Design and synthesis of cleavable biotinylated dideoxynucleotides for DNA sequencing by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Knowledge of the anti-drug antibody (ADA) status is necessary in early research studies. Because specific assay materials are sparse and time is pressing, a generic assay format with drug tolerance for detection of ADAs in serum samples from mice exposed to immunoglobulin G (IgG) or antigen-binding fragments (Fabs) is highly desirable. This article describes a generic immune complex assay in the sandwich enzyme-linked immunosorbent assay (ELISA) format based on (i) transformation of free ADAs to immune complexes by preincubation with excess drug, (ii) the use of a murine anti-human Fab constant domain Fab as capture reagent, (iii) detection of the immune complexes by a peroxidase-labeled rabbit anti-murine Fc antibody, and (iv) ADA-positive control conjugates consisting of human Fab and murine IgG. Results of the experiments suggest that the generic immune complex assay for mouse serum samples was at least equivalent to specific ADA immune assays and even superior regarding drug tolerance. The generic immune complex assay confers versatility as it detects ADAs in complex with full-length IgG as well as with Fabs independent of the target specificity in mouse serum samples. These features help to save the sparse amounts of specific antibodies available in early research and development and speed up drug candidate selection.     

Multiplex single-nucleotide polymorphism genotyping by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

A robust high-throughput single-nucleotide polymorphism (SNP) genotyping method is reported, which applies allele-specific extension to achieve allelic discrimination and uses matrix-assisted laser desorption/ionization time-of-flight mass spectrometry to measure the natural molecular weight difference of oligonucleotides for determination of the base in a single-nucleotide polymorphic location. Tenfold PCR is performed successfully by carefully designing the primers and adjusting the conditions of PCR. In addition, two ways used for PCR product purification are compared and the matrix used in mass spectrometry for high-throughput oligonucleotide analysis is evaluated. The result here shows that the method is very effective and suitable for high-throughput genotyping of SNPs.     

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.     

Quantitative determination of peptides using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

A method is described for the quantitative determination of peptides using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Known limitations imposed by crystal heterogeneity, peptide ionization differences, data handling, and protein quantification with MALDI-TOF mass spectrometry are addressed in this method with a "seed crystal" protocol for analyte-matrix formation, the use of internal protein standards, and a software package called maldi_quant. The seed crystal protocol, a new variation of the fast-evaporation method, minimizes crystal heterogeneity and allows for consistent collection of protein spectra. The software maldi_quant permits rapid and automated analysis of peak intensity data, normalization of peak intensities to internal standards, and peak intensity deconvolution and estimation for vicinal peaks. Using insulin proteins in a background of other unrelated peptides, this method shows an overall coefficient of variance of 4.4%, and a quantitative working range of 0.58-37.5 ng bovine insulin per spot. Coupling of this methodology to powerful analytical procedures such as immunoprecipitation is likely to lead to the rapid and reliable quantification of biologically relevant proteins and their closely related variants.     

Assay of protein tyrosine phosphatases by using matrix-assisted laser desorption ionization time-of-flight mass spectrometry

A nonradioactive assay for protein tyrosine phosphatases (PTPs), employing a tyrosine-phosphorylated peptide as a substrate, has been developed and applied to analyze purified enzymes, cell extracts, and immunoprecipitates. The reaction was followed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) in a linear and positive ion mode with delayed extraction. MALDI-TOF MS detects a loss of peptide mass by 80 Da as a result of dephosphorylation and, more importantly, it yields phospho-peptide to dephosphorylated product peak intensity ratios proportional to their concentration ratios. A strong bias of the MALDI-TOF MS toward detection of the non-phospho-peptide allows accurate detection of small fractions of dephosphorylation. The method is highly sensitive and reproducible. It can be applied to general assays of protein phosphatases with various phospho-peptides as substrates.     

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.     

Characterization of DNA glycosylase activity by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

The DNA of all organisms is persistently damaged by endogenous reactive molecules. Most of the single-base endogenous damage is repaired through the base excision repair (BER) pathway that is initiated by members of the DNA glycosylase family. Although the BER pathway is often considered to proceed through a common abasic site intermediate, emerging evidence indicates that there are likely distinct branches reflected by the multitude of chemically different 3′ and 5′ ends generated at the repair site. In this study, we have applied matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI–TOF–MS) to the analysis of model DNA substrates acted on by recombinant glycosylases. We examine the chemical identity of several possible abasic site and nicked intermediates generated by monofunctional and bifunctional glycosylases. Our results suggest that the intermediate from endoIII/Nth might not be a simple β-elimination product as described previously. On the basis of ¹⁸O incorporation experiments, we propose a new mechanism for the endoIII/Nth family of glycosylases that may resolve several of the previous controversies. We further demonstrate that the use of an array of lesion-containing oligonucleotides can be used to rapidly examine the substrate preferences of a given glycosylase. Some of the lesions examined here can be acted on by more than one glycosylase, resulting in a spectrum of damaged intermediates for each lesion, suggesting that the sequence and coordination of repair activities that act on these lesions may influence the biological outcome of damage repair.     

