Highly multiplexed genotyping of coronary artery disease-associated SNPs using MALDI-TOF mass spectrometry

Highly multiplexed genotyping methods are needed to support a comprehensive analysis of single nucleotide polymorphisms (SNPs) in coronary artery disease (CAD)-related genes. In this study we evaluated chip-based MALDI-TOF mass spectrometry for multiplexed genotyping of SNPs associated with CAD. Our analysis included 14 healthy Japanese individuals and 19 Japanese patients with myocardial infarction whose first attack occurred before age 50. We selected 29 candidate genes involved in 1) the renin-angiotensin system, 2) lipid metabolism, 3) cytokines and adhesion molecules, 4) growth factors, and 5) the coagulation-fibrinolysis system. Genotyping of candidate SNPs was performed by MALDI-TOF MS using a MassARRAY system, and 4-plex analysis was achieved at a maximum. All 39 SNPs determined by the fluorescent dye-terminator cycle sequencing method from four randomly selected patients were found to be in complete agreement with the results obtained from MassARRAY system. Significant differences were observed in the -1965delG of PAI1 (SERPINE1) with respect to allelic frequency, the G>A in the promoter region SNP in SM22 (TAGLN) for dominant genotype, and in two other SNPs (C>T in intron 1 of HGF, and -1965delG of PAI1) for recessive genotype. Three SNPs (803T>C of AGT, 677CT of MTHFR, 190T>C of ADRB3) showed weak differences in allelic frequency. MALDI-TOF-MS provided high performance with a multiplex assay design for analysis of CAD-related SNPs by increasing the throughput while maintaining a high level of accuracy.     

MALDI-TOF MS and TaqMan assisted SNP genotyping of DNA isolated from formalin-fixed and paraffin-embedded tissues (FFPET)

Formalin-fixed paraffin-embedded tissues (FFPET) from archived clinical samples provide an invaluable source for large-scale molecular genetic studies. Pharmacogenetic investigations that require long-term clinical follow-up data of patients may particularly benefit from FFPET analysis. Matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and TaqMan-based (Thermus aquaticus polymerase) methodologies have become standard genotyping procedures. However, no data are available on the applicability of MALDI-TOF MS to the genotyping of low quality DNA, as it is usually obtained from FFPET, and data from TaqMan genotyping are limited. We isolated constitutional DNA from 274 FFPET samples (229 patients with breast cancer and 45 patients with benign breast diseases) and genotyped 15 polymorphic loci in 10 genes. Nine SNPs were genotyped by MALDI-TOF MS, and six were genotyped by the TaqMan methodology. We established rates for successful allele assignment for all FFPET, for FFPET prepared prior to 1990, and for FFPET prepared post-1990. Both methodologies showed high success rates ranging between 70.9 and 99.6% (mean: 91.8%) for MALDI-TOF MS and between 82.3 and 97.7% (mean: 91.0%) for TaqMan genotyping. No significant differences in genotyping performances for FFPET prepared prior to 1990 or post-1990 were observed. With the exception of one, all other genotype frequencies were in Hardy-Weinberg equilibrium. Furthermore, genotype frequencies matched those observed in a German breast cancer population and other Caucasian populations. Our study shows for the first time that MALDI-TOF MS and TaqMan genotyping procedures provide reliable data, and are therefore applicable in studies that require large scale FFPET genotyping.     

Large-scale determination of SNP allele frequencies in DNA pools using MALDI-TOF mass spectrometry

One of the major challenges in the near future is the identification of genes that contribute to complex disorders. Large scale association studies that utilize a dense map of single nucleotide polymorphisms (SNPs) have been considered as a valuable tool for this purpose. However, genome-wide screens are limited by costs of genotyping thousands of SNPs in a large number of individuals. Here we present a pooling strategy that enables high-throughput SNP validation and determination of allele frequencies in case and control populations. Quantitative analysis of allele frequencies of SNPs in DNA pools is based on matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry of primer extension assays. We demonstrate the accuracy and reliability of this approach on pools of eight previously genotyped individuals with an allele frequency representation in the range of 0.1 to 0.9. The accuracy of measured allele frequencies was shown in DNA pools of 142 to 186 individuals using additional markers. Allele frequencies determined from the pooled samples deviate from the real frequencies by about 3%. The described method reduces costs and time and enables genotyping of up to thousands of samples by taking advantage of the high-throughput MALDI-TOF technology.     

