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.     

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.     

High-throughput killer cell immunoglobulin-like receptor genotyping by MALDI-TOF mass spectrometry with discovery of novel alleles

The killer cell immunoglobulin-like receptors (KIR) interact with major histocompatibility complex (MHC) class I ligands to regulate the functions of natural killer cells and T cells. Like human leukocyte antigens class I, human KIR are highly variable and correlated with infection, autoimmunity, pregnancy syndromes, and transplantation outcome. Limiting the scope of KIR analysis is the low resolution, sensitivity, and speed of the established methods of KIR typing. In this study, we describe a first-generation single nucleotide polymorphism (SNP)-based method for typing the 17 human KIR genes and pseudogenes that uses analysis by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. It is a high-throughput method that requires minute amounts of genomic DNA for discrimination of KIR genes with some allelic resolution. A study of 233 individuals shows that the results obtained by the SNP-based KIR/MALDI-TOF method are consistent with those obtained with the established sequence-specific oligonucleotide probe or sequence-specific polymerase chain reaction methods. The added sensitivity of the KIR/MALDI-TOF method allowed putative novel alleles of the KIR2DL1, KIR3DL1, KIR2DS5, and KIR2DL5 genes to be identified. Sequencing the KIR2DL5 variant proved it was a newly discovered allele, one that appears associated with Hispanic and Native American populations. This KIR/MALDI-TOF method of KIR typing should facilitate population and disease-association studies that improve knowledge of the immunological functions of KIR-MHC class I interactions.     

SNP genotyping using alkali cleavage of RNA/DNA chimeras and MALDI time-of-flight mass spectrometry

Single nucleotide polymorphisms (SNPs) are now widely used for many DNA analysis applications such as linkage disequilibrium mapping, pharmacogenomics and traceability. Many methods for SNP genotyping exist with diverse strategies for allele-distinction. Mass spectrometers are used most commonly in conjunction with primer extension procedures with allele-specific termination. Here we present a novel concept for allele-preparation for SNP genotyping. Primer extension is carried out with an extension primer positioned immediately upstream of the SNP that is to be genotyped, a complete set of four ribonucleotides and a ribonucleotide incorporating DNA polymerase. The allele-extension products are then treated with alkali, which results in the cleavage immediately after the first added ribonucleotide. In addition, to obtain fragments easily detectable by mass spectrometry, we have included a ribonucleotide in the primer usually at the fourth nucleotide from the 3′ terminus. The method was tested on four SNPs each with a different combination of nucleotides. The advantage over other mass spectrometry-based SNP genotyping assays is that this one only requires a PCR, a primer extension reaction with a universal extension mix and an inexpensive facile cleavage reaction, which makes it overall very cost effective and easy in handling.     

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.     

Hierarchical high-throughput SNP genotyping of the human Y chromosome using MALDI-TOF mass spectrometry

We have established the use of a primer extension/mass spectrometry method (the PinPoint assay) for high-throughput SNP genotyping of the human Y chromosome. 118 markers were used to define 116 haplogroups and typing was organised in a hierarchical fashion. Twenty multiplex PCR/primer extension reactions were set up and each sample could be assigned to a haplogroup with only two to five of these multiplex analyses. A single aliquot of one enzyme was found to be sufficient for both PCR and primer extension. We observed 100% accuracy in blind validation tests. The technique thus provides a reliable, cost-effective and automated method for Y genotyping, and the advantages of using a hierarchical strategy can be applied to any DNA segment lacking recombination.     

Single-nucleotide polymorphism analysis by MALDI-TOF mass spectrometry

Single-nucleotide polymorphisms (SNPs) have great potential for use in genetic-mapping studies, which locate and characterize genes that are important in human disease and biological function. For SNPs to realize their full potential in genetic analysis, thousands of different SNP loci must be screened in a rapid, accurate and cost-effective manner. Matrix-assisted laser desorption-ionization-time-of-flight (MALDI-TOF) mass spectrometry is a promising tool for the high-throughput screening of SNPs, with future prospects for use in genetic analysis.     

DNA sequencing using biotinylated dideoxynucleotides and mass spectrometry

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MS) has been explored widely for DNA sequencing. The major requirement for this method is that the DNA sequencing fragments must be free from alkaline and alkaline earth salts as well as other contaminants for accurately measuring the masses of the DNA fragments. We report here the development of a novel MS DNA sequencing method that generates Sanger-sequencing fragments in one tube using biotinylated dideoxynucleotides. The DNA sequencing fragments that carry a biotin at the 3′-end are made free from salts and other components in the sequencing reaction by capture with streptavidin-coated magnetic beads. Only correctly terminated biotinylated DNA fragments are subsequently released and loaded onto a mass spectrometer to obtain accurate DNA sequencing data. Compared with gel electrophoresis-based sequencing systems, MS produces a very high resolution of DNA-sequencing fragments, fast separation on microsecond time scales, and completely eliminates the compressions associated with gel electrophoresis. The high resolution of MS allows accurate mutation and heterozygote detection. This optimized solid-phase DNA-sequencing chemistry plus future improvements in detector sensitivity for large DNA fragments in MS instrumentation will further improve MS for DNA sequencing.     

