Discrimination of Aspergillus isolates at the species and strain level by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry fingerprinting

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI–TOF MS) was used to generate highly reproducible mass spectral fingerprints for 12 species of fungi of the genus Aspergillus and 5 different strains of Aspergillus flavus. Prior to MALDI–TOF MS analysis, the fungi were subjected to three 1-min bead beating cycles in an acetonitrile/trifluoroacetic acid solvent. The mass spectra contain abundant peaks in the range of 5 to 20 kDa and may be used to discriminate between species unambiguously. A discriminant analysis using all peaks from the MALDI–TOF MS data yielded error rates for classification of 0 and 18.75% for resubstitution and cross-validation methods, respectively. If a subset of 28 significant peaks is chosen, resubstitution and cross-validation error rates are 0%. Discriminant analysis of the MALDI–TOF MS data for 5 strains of A. flavus using all peaks yielded error rates for classification of 0 and 5% for resubstitution and cross-validation methods, respectively. These data indicate that MALDI–TOF MS data may be used for unambiguous identification of members of the genus Aspergillus at both the species and strain levels.     

Fungal pigments inhibit the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis of darkly pigmented fungi

The analysis of urinary acylglycines is an important biochemical tool for the diagnosis of many organic acidemias and mitochondrial fatty acid β-oxidation defects. A new rapid analytical method has been developed for quantification of acylglycines in urine by liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS). The method requires a simple sample preparation avoiding derivatization. It has high sensitivity, specificity, and throughput capability, and it requires minimal instrument maintenance. The use of chromatographic separation allows us to identify and quantify isomeric compounds that cannot be solved by appropriate multiple reaction monitoring (MRM) transitions. Urinary concentrations of the different acylglycines were determined using deuterated internal standards. The reference interval for the various metabolites was established using 120 healthy controls. The diagnostic usefulness of the method was demonstrated in three patients with propionic acidemia (PA), one patient with isovaleric acidemia (IVA), two patients with beta ketothiolase deficiency (BKTD), one patient with short branched chain amino acid deficiency (SBCAD), four patients with medium chain acyl-coenzyme A dehydrogenase deficiency (MCADD), one patient with isobutyryl-coenzyme A dehydrogenase deficiency (IBDHD), and one patient with multiple acyl-coenzyme A dehydrogenase deficiency (MADD).     

Determination of prolidase activity using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Proline-containing peptides of the X-proline type are cleaved by the dipeptidase prolidase. The classical method of prolidase assay relied on the colorimetric estimation of the liberated proline with ninhydrin using acidic media and heat. This method, however, gave inconsistent results due to the nonspecificity of the ninhydrin color reaction. We report here a method for the detection of the liberated proline using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Human sera were incubated with a mixture containing the dipeptide glycyl-proline in Tris-HCl supplemented with manganese at 37 degrees C for 24h. The samples were precipitated with trifluoroacetic acid and centrifuged. An aliquot of the supernatant was mixed with an equal volume of ferulic acid solution. An aliquot from this mixture was spotted on a stainless steel mass spectrometry grid and analyzed using MALDI-TOF mass spectrometry. The activity of the enzyme was determined by the complete disappearance of the glycyl-proline peak with the concomitant appearance of the proline peak and can be expressed in terms of the ratio of the area beneath the proline to the area beneath the glycyl-proline peak. Subjects homozygous for prolidase deficiency had a ratio ranging from 0.006 to 0.04 while obligatory heterozygotes had a ratio ranging from around 1.1 to 2.4. Normal subjects had ratios ranging from 9 to 239. Using this method we have unambiguously identified subjects with homozygous or heterozygous prolidase deficiency. In addition to the advantage of rapid sample preparation time, this method is highly specific, reproducible, and sensitive.     

