mode, multiple ion forms were observed for flavonol glycosides, including [M + H](+), [M + Na](+), [M + K](+), and [M - H + Na + K](+), with further fragmentation through loss of glycosidic residues. The negative mode for all flavonol glycosides resulted in [M - H](-) ion formation without detectable fragmentation. The multiple ions in the positive mode gave more information on individual flavonol glycoside structures than the negative mode. Flavonol glycosides showed similar intensities or responses in the positive mode, while kaempferol glycosides exhibited much less response than quercetin glycosides in negative mode.

The determination of non-ionic surfactants in surface waters by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry

Matrix-assisted time-of-flight mass spectrometry (MALDI-TOFMS) has been shown to be a useful tool for the analysis of a wide range of biological and synthetic polymers. This has included the analysis of some commercial formulations of some non-ionic surfactants. But, as yet, no one has reported the use of MALDI-TOFMS for the analysis of surfactants in environmental matrices. Here we report the use of MALDI-TOFMS for the qualitative and quantitative determination of nonylphenol ethoxylate surfactants in environmental surface waters. Following extraction/preconcentration by C18 solid phase extraction, the sample is mixed with a standard MALDI matrix (2,5-dihydroxybenzoic acid). The mixture is then crystallised on a stainless steel target for introduction into the mass spectrometer. The limit of detection is 40 µg/L based on a 250 mL sample. Copyright © 1999 John Wiley & Sons, Ltd.

Sample preparation strategies for low-volume determinations inIn vitro studies of proteins by use of MALDI-TOF MS

A flow-through microdispenser, manufactured from micromachined silicon and based on a piezoelectric element, has been used as an interface for high-density target sample deposition in matrix assisted laser-desorption ionization-time-of-flight mass spectrometry (MALDI-TOF). The dispenser has been used for sample preparation in MALDI-TOF MS analysis proteins. Single 110-picoliter sample droplets containing between 200 and 300 attomol proteins can be determined. The sensitivity of analysis of a dilute protein was increased by use of pre-concentration procedure in which the protein solution was dispensed as four sequences each of 200 droplets.