Single-molecule analysis for molecular haplotyping

In the genome era, there is great hope that genetic approaches such as linkage equilibrium mapping can be used to study common human disorders using a case-control population association study design. Ideally, the parental chromosomes are marked so that chromosomal regions in the form of haplotypes are compared in these studies to increase the power of association. Determining the haplotypes in a diploid individual is a major technical challenge in genetic studies of complex traits. A molecular approach to haplotyping is therefore highly desirable. Recent advances in DNA preparation, separation, labeling, and image analysis provide hope that a strategy of using a three-dye system coupled with DNA distance measurements between alleles will yield haplotype information of sufficiently high quality for genetic studies. In this work, we present the outline of the major challenges one must meet in developing a robust strategy for SNP detection and molecular haplotyping using single molecule analysis.     

Novel Plexor SNP genotyping technology: comparisons with TaqMan and homogenous MassEXTEND MALDI-TOF mass spectrometry

Analysis of SNPs for association, linkage, haplotype, and pharmacogenetic studies has led to a dramatic increase in the number and evolution of medium- to high-throughput genotyping technologies. This study introduces Plexor as a new method for medium-throughput (single SNP) genotyping. We compare this fluorescent-based chemistry for call rate, accuracy, affordability, throughput, and overall efficiency against two commonly used technologies. These include fluorescent-based TaqMan allelic discrimination for single SNP analysis (medium-throughput) and the homogenous MassEXTEND (hME) chemistry using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry for multiple SNP analysis (high-throughput). Analysis of 11 SNPs, including all six possible nucleotide substitutions, showed Plexor to be highly comparable for both call rate (94.7%) and accuracy (99.2%) to the TaqMan (94.6% and 99.8%, respectively) and hME (91.9% and 98.1%, respectively) chemistries. We demonstrate that this novel method is an efficient, cost-effective alternative to TaqMan genotyping commonly used in diagnostic settings.     

Increased forensic efficiency of DNA fingerprints through simultaneous resolution of length and nucleotide variability by high-performance mass spectrometry

Short tandem repeat (STR) typing is the most powerful method for determining the origin of a sample for a number of molecular disciplines such as medical genetics, population genetics, tumor analysis, transplantation medicine, or forensic crime scene analysis. STR alleles are routinely differentiated based upon their fragment size by electrophoresis under denaturing conditions, which does not take nucleotide variability into consideration. This simplification leads to loss of biological information as the nature of the individual sequence motifs that build an STR is not described. An alternative detection platform would be mass spectrometry, which captures the underlying sequence variation by comparing the molecular masses of DNA fragments. Here, we demonstrate that the combination of ion-pair reversed-phase high-performance liquid chromatography and electrospray ionization quadrupole time-of-flight mass spectrometry (ICEMS) is able to simultaneously detect length and nucleotide variability in STRs. Overall, 21 forensically relevant STRs that are also used in other scientific fields were screened in an Austrian population sample for the occurrence of nucleotide variability within or close to the repeat region. A total of 11 of the investigated loci (SE33, D2S1338, vWA, D21S11, D3S1358, D16S539, D8S1179, D7S820, D13S317, D5S818, and D2S441) brought additional allele (sequence) variants. Forensic efficiency, as determined by typical statistical parameters, was significantly increased by 20 to 30%. The beauty of ICEMS-STR-analysis is the fact that it represents one of the few technological advancements that allows direct comparison of newly generated data with existing data such as stored in DNA databases, which have a retarding effect on new developments.     