Protein phosphorylation analysis by site-specific arginine-mimic labeling in gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Although recent advances in gel electrophoresis and mass spectrometry have greatly facilitated separation, purification, and identification of proteins, significant challenges remain in relation to phosphoprotein analysis. Here we introduce a powerful method for analysis of protein phosphorylation in which phosphorylation sites are labeled with guanidinoethanethiol (GET) by beta-elimination/Michael addition prior to proteolysis and mass spectrometry (MS) analysis. This technique is especially useful in conjunction with gel-based technology in that all of the processes involved, including GET labeling, washing, and phosphospecific enzymatic hydrolysis, can be carried out in excised gel slices, thereby minimizing sample loss and contamination. The novel GET tag, which has a highly basic guanidine group, increases the peak intensities for the GET-labeled tryptic peptides by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS. In addition, phosphospecific proteolytic cleavage occurs at guanidinoethylcysteine (Gec) residue, which is arginine-mimic formed by GET tagging of phosphorylated serine residues. Thus, GET tagging is especially useful in analysis of long tryptic phosphopeptides. To illustrate the utility of the in-gel GET tagging and digestion approach, we used it to precisely analyze the phosphorylation sites of human glutathione S-transferase P1 (GSTP1), an enzyme involved in phase II metabolism of many carcinogens and anticancer drugs. The in-gel GET tagging/digestion technique significantly enhances the analytical potential of gel electrophoresis/MS in studies of proteome phosphorylation.     

Structural and quantitative analysis of N-linked glycans by matrix-assisted laser desorption ionization and negative ion nanospray mass spectrometry

Negative ion electrospray (ESI) fragmentation spectra derived from anion-adducted glycans were evaluated for structural determination of N-linked glycans and found to be among the most useful mass spectrometric techniques yet developed for this purpose. In contrast to the more commonly used positive ion spectra that contain isobaric ions formed by losses from different regions of the molecules and often lead to ambiguous deductions, the negative ion spectra contain ions that directly reflect structural features such as the branching pattern, location of fucose, and the presence of bisecting GlcNAc. These structural features are sometimes difficult to determine by traditional methods. Furthermore, the spectra give structural information from mixtures of isomers and from single compounds. The method was evaluated with well-characterized glycans from IgG and used to explore structures of N-linked glycans released from serum glycoproteins with the aim of identifying biomarkers for cancer. Quantities of glycans were measured by ESI and by matrix-assisted laser desorption ionization mass spectrometry; each technique produced virtually identical results for the neutral desialylated glycans.     

Convenient and rapid analysis of linkage isomers of fucose-containing oligosaccharides by matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight mass spectrometry

Matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight mass spectrometry (MALDI-QIT-TOF MS) was used to analyze three pyridylamino (PA)-fucosyloligosaccharides isolated from human milk: lacto-N-fucopentaose (LNFP) I [Fucα1-2Galβ1-3GlcNAcβ1-3Galβ1-4Glc-PA], LNFP II [Galβ1-3(Fucα1-4)GlcNAcβ1-3Galβ1-4Glc-PA], and LNFP III [Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4Glc-PA]. These oligosaccharides are linkage isomers. MALDI-QIT-TOF MS provides MSⁿ spectra, which we used to characterize these PA-oligosaccharides. MS/MS/MS analysis of the non-reducing end tri-saccharide ions generated by MS/MS was able to distinguish these oligosaccharide isomers. The MALDI-QIT-TOF MS is a very convenient and rapid method, therefore, it would be useful for high throughput structural analyses of various types of pyridylaminated oligosaccharide isomers.     

A study on in vitro glycation processes by matrix-assisted laser desorption ionization mass spectrometry

The number of glucose molecules condensed on glycated bovine serum albumin have been easily determined by means of matrix-assisted laser desorption/ionization mass spectrometry. Measurements were carried out on samples from incubation of the proteins with glucose at different concentrations (0.02 M, 0.2 M, 2 M and 5 M). A clear increase in molecular mass of BSA with respect to incubation time is detected. In contrast to what is observed with fluorescence, the plots of molecular mass increase vs. incubation time show tha occurrence of a steady state, corresponding to the complete saturation of all the protein sites against glucose. Comparison of fluorescence and molecular mass data reveals that some further reactions, different from condensation, must take place, which could be in principle either intramolecular or originated by reactivity of modified condensed gluocse moieties vs. free glucose.     