Panel of X-linked single-nucleotide polymorphic markers for DNA identification (XSNPid) based on multiplex genotyping by multilocus PCR and MALDI-TOF mass spectrometry

Human genetic markers linked with the X chromosome (X-linked) are used in the field of population and medical genetics, as well as for DNA identification of individuals in forensic science and forensic medicine. We proposed an XSNPid panel that consists of 66 unlinked single nucleotide X chromosome markers and developed a protocol for their multiplex genotyping using multilocus PCR and MALDI-TOF mass spectrometry. The XSNPid panel is genotyped within two multiplexes (36 and 30 markers). The developed protocol provides an efficient genotype reading; the fraction of determined genotypes is 98.29%. The high level of gene diversity (0.461) for the X-linked SNPs included in the panel is characteristic of the Russian population. A total of 63 out of 66 markers that provide a high efficiency of genotyping and independent inheritance are suitable for DNA identification purposes. The XSNPid panel is characterized by a very high discriminating ability when studying the Russian population. The probability of genotype coincidence in two unrelated individuals is 9 × 10^–27 for women and 2 × 10^–18 for men. Also, the XSNPid panel has a greater multiplex capacity in addition to a higher discriminating ability compared to the other closest analogues of the X chromosome SNP sets, which makes it more cost effective and less time consuming. The XSNPid panel is a convenient tool, not only for individual DNA identification, but also for population genetic studies.     

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.     

Direct N-terminal sequencing of polypeptides using a thermostable bacterial aminopeptidase and MALDI-TOF mass spectrometry

Mass spectrometry-based amino acid sequencing is currently based almost entirely on collision-induced peptide fragmentation and analyses. Here, we describe a single-stage MS-based technique for amino acid sequencing involving partial, heterogenous digestion of a peptide by a processive, non-specific, heat-loving Bacillus subtilis-derived aminopeptidase (BsuAP), which acts optimally at 70 °C and allows ‘single-shot’ sequencing to be carried out through simultaneous accumulation, and detection of sub-populations of peptides of progressively reducing length.     

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.     

Applications of mass spectrometry to DNA sequencing.

The ability of the mass spectrometer to analyze collectively the masses of DNA fragments that are produced in the Sanger procedure for sequencing may allow the gel electrophoresis step to be eliminated. On the other hand, if gel electrophoresis is required, the use of resonance ionization spectroscopy coupled to a mass spectrometer may enable much faster analysis of DNA bands labeled with stable isotopes. Other combinations of labeling of the DNA and its mass spectrometric analysis with or without gel electrophoresis are also considered. Recent advances in these areas of mass spectrometry are reviewed.     

Capture of assay template by multiplex PCR of long amplicons for genotyping SNPs and InDels with MALDI-TOF mass spectrometry

Mis-priming associated with uncharacterised single nucleotide polymorphisms (SNPs) may lead to failure of PCR for genotyping. This is particularly troublesome in high-throughput SNP genotyping applications relying on multiplex PCR (2–40-plex) generating many short amplicons (80–120 bp) of similar size, an approach best suited for whole genome scans. However, if the target SNPs are clustered within a few target genes one option to ameliorate this is to increase the amplicon length, effectively reducing the potential for primer/template interactions and mis-priming. We tested this approach in a diverse population of 372 Eucalyptus pilularis individuals (π = 8.11 × 10⁻³, H _e = 0.75) using a modified Sequenom iPLEX gold assay. Four candidate genes (MYB1, MYB2, CAD and CCR) were amplified in a single long range multiplex capture PCR generating 6 long amplicons ranging in size from 907 to 2,225 bp. This contrasts with the standard approach which would have required the amplification of 98 short amplicons in 4 multiplex reactions. These 6 long amplicons provided the assay template for 98 assays (87 SNP and 11 InDel) within the 4 candidate genes. Reaction results indicated that longer amplicons could provide a suitable template for genotyping assays, with 90.8% of assays functional and 84.3% of assays suitable for downstream analysis. Additional advantages of this approach were the capacity for troubleshooting using gel electrophoresis and savings of 94% in capture primer synthesis costs. This approach will have the greatest relevance for candidate gene approaches for association testing in uncharacterised populations of organisms with high sequence diversity.     

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.     

Multiplex SNP genotyping by MALDI-TOF mass spectrometry: Frequencies of 56 immune response gene SNPs in human populations

Single-nucleotide polymorphisms (SNPs) are the most common type of genetic polymorphisms. Despite the progress in sequencing and postgenomic technologies, targeted SNP genotyping continues to be in highest demand in the approach to human and medical genetics. In this work, we describe the application of multiple SNP genotyping by MALDI-TOF mass spectrometry for analysis of genetic diversity of immune response genes in human populations. It was shown that MALDI-TOF mass spectrometry is a rapid, accurate, and efficient method of medium-scale SNP genotyping. Allele frequencies of 56 SNPs in 41 genes implicated in the regulation of immune response were similar in four populations studied (Russians, Komi, Khanty, and Buryats). These populations had similar levels of genetic diversity and were clustered according to their geographic location. The cost efficiency of MALDI-TOF mass spectrometry was evaluated compared to real-time PCR technology.