Detection and assignment of TP53 mutations in tumor DNA using peptide mass signature genotyping

This report describes the application of a new approach to tumor genotyping called peptide mass signature genotyping (PMSG) that is particularly suited to detecting minority sequences in a DNA sample. Detecting minority sequences is essential for accurate tumor genotyping because tumor resections are generally a mixture of malignant and non-malignant cells, with the mutations of interest often outnumbered by the corresponding wild-type alleles. To explore the suitability of PMSG for tumor genotyping, 25 human squamous cell carcinomas of the head and neck, as well as a set of cell lines derived from those tumors, were analyzed for mutations in exons 5 to 8 of the TP53 gene, the exons that encode the DNA-binding domains of the p53 protein. PMSG identified mutations in 11 tumor DNA samples, whereas dideoxy sequencing of the same samples detected mutations in only four. Currently, PMSG can be used to detect mutations that are present in only 20% of the sample DNA, and we expect that this threshold will be lowered significantly as the PMSG process is improved. Hum Mutat 22:158-165, 2003.     

Mutation scanning by ion-pair reversed-phase high-performance liquid chromatography-electrospray ionization mass spectrometry (ICEMS)

Partially denaturing high-performance liquid chromatography has emerged as the most sensitive physical mutation scanning method. However, there are a few reports of mutations missed or only detectable at unique temperatures. The combined use of ion-pair reversed-phase high-performance liquid chromatography under completely denaturing conditions and electrospray ionization quadrupole ion trap mass spectrometry (ICEMS) obviates the need for selecting appropriate temperatures for resolving heteroduplices and allows the discrimination of different alleles even when they co-elute due to distinct mass differences between nucleobases. This was demonstrated for the detection of four mutations (259G>A, 286A>G, 300T>G, and 331+1G>A) in exon 5 and intron 5 of BRCA1, respectively. Current mass resolution of quadrupole ion trap mass spectrometers limits the identification of single A>T or T>A transversions with a mass difference of 9 Da to fragments <80 base pairs (bp). The presence of all other mutations can be detected in fragments up to approximately 105 bp. The approach may prove particularly useful in the mutational scanning of AT- or GC-rich sequences that are recalcitrant to most other methods.     

Polymorphism analysis within the HLA-A locus by universal oligonucleotide array

Human leukocyte antigen (HLA) class I genes present some of the most complex single nucleotide polymorphism (SNP) patterns in the human genome. HLA typing is therefore extremely challenging. In this article, we use the ligation detection reaction (LDR) combined with a universal array (UA) as a robust and efficient method to analyze SNPs within the HLA-A region that includes HLA-A alleles of interest for immunotherapy in tumor diseases. The LDR, combined with a UA platform, has been optimized for the detection of 27 alleles distributed within exons 2 and 3 of HLA-A. The assay involves the amplification by PCR of the HLA-A genomic region (1,900 bp), the cycled ligation reaction, followed by the capture of ligated products through hybridization onto a UA. Each slide was designed to allow the detection of up to eight samples in parallel. The PCR/LDR/UA HLA-A assay was evaluated by analyzing 62 individuals (31 homozygous and 31 heterozygous) previously typed by direct sequencing. We demonstrate that the microarray genotyping procedure described here is a robust and efficient method for unambiguous detection of HLA alleles. HLA genotyping by PCR/LDR/UA is in perfect agreement with typing obtained by direct sequencing. Our results clearly demonstrate that the combination of enzymatic processing (LDR) and a demultiplexing hybridization onto a UA is a robust tool for SNP discrimination within the highly polymorphic HLA region. We demonstrate the specificity and efficiency of such an approach, suggesting the feasibility of a PCR/LDR/UA low resolution HLA typing procedure.     

Multiplex MassARRAY spectrometry (iPLEX) produces a fast and economical test for 56 familial hypercholesterolaemia-causing mutations

