Single-nucleotide polymorphisms: analysis by mass spectrometry

Matrix-assisted laser desorption-ionization (MALDI) mass spectrometry has evolved as a powerful method for analyzing nucleic acids. Here we provide protocols for genotyping single-nucleotide polymorphisms (SNPs) by MALDI based on PCR and primer extension to generate allele-specific products. Furthermore, we present three different approaches for sample preparation of primer-extension products before MALDI analysis and discuss their potential areas of application. The first approach, the 'GOOD' assay, is a purification-free procedure that uses DNA-modification chemistry, including alkylation of phosphorothioate linkages in the extension primers. The other two approaches use either solid-phase extraction or microarray purification for the purification of primer-extension products. Depending on the reaction steps of the various approaches, the protocols take about 6-8 hours.     

Quantitative approach to single-nucleotide polymorphism analysis using MALDI-TOF mass spectrometry

Pooling of DNA samples before genotyping is a valuable means of streamlining large-scale genotyping efforts in disease association studies, single-nucleotide polymorphism (SNP) validation or mutant allele screening programs. In this report, we explore the application of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to quantitative analysis of SNPs. The measurements are based on MALDI-TOF MS analysis of primer extension assays performed on standard mixtures of pooled PCR products at several test loci. The inherent high molecular weight resolution of MALDI-TOF MS conveys high specificity and good signal-to-noise ratio for performing accurate quantitation. The methods described maximize the sensitivity and quantitative capacity of MALDI-TOF MS while preserving the throughput and economic advantages of the MALDI-TOF platform. Using the format described, we demonstrate that allele frequencies as low as 5% can be detected quantitatively and unambiguously.     

Single-nucleotide polymorphism analysis by pyrosequencing

There is a growing demand for high-throughput methods for analysis of single-nucleotide polymorphic (SNP) positions. Here, we have evaluated a novel sequencing approach, pyrosequencing, for such purposes. Pyrosequencing is a sequencing-by-synthesis method in which a cascade of enzymatic reactions yields detectable light, which is proportional to incorporated nucleotides. One feature of typing SNPs with pyrosequencing is that each allelic variant will give a unique sequence compared to the two other variants. These variants can easily be distinguished by a pattern recognition software. The software displays the allelic alternatives and allows for direct comparison with the pyrosequencing raw data. For optimal determination of SNPs, various protocols of nucleotide dispensing order were investigated. Here, we demonstrate that typing of SNPs can efficiently be performed by pyrosequencing using an automated system for parallel analysis of 96 samples in approximately 5 min, suitable for large-scale screening and typing of SNPs.     

MALDI-TOF mass spectrometry

Matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) has developed during the past decade into a versatile tool for biopolymer analysis. The aim of this review is to summarize this development and outline the applications, which have been enabled for routine use in the field of nucleic acid analysis. These include the anlaysis of mutations, the resequencing of amplicons with a known reference sequence, and the quantitative analysis of gene expression and allelic frequencies in complex DNA mixtures.     

Identification of fungal microorganisms by MALDI-TOF mass spectrometry

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has emerged as a reliable tool for fast identification and classification of microorganisms. In this regard, it represents a strong challenge to microscopic and molecular biology methods. Nowadays, commercial MALDI systems are accessible for biological research work as well as for diagnostic applications in clinical medicine, biotechnology and industry. They are employed namely in bacterial biotyping but numerous experimental strategies have also been developed for the analysis of fungi, which is the topic of the present review. Members of many fungal genera such as Aspergillus, Fusarium, Penicillium or Trichoderma and also various yeasts from clinical samples (e.g. Candida albicans) have been successfully identified by MALDI-TOF MS. However, there is no versatile method for fungi currently available even though the use of only a limited number of matrix compounds has been reported. Either intact cell/spore MALDI-TOF MS is chosen or an extraction of surface proteins is performed and then the resulting extract is measured. Biotrophic fungal phytopathogens can be identified via a direct acquisition of MALDI-TOF mass spectra e.g. from infected plant organs contaminated by fungal spores. Mass spectrometric peptide/protein profiles of fungi display peaks in the m/z region of 1000–20 000, where a unique set of biomarker ions may appear facilitating a differentiation of samples at the level of genus, species or strain. This is done with the help of a processing software and spectral database of reference strains, which should preferably be constructed under the same standardized experimental conditions.     