基质辅助激光解析电离飞行时间质谱在医学真菌领域的应用进展

基质辅助激光解析电离飞行时间质谱（matrix-assisted laser desorption/ionization time-of-flight mass spectrometry,MALDI-TOF MS）是近年来新兴的微生物检测技术,通过核糖体蛋白分析实现对真菌快速、准确鉴定。本文针对MALDI-TOF MS用于致病真菌鉴定、分类、体外抗真菌药物敏感性检测以及临床微生物样本直接检测等方面作一综述。     

High-throughput proteomics using matrix-assisted laser desorption/ ionization mass spectrometry

It has become evident that the mystery of life will not be deciphered just by decoding its blueprint, the genetic code. In the life and biomedical sciences, research efforts are now shifting from pure gene analysis to the analysis of all biomolecules involved in the machinery of life. One area of these postgenomic research fields is proteomics. Although proteomics, which basically encompasses the analysis of proteins, is not a new concept, it is far from being a research field that can rely on routine and large-scale analyses. At the time the term proteomics was coined, a gold-rush mentality was created, promising vast and quick riches (i.e., solutions to the immensely complex questions of life and disease). Predictably, the reality has been quite different. The complexity of proteomes and the wide variations in the abundances and chemical properties of their constituents has rendered the use of systematic analytical approaches only partially successful, and biologically meaningful results have been slow to arrive. However, to learn more about how cells and, hence, life works, it is essential to understand the proteins and their complex interactions in their native environment. This is why proteomics will be an important part of the biomedical sciences for the foreseeable future. Therefore, any advances in providing the tools that make protein analysis a more routine and large-scale business, ideally using automated and rapid analytical procedures, are highly sought after. This review will provide some basics, thoughts and ideas on the exploitation of matrix-assisted laser desorption/ ionization in biological mass spectrometry – one of the most commonly used analytical tools in proteomics – for high-throughput analyses.     

Liquid ultraviolet matrix-assisted laser desorption/ionization -- mass spectrometry for automated proteomic analysis

We have combined several key sample preparation steps for the use of a liquid matrix system to provide high analytical sensitivity in automated ultraviolet -- matrix-assisted laser desorption/ionisation -- mass spectrometry (UV-MALDI-MS). This new sample preparation protocol employs a matrix-mixture which is based on the glycerol matrix-mixture described by Sze et al. The low-femtomole sensitivity that is achievable with this new preparation protocol enables proteomic analysis of protein digests comparable to solid-state matrix systems. For automated data acquisition and analysis, the MALDI performance of this liquid matrix surpasses the conventional solid-state MALDI matrices. Besides the inherent general advantages of liquid samples for automated sample preparation and data acquisition the use of the presented liquid matrix significantly reduces the extent of unspecific ion signals in peptide mass fingerprints compared to typically used solid matrices, such as 2,5-dihydroxybenzoic acid (DHB) or alpha-cyano-hydroxycinnamic acid (CHCA). In particular, matrix and low-mass ion signals and ion signals resulting from cation adduct formation are dramatically reduced. Consequently, the confidence level of protein identification by peptide mass mapping of in-solution and in-gel digests is generally higher.     

Quantification of bifunctional diethylenetriaminepentaacetic acid derivative conjugation to monoclonal antibodies by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

The results of the characterization of a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry-based method that was developed to establish the stoichiometry of CHX-A'-diethylenetriaminepentaacetic acid (DTPA) or benzyl-DTPA conjugated to a recombinant immunoglobulin G (IgG) are reported. This simple method does not require an accurate measurement of the sample protein concentration to accurately quantify the number of DTPA conjugated. It is also not necessary to thoroughly remove nonconjugated DTPA from the sample. The average number of moles of DTPA attached per mole of IgG was calculated from the difference in the observed masses of DTPA-IgG and nonconjugated IgG divided by the molecular weight of the DTPA derivative. As more DTPA is attached, the [M+H](+) peak of DTPA-IgG becomes broader and noisier. Also, the signal intensity in the mass spectrum decreases, apparently due to the increase in the heterogeneity in the number of DTPA attached to each molecule of IgG. The standard deviation of the measured mass and that of the stoichiometry of the DTPA attached per IgG increased as more DTPA was attached. The standard deviation, expressed as coefficient of variation for samples with 2 to 4 mol of DTPA attached per mole of IgG, was 8 to 9%.     