Phyloproteomics: species identification of Enterobacteriaceae using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

To evaluate matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF MS) as a tool for rapid identification of common clinical bacterial isolates, we analyzed 25 carefully selected isolates of pathogenic Escherichia coli (E. coli) and additional Enterobacteriaceae members. Organisms were prepared according to clinical microbiological protocols and analyzed with minimal additional processing. Spectra were reproducible from preparation to preparation and comprised 40-100 peaks primarily representing intracellular proteins with masses up to 25 kDa. Spectra of 14 genetically diverse bacteremic isolates of E. coli were compared with isolates representing other genera within the Enterobacteriaceae family. Using a new spectrum comparison algorithm, E. coli isolates were closely related to each other and were readily distinguishable from other Enterobacteriaceae, including Salmonella and Shigella. Presently, the methodology permits the analysis of 40 unknown isolates per hour per instrument. These results suggest that MALDI-ToF MS offers a rapid and reliable approach for performing phyloproteomics i.e., identification of unknown bacterial isolates based on similarities within protein biomarker databases.     

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.     

Rapid discrimination of environmental Vibrio by matrix-assisted laser desorption ionization time-of-flight mass spectrometry

The aim of this study was to discriminate 30 Vibrio strains isolated from two wastewater treatment plants from Agadir, Morocco by two molecular typing methods, pulsed-field gel electrophoresis (PFGE) and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Out of the 30 strains of Vibrio examined in this study, 5 isolates could not be typed by PFGE and consistently appeared as a smear on the gel. In general, high genetic biodiversity among the Vibrio strains was found regardless to the isolation source. The results of MALDI TOF analysis show a high congruence of strain grouping demonstrating the accuracy and reliability of MALDI-TOF MS.     

Mass determination of major plasma proteins by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) serves as a rapid and accurate means to determine masses of proteins independent of their shapes or interactions with other molecules. It provides one of the most fundamental characterizations of major plasma proteins. Purified proteins in saline or serum specimens were prepared for analysis by dilution, mixing with a solution of sinapinic acid, and drying on a target plate. Specimens were analyzed in a linear TOF mode with external calibration. Analyses of 24 purified plasma proteins showed predominance of singly charged ions with lesser amounts of dimer and doubly charged monomer, and provided measured masses for these proteins. A number of proteins, including albumin, transferrin, apolipoproteins A-I, A-II, C-I, C-II, and C-III, and prealbumin, could be analyzed directly in serum with appropriate dilution. Measured values for masses of major plasma proteins will assist in analysis of serum and plasma. It is possible to analyze a number of components by MALDI-TOF/MS directly in diluted serum. Extremely simple sample preparation techniques may be useful in analyzing structural variation of several major plasma proteins, particularly those with masses <30 kDa, including a number of apolipoproteins and markers of nutritional status or acute phase responses.     

Structural analysis of cartilage proteoglycans and glycoproteins using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Cartilage extracellular matrix molecules synthesized and maintained by chondrocytes form a strong, elastic tissue functioning to cushion and protect the subchondral bone. Osteoarthritis is characterized by degradation of cartilage extracellular matrix molecules resulting in fibrillation, irreversible erosion, and eventual failure of the tissue. With recent interest in the degradation of cartilage extracellular matrix molecules, a need for more detailed structural information exists. Posttranslational modifications are believed to play a role in determining the susceptibility of these molecules to proteolytic degradation during the development of osteoarthritis. The purpose of this paper is to show how the application of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry to extracellular matrix protein and proteoglycan structure will help elucidate problems in extracellular matrix biochemistry. Methodological issues relating to the high molecular weight, polydispersity, and high degree of posttranslational modification of these molecules are discussed. MALDI-TOF mass spectrometry provides an improved level of detail for extracellular matrix protein and proteoglycan structure and is useful in addressing issues surrounding the causes of degradation during osteoarthritis.     

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.     

Label-free measurement of histone lysine methyltransferases activity by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Histone lysine methyltransferases (HKMTs) are enzymes that play an essential role in epigenetic regulation. Thus, identification of inhibitors specifically targeting these enzymes represents a challenge for the development of new antitumor therapeutics. Several methods for measuring HKMT activity are already available. Most of them use indirect measurement of the enzymatic reaction through radioactive labeling or antibody-recognized products or coupled enzymatic assays. Mass spectrometry (MS) represents an interesting alternative approach because it allows direct detection and quantification of enzymatic reactions and can be used to determine kinetics and to screen small molecules as potential inhibitors. Application of mass spectrometry to the study of HKMTs has not been fully explored yet. We describe here the development of a simple reliable label-free MALDI-TOF MS-based assay for the detection and quantification of peptide methylation, using SET7/9 as a model enzyme. Importantly, the use of expensive internal standard often required in mass spectrometry quantitative analysis is not necessary in this assay. This MS assay allowed us to determine enzyme kinetic parameters as well as IC₅₀ for a known inhibitor of this enzyme. Furthermore, a comparative study with an antibody-based immunosorbent assay showed that the MS assay is more reliable and suitable for the screening of inhibitors.     

Improving sample treatment for in-solution protein identification by peptide mass fingerprint using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Three ultrasonic energy sources were studied to speed up the sample treatment for in-solution protein identification by peptide mass fingerprint using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Protein reduction, alkylation, and enzymatic digestion steps were done in 15 min. Nine proteins, including zinc resistance-associated protein precursor from Desulfovibrio desulfuricans strain G20 and split-soret cytochrome c from D. desulfuricans ATCC27774 were successfully identified with the new protocol.