Analysis of glycated insulin by MALDI-TOF mass spectrometry

Non-enzymatic glycation of protein is mediated via an interaction between the aldehyde group of a reducing sugar and available alpha- or epsilon-amino moieties of the protein. The above event can alter the biological activity of the protein and therefore, it is of particular interest to monitor the glycation of proteins having important functional roles in metabolism. In the present study, matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) has been used to determine the non-enzymatic glycation of bovine insulin. The degree of insulin glycation was increased in both concentration- and time-dependent manner in relation to exposure to glucose, and the event was more pronounced for monoglycation reaction than that noticed for the diglycation of the hormone. Enzymatic digestion of insulin preparations with endoproteinase Glu C has revealed that each of the B 1-13 and B 22-30 peptide fragments of glycated insulin contains a site of binding of a single glucose molecule. Finally, attempt has been made in order to increase the sensitivity of the glycation assay through efficient enrichment of the glycated insulin on magnetic beads containing immobilized 3-aminophenylboronic acid (APBA) on their surface.     

Tyrosine residues modification studied by MALDI-TOF mass spectrometry

Amino acid residue-specific reactivity in proteins is of great current interest in structural biology as it provides information about solvent accessibility and reactivity of the residue and, consequently, about protein structure and possible interactions. In the work presented tyrosine residues of three model proteins with known spatial structure are modified with two tyrosine-specific reagents: tetranitromethane and iodine. Modified proteins were specifically digested by proteases and the mass of resulting peptide fragments was determined using matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry. Our results show that there are only small differences in the extent of tyrosine residues modification by tetranitromethane and iodine. However, data dealing with accessibility of reactive residues obtained by chemical modifications are not completely identical with those obtained by nuclear magnetic resonance and X-ray crystallography. These interesting discrepancies can be caused by local molecular dynamics and/or by specific chemical structure of the residues surrounding.     

High-resolution human papillomavirus genotyping by MALDI-TOF mass spectrometry

We describe a matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry (MS)-based assay for human papillomavirus (HPV) genotyping--the restriction fragment mass polymorphism (RFMP) assay, which is based on mass measurement of genotype-specific oligonucleotide fragments generated by TypeIIS restriction endonuclease cleavage after recognition sites have been introduced by PCR amplification. The use of a TypeIIS restriction enzyme makes the RFMP assay independent of sequence and applicable to a wide variety of HPV genotypes, because these enzymes have cleavage sites at a fixed distance from their recognition sites. After PCR amplification, samples are subjected to restriction enzyme digestion with FokI and BtsCI and desalting using Oasis purification plates, followed by analysis by MALDI-TOF MS. Overall, the protocol is simple, takes approximately 4-4.5 h and can accurately detect and identify at least 74 different HPV genotypes.     

Direct analysis of the kinetic profiles of organophosphate-acetylcholinesterase adducts by MALDI-TOF mass spectrometry

A sensitive matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry procedure has been established for the detection and quantitation of acetylcholinesterase (AChE) inhibition by organophosphate (OP) compounds. Tryptic digests of purified recombinant mouse AChE (mAChE) were fractionally inhibited by paraoxon to form diethyl phosphoryl enzyme. The tryptic peptide of mAChE that contains the active center serine residue resolves to a molecular mass of 4331.0 Da. Phosphorylation of the enzyme by paraoxon results in covalent modification of the active center serine and a corresponding increase in molecular mass of the tryptic peptide by 136 Da. The relative abundance of AChE peptides containing a modified active center serine strongly correlates with the fractional inhibition of the enzyme, achieving a detection range of phosphorylated to nonphosphorylated enzyme of 5-95%. Modifications of AChE by OP compounds resulting in dimethyl, diethyl, and diisopropyl phosphoryl adducts have been monitored with subpicomole amounts of enzyme. The individual phosphorylated adducts of AChE that result from loss of one alkyl group from the inhibited enzyme (the aging reaction) and the reappearance of unmodified AChE (spontaneous reactivation) have been resolved by the kinetic profiles and relative abundance of species. Further, the tryptic peptide containing the active center serine of AChE, isolated from mouse brain by anion-exchange and affinity chromatography, has been monitored by mass spectrometry. Native brain AChE, purified from mice treated with sublethal doses of metrifonate, has demonstrated that enzyme modifications resulting from OP exposure can be detected in a single mouse brain. For dimethyl phosphorylated AChE, OP exposure has been monitored by the ratio of tryptic peptide peaks that correspond to unmodified (uninhibited and/or reactivated), inhibited, and aged enzyme.     