Evaluating factor XIII specificity for glutamine-containing substrates using a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry assay

Activated factor XIII (FXIIIa) catalyzes the formation of γ-glutamyl-ε-lysyl cross-links within the fibrin blood clot network. Although several cross-linking targets have been identified, the characteristic features that define FXIIIa substrate specificity are not well understood. To learn more about how FXIIIa selects its targets, a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI–TOF MS)-based assay was developed that could directly follow the consumption of a glutamine-containing substrate and the formation of a cross-linked product with glycine ethylester. This FXIIIa kinetic assay is no longer reliant on a secondary coupled reaction, on substrate labeling, or on detecting only the final deacylation portion of the transglutaminase reaction. With the MALDI–TOF MS assay, glutamine-containing peptides derived from α₂-antiplasmin, Staphylococcus aureus fibronectin binding protein A, and thrombin-activatable fibrinolysis inhibitor were examined directly. Results suggest that the FXIIIa active site surface responds to changes in substrate residues following the reactive glutamine. The P₋₁ substrate position is sensitive to charge character, and the P₋₂ and P₋₃ substrate positions are sensitive to the broad FXIIIa substrate specificity pockets. The more distant P₋₈ to P₋₁₁ region serves as a secondary substrate anchoring point. New knowledge on FXIIIa specificity may be used to design better substrates or inhibitors of this transglutaminase.     

基质辅助激光解吸电离飞行时间质谱在微生物鉴定中的研究进展

与传统的微生物鉴定技术相比,基质辅助激光解吸电离飞行时间质谱(matrix-assisted laser desorption ionization time-of-flight mass spectrometry, MALDI-TOF MS)是一种准确、可靠和快速的鉴定和分型的技术。本文通过检索近年来国内外相关研究论文,总结最新的研究进展,发现MALDI-TOF MS在临床病原微生物、食源性微生物以及环境微生物等鉴定中有较大的优势,加快了微生物鉴定的进程,同时探索该技术在新领域的最新进展和面临的挑战,以期为我国基质辅助激光解吸电离飞行时间质谱技术的发展提供参考。     

Enhancement of ovarian tumor classification by improved reproducibility in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of serum glycans

The serum N-glycome is a promising source of biomarker discovery. Matrix-assisted laser desorption/ionization time-of-flight (MALDI–TOF) mass spectrometry (MS) profiling of serum N-glycans was attempted for differentiating borderline ovarian tumor from benign cases, for which a low data spread is essential. An experimental protocol using matrix-prespotted MALDI plates and fast vacuum drying of the loaded N-glycan samples was developed, thereby minimizing the intensity variations in the replicates to an average relative standard deviation (RSD) of 3.96% for the highest N-glycan peak (m/z 1485.53) of the Sigma–Aldrich serum standard. When applied to sera of ovarian tumors, this procedure exhibited an average RSD of 5.74% for m/z 1485.53 and of 7.28% for all MS peaks. This improved reproducibility combined with the OVA-Beyond^® screening software resulted in 75.1% and 79.4% correct classification for benign and borderline tumor samples, respectively, while the classification rates by the conventional ovarian tumor marker CA-125 were 54.4% and 53.1%, respectively. Both true positive rate and true negative rate fluctuated with small numbers of markers and converged as the number of markers increased. Cross-validations were performed in comparison with CA-125. These results suggest that our optimized process for MALDI–TOF MS of the serum glycome has a great potential for the screening of early stage ovarian cancer.     