Familial hypercholesterolaemia (FH) is a common single gene disorder, pre-disposing to cardiovascular disease, which is most commonly caused by mutations in the LDL-receptor (LDLR) gene. About 5% of patients carry the p.R3527Q (previously R3500Q) mutation in the apolipoprotein B (APOB) gene and 2% carry the p.D374Y mutation in the PCSK9 gene, but the lack of high-throughput methods make routine genetic diagnosis difficult. In this study, we developed an iPLEX MassARRAY Spectrometry mutation test to identify 56 mutations (54 in the LDLR gene, 1 in the APOB gene and 1 in the PCSK9 gene). The iPLEX test was verified by analysing 150 DNA samples from FH patients with a previously characterized mutation and 96 no-mutation control samples. Mutations were identified in all 150 FH mutation-positive samples using the iPLEX assay, with 96% directly called by the software. The false-positive rate in no-mutation control samples was 0.015%. The overall specific mutation assay failure rate was 2.1%. In the UK, this gives an average detection rate of 75%.The FH iPLEX test is not only designed for large-scale targeted population screening for FH mutations, such as lipid clinic patients, but can also be used for population screening. The assay can easily be developed further to include additional FH-causing mutations, thus increasing the sensitivity of the diagnostic assay.     

Use of MALDI-TOF mass spectrometry to analyze the molecular profile of Pseudomonas aeruginosa biofilms grown on glass and plastic surfaces

Biofilms are microbial sessile communities attached to surfaces that are known for causing many medical problems. A bacterial biofilm of clinical relevance is formed by the gram-negative bacteria Pseudomonas aeruginosa. During the formation of a biofilm, the initial adhesion of the cells is of crucial importance, and the characteristics of the contact surface have great influence on this step. In the present study, we aimed to use matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) profiling as a new methodology to monitor P. aeruginosa biofilm development. Biofilms were grown within polypropylene tubes containing a glass slide, and were harvested after 3, 5, 7, 9, or 12 days of inoculation. Planktonic cells were obtained separately by centrifugation as control. Two independent MALDI-TOF experiments were performed, one by collecting biofilms from both the glass slide and the polypropylene tube internal surface, and the other by acquiring biofilms from these surfaces separately. Scanning electron microscopy (SEM) and atomic force microscopy (AFM) were used to evaluate the morphological progression of the biofilm. The molecular results showed that MALDI profiling is able not only to distinguish between different biofilm stages, but it is also appropriate to indicate when the biofilm cells are released at the dispersion stage, which occurred first on polypropylene surface. Finally, the present study pointed out that MALDI profiling may emerge as a promising tool for the clinical diagnostic and prognostic workup of biofilms formation and control.     

An integrated microfluidic chip for the analysis of biochemical reactions by MALDI mass spectrometry

Using an integrated microfluidic chip combined with mass spectrometry is an attractive method for parallel and multiple analyses because of its inherent simplicity, low sample consumption, and high sensitivity. To realize an effective microfluidic chip for the rapid analysis of biochemical reactions by matrix assisted laser desorption/ionization (MALDI)–mass spectrometry (MS), the basic operations on microfluids, namely loading, metering, cutting, transporting, mixing, and injecting, must be integrated. This study describes an integrated microfluidic chip with MALDI–MS that performs the on-chip analysis of biochemical reactions, such as enzymatic reactions. For on-chip multiple reactions, we present sequential fluidic manipulations with nanoliter-sized droplets, based on the precise control of wettability and the capillary pressure of a microchannel. The microfluidic chip we have developed successfully performed biochemical reactions and can dispense a droplet of a few hundred nanoliters on the MALDI target plate according to the designed multiple reaction procedure. Finally, the MS spectrum showed accurate and clear characteristic peaks for reaction products. Our investigations into reaction efficiency showed that the microfluidic chip could reduce the reaction time to one third, and the volume to one hundredth, of off-chip methods using conventional labware such as the micropipette and Eppendorf tube.     

Pyrosequencing: an accurate detection platform for single nucleotide polymorphisms

Pyrosequencing, a non-electrophoretic method for DNA sequencing, is emerging as a popular platform for analysis of single nucleotide polymorphisms (SNPs). This technology has the advantage of accuracy, ease-of-use, and high flexibility for different applications. Here, we review the methodology and the use of this technique for SNP genotyping, SNP discovery, haplotyping, and allelic frequency studies. In addition, we describe new schemes for template preparation and multiplexing as an effort for cost reduction in large-scale studies.     