Quantitative analysis of denatured collagen by collagenase digestion and subsequent MALDI-TOF mass spectrometry

Collagens are the most abundant proteins in vertebrate tissues and constitute significant moieties of the extracellular matrix (ECM). The determination of the collagen content is of relevance not only in the field of native tissue research, but also regarding the quality assessment of bioengineered tissues. Here, we describe a quantitative method to assess small amounts of collagen based on MALDI-TOF (matrix-assisted laser desorption/ionization time-of-flight) mass spectrometry (MS) subsequent to digestion of collagen with clostridial collagenase (clostridiopeptidase A) in order to obtain characteristic oligopeptides. Among the resulting peptides, Gly-Pro-Hyp, which is highly indicative of collagen, has been used to assess the amount of collagen by comparing the Gly-Pro-Hyp peak intensities with the intensities of a spiked tripeptide (Arg-Gly-Asp). The approach presented herein is both simple and convenient and allows the determination of collagen in microgram quantities. In tissue samples such as cartilage, the actual collagen content has additionally been determined for comparative purposes by nuclear magnetic resonance spectroscopy subsequent to acidic hydrolysis. Both methods give consistent data within an experimental error of ±10%. Although the differentiation of the different collagen types cannot be achieved by this approach, the overall collagen contents of tissues can be easily determined.     

Identification of Gram-positive anaerobic cocci by MALDI-TOF mass spectrometry

Gram-positive anaerobic cocci (GPAC) are part of the commensal microbiota of humans and are a phylogenetically heterogeneous group of organisms. To evaluate the suitability of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for the identification of GPAC, a database was constructed, using reference strains of commonly encountered GPAC and clinical isolates of which the sequence of the 16S rRNA gene was determined. Subsequently, the database was validated by identifying 107 clinical isolates of GPAC. Results were compared with the identifications obtained by 16S sequencing or fluorescent in situ hybridization (FISH). Strains belonging to the same species grouped together, in most cases, by MALDI-TOF MS analyses. Strains with sequence similarities less than 98% to their closest relatives, formed clusters distinct from recognized species in the MALDI-TOF MS dendrogram and, therefore could not be identified. These strains probably represent new species. Only three clinical isolates (2 strains of Finegoldia magna and 1 strain of Anaerococcus vaginalis) could not be identified. For all the other GPAC strains (96/107), reliable identifications were obtained. Therefore, we concluded that MALDI-TOF MS is an excellent tool for the identification of phylogenetically heterogeneous groups of micro-organisms such as GPAC.     

Delineation of Stenotrophomonas spp. by multi-locus sequence analysis and MALDI-TOF mass spectrometry

The genus Stenotrophomonas is genetically and phenotypically heterogeneous. Of the nine species now accepted, only S. maltophilia is of clinical importance. Based on DNA-sequences of seven house keeping genes, it encompasses genogroups of DNA-similarity below 97% that predominantly comprise strains of environmental origin. Therefore, in order to unravel the uneven distribution of environmental isolates within genogroups and reveal genetic relationships within the genus, there is need for an easy and reliable approach for the identification and delineation of Stenotrophomonas spp. In this first study, a multi-locus sequence analysis (MLSA) with seven housekeeping genes (atpD, gapA, guaA, mutM, nuoD, ppsA and recA) was applied for analysis of 21 S. maltophilia of environmental origin, Stenotrophomonas spp. and related genera. The genotypic findings were compared with the results of matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) analyses. Our MLSA provided reliable inter- and intra-species discrimination of all tested isolates that correlated with the MALDI-TOF mass spectrometry data. One distantly related genogroup of environmental S. maltophilia strains needs to be reclassified as S. rhizophila. However, there are still remaining delineated S. maltophilia genogroups of predominantly environmental origin. Our data provide further evidence that ‘Pseudomonas’ beteli is a heterotypic synonym of S. maltophilia. Based on MLSA and MALDI-TOF data, Stenotrophomonas sp. (DSM 2408) belongs to S. koreensis.     

Solid-supported enzymatic synthesis of pectic oligogalacturonides and their analysis by MALDI-TOF mass spectrometry

Solid-phase biosynthetic reactions, followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis (MALDI-TOF), was used to gain insight into the biosynthesis of pectin oligomers. Sepharose supports bearing long pectic oligogalacturonides (OGAs) anchored through a disulfide-containing cleavable linker, were prepared. The OGAs (degrees of polymerization of 13 and 14) were efficiently immobilized through the reducing end via formation of an oxime linkage. These OGA-derivatized matrices were subsequently employed in novel solid-phase enzymatic reactions, with the pectin biosynthetic enzyme, alpha-1,4-galacturonosyltransferase, GalAT (solubilized from Arabidopsis thaliana) and the glycosyl donor, uridine diphosphate-galacturonic acid (UDP-GalA). Solid-supported biosynthesis was followed by cleavage of the immobilized OGAs and direct analysis of the products released into the liquid phases by MALDI-TOF mass spectrometry. In time course studies conducted with an immobilized (alpha-D-GalA)14 and limiting amounts of the glycosyl donor, the predominant product was an OGA extended by one GalA residue at the non-reducing end (i.e., (GalA)15). When UDP-GalA was added in approximately excess compared to immobilized (GalA)13, OGAs up to the 16-mer were synthesized, confirming the non-processivity of the GalAT in vitro.     