Ribosomal protein profiling by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for phylogenety-based subspecies resolution of Bifidobacterium longum

The taxonomic positions of the subspecies of Bifidobacterium longum (B. longum subsp. longum, subsp. infantis, and subsp. suis) have been controversial. A current proposal is that the former two species “B. infantis” and “B. suis” be unified with B. longum and all three reclassified as three subspecies. To test this proposal, ribosomal protein profiling as observed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was applied to the classification of 17 strains of B. longum, including three subspecies. Among 41 different kinds of ribosomal proteins selected as biomarkers whose masses were calculated from their amino acid sequences, 31-41 ribosomal proteins were observed in sample strains with the same masses as the references. The high matching rate indicates high conservation of ribosomal proteins within the sample strains, and therefore strongly supports the unification of the former species. However, the masses of some ribosomal proteins varied within species. The phylogenetic tree constructed from the profiles of ribosomal proteins matched the references, showing a clear cluster of the subsp. longum and the subsp. infantis strains. This result supports the proposal to reclassify B. longum into subsp. longum and subsp. infantis. The subsp. suis strains formed an individual sub-cluster within the infantis cluster. However, their ribosomal proteins have both characters of longum and infantis types. This result suggests that the taxonomic position of the subsp. suis should be reconsidered.     

Structural characterization of N-linked oligosaccharides on monoclonal antibody cetuximab by the combination of orthogonal matrix-assisted laser desorption/ionization hybrid quadrupole-quadrupole time-of-flight tandem mass spectrometry and sequential enzymatic digestion

Cetuximab is a novel therapeutic monoclonal antibody with two N-glycosylation sites: a conserved site in the CH2 domain and a second site within the framework 3 of the variable portion of the heavy chain. The detailed structures of these oligosaccharides were successfully characterized using orthogonal matrix-assisted laser desorption/ionization hybrid quadrupole-quadrupole time-of-flight mass spectrometry (oMALDI Qq-TOF MS) and tandem mass spectrometry (MS/MS) in combination with exoglycosidase digestion. The N-linked oligosaccharides were released by treatment with N-glycanase F, reductively aminated with anthranilic acid, and fractionated by normal phase high-performance liquid chromatography (NP-HPLC). The fluorescent-labeled oligosaccharide pool and fractions were analyzed by oMALDI Qq-TOF MS and MS/MS in negative ion mode. Each fraction was further digested with an array of exoglycosidase mixtures, and subsequent MALDI TOF MS analysis of the resulting products yielded information about structural features of the oligosaccharide. The combined data revealed the presence of 21 distinct oligosaccharide structures in cetuximab. These oligosaccharides differ mainly in degree of sialylation with N-glycolyl neuraminic acid and extent of galactosylation (zero-, mono-, di-, and alpha(1-3)-galactosidase). The individual oligosaccharides were further assigned to the specific sites in the Fab and Fc regions of the antibody. This study represents a unique approach in that MS/MS data were used to identify and confirm the oligosaccharide structures of a protein.     

Metabolite profiling of plant carotenoids using the matrix-assisted laser desorption ionization time-of-flight mass spectrometry

Although modern MS has facilitated the advent of metabolomics, some natural products such as carotenoids are not readily compatible to detection by MS. In the present article, we describe how matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI/TOF-MS) can be utilized to acquire mass spectra of carotenoids effectively. The procedure is sensitive (pmole range), reduces 'spot to spot' variation and provides high mass accuracy, thus aiding identification. The technique has been applied in vivo to the analysis of carotenoids in isolated plant cells and in vitro to three applications: (i) to show compatibility with purification methods such as LC, TLC and HPLC; (ii) for the rapid identification and quantification (by isotope dilution) of carotenoids present in crude extracts from plant tissues and whole cells; (iii) simultaneous semi-quantitative determination of carotenoids metabolites (m/z values) in crude plant extracts. Multivariate analysis of the recorded m/z values shows the effectiveness of the procedure in distinguishing genotypes from each other. In addition, the utility of the technique has been demonstrated on two mutant tomato populations, to determine alterations in carotenoid content, and a comparison made with traditional HPLC-photodiode array analysis. These data show that MALDI/TOF-MS can be used to rapidly profile, identify and quantify plant carotenoids reproducibly, as well as detecting other metabolites (m/z) in complex biological systems.