Serum Peptidome Profiling in Patients with Lung Cancer

Serum peptide profiling is a promising approach for classification of cancer versus noncancer samples. In this study, we aimed to search for discriminating peptide patterns in serum samples between lung cancer patients and healthy controls. The magnetic beads‐based weak cation‐exchange chromatography followed by matrix‐assisted laser desorption and ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) was used in this study to identify patients with lung cancer. In total, serum samples from 64 lung cancer patients (32 for training set and 32 for testing set), 64 healthy controls (32 for training set and 32 for testing set), and 10 COPD patients (for disease control) were analyzed in this study. The mass spectra data analyzed with ClinProTools software was used to distinguish between cancer patients and healthy individuals based on three different algorithm models (GA, SNN, and QC). In the training set, patients with lung cancer could be identified with the mean sensitivity of 98.9% and specificity of100%. Similar results could be obtained from testing set, showing 87% sensitivity and 84.8% specificity. Screening for serum peptide patterns using MALDI‐TOF MS showed high sensitivity and specificity in identifying patients with lung cancer. Anat Rec, 2010. © 2010 Wiley‐Liss, Inc.     

Features of TAP-independent MHC class I ligands revealed by quantitative mass spectrometry

TAP is responsible for transferring cytosolic peptides into the ER, where they can be loaded onto MHC molecules. Deletion of TAP results in a drastic reduction of MHC class I surface expression and alters the presented peptide pattern. This key molecule in antigen processing is tackled by several viruses and lost in some tumors, rendering the altered cells less vulnerable to T cell-based immune surveillance. Using the TAP-deficient cell line LCL721.174 and its TAP-expressing progenitor cell line LCL721.45, we identified and quantified more than 160 HLA ligands, 50 of which were presented TAP-independently. Peptides which were predominantly presented on the TAP-deficient LCL721.174 cell line had a decreased MHC binding affinity according to their SYFPEITHI and BIMAS score. About half of the identified TAP-independently presented peptides were not derived from signal sequences and may partly be generated by the proteasome. Furthermore, we have excluded the possibility that differences in HLA ligand presentation between LCL721.45 and LCL721.174 were due to varying expression of the source proteins or due to changes in the antigen loading complex. Features of peptides presented independently of TAP as well as proteasomal contribution to their generation provide an insight into basic immunological mechanisms.     

High-throughput multiplex SNP genotyping with MALDI-TOF mass spectrometry: practice, problems and promise

Single nucleotide polymorphisms (SNPs) are currently being identified and mapped at a remarkable pace, providing a rich genetic resource with vast potential for disease gene discovery, pharmacogenetics, and understanding the origins of modern humans. High-throughput, cost effective genotyping methods are essential in order to make the most advantageous and immediate use of these SNP data. We have incorporated the use of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) in our laboratory as a tool for differentiating genotypes based on the mass of the variant DNA sequence, and have utilized this method for production scale SNP genotyping. We have combined a 4 microl PCR amplification reaction using 3 ng of genomic DNA with a secondary enzymatic reaction (mini-sequencing) containing oligonucleotide primers that anneal immediately upstream of the polymorphic site, dideoxynucleotides, and a thermostable polymerase used to extend the PCR product by a single base pair. Mass spectrometry (MS) analysis of mini-sequencing reactions was performed using a MALDI-TOF instrument (Voyager-DE, Perseptive Biosystems, Framingham, MA). We performed both single and multiplex PCR and mini-sequencing reactions, and genotyped seven different variant sites in a random sample of 989 individuals. Genotypes generated with MS methods were compared with genotypes produced using a 5' exonuclease fluorescence-based assay (Taqman, Applied Biosystems, Foster City, CA) and a gel-based genotyping protocol. Because multiple polymorphisms can be detected in a single reaction, the MS technique provides a cost-effective and efficient method for high-throughput genotyping.     