Towards GAG glycomics: analysis of highly sulfated heparins by MALDI-TOF mass spectrometry

Glycomics is a developing field that provides structural information on complex populations of glycans isolated from tissues, cells and organs. Strategies employing matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) are central to glycomic analysis. Current MALDI-based glycomic strategies are capable of efficiently analyzing glycoprotein and glycosphingolipid glycomes but little attention has been paid to devising glycomic methodologies suited to the analysis of glycosaminoglycan (GAG) polysaccharides which pose special problems for MALDI analysis because of their high level of sulfation and large size. In this paper, we describe MALDI strategies that have been optimized for the analysis of highly sulfated GAG-derived oligosaccharides. A crystalline matrix norharmane, as well as an ionic liquid 1-methylimidazolium alpha-cyano-4-hydroxycinnamate (ImCHCA), have been used for the analysis of heparin di-, tetra-, hexa- and decasaccharides carrying from 2 to 13 sulfate groups. Information about the maximum number of sulfate groups is obtained using the ionic liquid whereas MALDI-TOF/TOF MS/MS experiments using norharmane allowed the determination of the nature of the glycosidic backbone, and more precise information about the presence and the position in the sequence of N-acetylated residues.     

Direct analysis of neuropeptides by in situ MALDI-TOF mass spectrometry in the rat brain

The measure of neuropeptides is an important tool in biology to better define endocrine and neuroendocrine function. Traditionally most methods have relied on the development of specific antibodies. Newer molecular methodologies have used measures of gene expression of neuropeptide precursors, such as Northern blot, PCR or in situ hybridization analysis. Matrix-assisted laser desorption/ionization (MALDI) mass analysis is a novel powerful technique for investigation of neuropeptides. Multiple peptides and peptide forms can be detected simultaneously and with great sensitivity in tissue extracts or partially purified samples. We have now adapted a MALDI methodology for the direct measurement of neuropeptides on fresh tissue sections of rat brains. We have validated the method by examining peptidergic mass profiles of the supraoptic nucleus (SON) and caudate putamen hypothalamic regions. Interestingly, mass profiles showed that vasopressin, which is specifically present in the SON, is modulated when animals are treated with lipopolysaccharides. MALDI-MS on brain slides is a novel complementary technique for neurobiologists and endocrinologists in order to investigate the dynamic and regional distribution of neuropeptides during physiological events.     

Lipid fingerprints of intact viruses by MALDI-TOF/mass spectrometry

A number of viruses contain lipid membranes, which are in close contact with capsid proteins and/or nucleic acids and have an important role in the viral infection process. In this study membrane lipids of intact viruses have been analysed by MALDI-TOF/MS with a novel methodology avoiding lipid extraction and separation steps. To validate the novel method, a wide screening of viral lipids has been performed analysing highly purified intact bacterial and archaeal viruses displaying different virion architectures. Lipid profiles reported here contain all lipids previously detected by mass spectrometry analyses of virus lipid extracts. Novel details on the membrane lipid composition of selected viruses have also been obtained. In addition we show that this technique allows the study of lipid distribution easily in subviral particles during virus fractionation. The possibility to reliably analyse minute amounts of intact viruses by mass spectrometry opens new perspectives in analytical and functional lipid studies on a wider range of viruses including pathogenic human ones, which are difficult to purify in large amounts.     

MALDI-TOF mass spectrometry: a versatile tool for high-performance DNA analysis

Matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) has developed during the past decade into a versatile tool for biopolymer analysis. The aim of this review is to summarize this development and outline the applications, which have been enabled for routine use in the field of nucleic acid analysis. These include the analysis of mutations, the resequencing of amplicons with a known reference sequence, and the quantitative analysis of gene expression and allelic frequencies in complex DNA mixtures.     

Analysis of four human microsporidian isolates by MALDI-TOF mass spectrometry

Spores of four species of microsporidia isolated from humans were analyzed by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) and specific biomarkers were found for each. The microsporidia analyzed included three species, Encephalitozoon cuniculi, Encephalitozoon hellem, and Encephalitozoon intestinalis and the fourth organism is the recently described Brachiola algerae. Whole spores, spore shells, and soluble fractions were applied directly to the MALDI target without further purification steps. MALDI-TOF MS analysis of both whole spores and soluble fractions of the four isolates revealed a group of unique, characteristic, and reproducible spectral markers in the mass range of 2,000-8,000 Da. Statistical analysis of the averaged centroided masses uncovered two distinct sets of unique peptides or biomarkers, one originated from whole spores and the other from soluble fractions, that can differentiate the four microsporidian species studied. MALDI-TOF MS analysis of whole organisms is a rapid, sensitive, and specific option to characterize microsporidian isolates and has the potential for several applications in parasitology.