Rapid detection of antibiotic resistance by MALDI-TOF mass spectrometry using a novel direct-on-target microdroplet growth assay

ObjectivesWe aimed to develop a universal phenotypic method, which allows easy and rapid antimicrobial susceptibility testing independently of underlying resistance mechanisms.MethodsWe established a novel direct-on-target microdroplet growth assay for the detection of antibiotic resistance within a few hours, which is based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The microorganisms were incubated with and without meropenem in nutrient broth as microdroplets directly on MALDI-TOF MS target. Subsequently, broth was separated from microbial cells by contacting the microdroplets with an absorptive material. The microorganisms grown in the presence of antibiotic were detected by MALDI-TOF MS. A total of 24 Klebsiella pneumoniae and 24 Pseudomonas aeruginosa isolates were used to assess performance for detection of meropenem resistance. The microdroplet volumes investigated were 2, 4, 6, 8 and 10 μL.ResultsThe best performance was achieved using 6-μL microdroplets. Applying this volume, all growth controls were successfully detected (definition of valid test), and all isolates were correctly categorized as susceptible or non-susceptible after an 18-h incubation. For K. pneumoniae, rate of valid tests, sensitivity and specificity all reached 100% after a 4-h incubation of 6-μL microdroplets. Using the same microdroplet volume for P. aeruginosa, incubation for 5 h resulted in 83.3% of valid tests with 100% sensitivity and 100% specificity.ConclusionsWe demonstrated easy, rapid and accurate resistance detection using carbapenem-resistant Gram-negative bacteria as an example. Our technology is suitable for automatization and expandable to further applications, e.g. simultaneous testing of multiple antibiotics as well as resistance determination directly from clinical samples.     

Solution-phase DNA mutation scanning and SNP genotyping by nanoliter melting analysis

Solution-phase, DNA melting analysis for heterozygote scanning and single nucleotide polymorphism (SNP) genotyping was performed in 10 nl volumes on a custom microchip. Human genomic DNA was PCR amplified in the presence of the saturating fluorescent dye, LCGreen Plus, and placed within microfluidic channels that were created between two glass slides. The microchip was heated at 0.1 degrees C/s with a Peltier device and viewed with an inverted fluorescence microscope modified for photomulitiplier tube detection. The melting data was normalized and the negative first derivative plotted against temperature. Mutation scanning for heterozygotes was easily performed by comparing the shape of the melting curve to homozygous standards. Genotyping of homozygotes by melting temperature (T(m)) required absolute temperature comparisons. Mutation scanning of ATM exon 17 and CFTR exon 10 identified single base change heterozygotes in 84 and 201 base-pair (bp) products, respectively. All genotypes at HFE C282Y were distinguished by simple melting analysis of a 40-bp fragment. Sequential analysis of the same sample on the gold-standard, commercial high-resolution melting instrument HR-1, followed by melting in a 10 nl reaction chamber, produced similar results. DNA melting analysis requires only minutes after PCR and is a simple method for genotyping and scanning that can be reduced to nanoliter volumes. Microscale systems for performing DNA melting reduce the reagents/DNA template required with a promise for high throughput analysis in a closed chamber without risk of contamination.     

Analysis by mass spectrometry of 100 cystic fibrosis gene mutations in 92 patients with congenital bilateral absence of the vas deferens

BACKGROUND: Limited mutation analysis for congenital bilateral absence of the vas deferens (CBAVD) has revealed only a minority of men in whom two distinct mutations were detected. We aimed to determine whether a more extensive mutation analysis would be of benefit in genetic counselling and prenatal diagnosis. METHODS: We studied a cohort of 92 men with CBAVD using mass spectrometry and primer oligonucleotide base extension to analyse an approximately hierarchical set of the most common 100 CF mutations. RESULTS: Analysis of 100 CF mutations identified 33/92 (35.9%) patients with two mutations and 29/92 (31.5%) with one mutation, compound heterozygosity accounting for 94% (31/33) of those with two mutations. This panel detected 12.0% more CBAVD men with at least one mutation and identified a second mutation in >50% of those considered to be heterozygotes under the two routine 25 mutation panel analyses. CONCLUSION: Compound heterozygosity of severe/mild mutations accounted for the vast majority of the CBAVD patients with two mutations, and underscores the value of a more extensive CF mutation panel for men with CBAVD. The CF100 panel enables higher carrier detection rates especially for men with CBAVD, their partners, partners of known CF carriers, and those with 'mild' CF with rarer mutations.