Solid phase capturable dideoxynucleotides for multiplex genotyping using mass spectrometry

We report an approach using solid phase capturable biotinylated dideoxynucleotides (biotin-ddNTPs) in single base extension for multiplex genotyping by mass spectrometry (MS). In this method, oligonucleotide primers that have different molecular weights and that are specific to the polymorphic sites in the DNA template are extended with biotin-ddNTPs by DNA polymerase to generate 3′-biotinylated DNA products. These products are then captured by streptavidin-coated solid phase magnetic beads, while the unextended primers and other components in the reaction are washed away. The pure extension DNA products are subsequently released from the solid phase and analyzed by matrix-assisted laser desorption/ionization time-of-flight MS. The mass of the extension products is determined using a stable oligonucleotide as a common internal mass standard. Since only the pure extension DNA products are introduced to the MS for analysis, the resulting mass spectrum is free of non-extended primer peaks and their associated dimers, which increases the accuracy and scope of multiplexing in single nucleotide polymorphism (SNP) analysis. The solid phase purification approach also facilitates desalting of the captured oligonucleotides, which is essential for accurate mass measurement by MS. We selected four biotin-ddNTPs with distinct molecular weights to generate extension products that have a 2-fold increase in mass difference compared to that with conventional ddNTPs. This increase in mass difference provides improved resolution and accuracy in detecting heterozygotes in the mass spectrum. Using this method, we simultaneously distinguished six nucleotide variations on synthetic DNA templates mimicking mutations in the p53 gene and two disease-associated SNPs in the human hereditary hemochromatosis gene.     

Surface-assisted laser desorption/ionization mass spectrometry

Laser desorption/ionization mass spectrometry (MS) is rapidly growing in popularity as an analytical characterization method in several fields. The technique shot to prominence using matrix-assisted desorption/ionization for large biomolecules (>700 Da), such as proteins, peptides and nucleic acids. However, because the matrix, which consists of small organic molecules, is also ionized, the technique is of limited use in the low-molecular-mass range (<700 Da). Recent advances in surface science have facilitated the development of matrix-free laser desorption/ionization MS approaches, which are referred to here as surface-assisted laser desorption/ionization (SALDI) MS. In contrast to traditional matrix-assisted techniques, the materials used for SALDI-MS are not ionized, which expands the usefulness of this technique to small-molecule analyses. This review discusses the current status of SALDI-MS as a standard analytical technique, with an emphasis on potential applications in proteomics.     

Surface-assisted laser desorption/ionization mass spectrometry

Laser desorption/ionization mass spectrometry (MS) is rapidly growing in popularity as an analytical characterization method in several fields. The technique shot to prominence using matrix-assisted desorption/ionization for large biomolecules (>700 Da), such as proteins, peptides and nucleic acids. However, because the matrix, which consists of small organic molecules, is also ionized, the technique is of limited use in the low-molecular-mass range (<700 Da). Recent advances in surface science have facilitated the development of matrix-free laser desorption/ionization MS approaches, which are referred to here as surface-assisted laser desorption/ionization (SALDI) MS. In contrast to traditional matrix-assisted techniques, the materials used for SALDI-MS are not ionized, which expands the usefulness of this technique to small-molecule analyses. This review discusses the current status of SALDI-MS as a standard analytical technique, with an emphasis on potential applications in proteomics.     

Optimizing sequence coverage for a moderate mass protein in nano-electrospray ionization quadrupole time-of-flight mass spectrometry

Sample pretreatment was optimized to obtain high sequence coverage for human serum albumin (HSA, 66.5 kDa) when using nano-electrospray ionization quadrupole time-of-flight mass spectrometry (nESI–Q-TOF–MS). Use of the final method with trypsin, Lys-C, and Glu-C digests gave a combined coverage of 98.8%. The addition of peptide fractionation resulted in 99.7% coverage. These results were comparable to those obtained previously with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI–TOF–MS). The sample pretreatment/nESI–Q-TOF–MS method was also used with collision-induced dissociation to analyze HSA digests and to identify peptides that could be employed as internal mass calibrants in future studies of modifications to HSA.     

The essence of DNA sample preparation for MALDI mass spectrometry

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry has become an important analytical technique in nucleic acid research. MALDI is used for quality control of oligonucleotides as well as for analyzing DNA markers. Sample preparation of nucleic acids is crucial for obtaining high-quality mass spectra. Sample purity, solvent content, suitable matrices, and substrate surfaces, as well as laboratory conditions affect spectra quality. This review presents essential information with regard to sample preparation, DNA modification chemistry, and DNA purification, along with a discussion of instrumental advances, which facilitate and extend the applicability of MALDI in genomics.     

The essence of DNA sample preparation for MALDI mass spectrometry

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry has become an important analytical technique in nucleic acid research. MALDI is used for quality control of oligonucleotides as well as for analyzing DNA markers. Sample preparation of nucleic acids is crucial for obtaining high-quality mass spectra. Sample purity, solvent content, suitable matrices, and substrate surfaces, as well as laboratory conditions affect spectra quality. This review presents essential information with regard to sample preparation, DNA modification chemistry, and DNA purification, along with a discussion of instrumental advances, which facilitate and extend the applicability of MALDI in genomics.     

Sample preparation for sequencing hits from one-bead-one-peptide combinatorial libraries by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Optimization of bead analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) after the screening of one-bead-one-peptide combinatorial libraries was achieved, involving the fine-tuning of the whole process. Guanidine was replaced by acetonitrile (MeCN)/acetic acid (AcOH)/water (H₂O), improving matrix crystallization. Peptide-bead cleavage with NH₄OH was cheaper and safer than, yet as efficient as, NH₃/tetrahydrofuran (THF). Peptide elution in microtubes instead of placing the beads in the sample plate yielded more sample aliquots. Successive dry layers deposit sample preparation was better than the dried droplet method. Among the matrices analyzed, α-cyano-4-hydroxycinnamic acid resulted in the best peptide ion yield. Cluster formation was minimized by the addition of additives to the matrix.     

DNA sequence analysis by hybridization with oligonucleotide microchips: MALDI mass spectrometry identification of 5mers contiguously stacked to microchip oligonucleotides

Matrix-assisted laser desorption ionization mass spectrometry (MALDI MS) has been applied to increase the informational output from DNA sequence analysis. It has been used to analyze DNA by hybridization with microarrays of gel-immobilized oligonucleotides extended with stacked 5mers. In model experiments, a 28 nt long DNA fragment was hybridized with 10 immobilized, overlapping 8mers. Then, in a second round of hybridization DNA–8mer duplexes were hybridized with a mixture of 10 5mers. The stability of the 5mer complex with DNA was increased to raise the melting temperature of the duplex by 10–15°C as a result of stacking interaction with 8mers. Contiguous 13 bp duplexes containing an internal break were formed. MALDI MS identified one or, in some cases, two 5mers contiguously stacked to each DNA–8mer duplex formed on the microchip. Incorporating a mass label into 5mers optimized MALDI MS monitoring. This procedure enabled us to reconstitute the sequence of a model DNA fragment and identify polymorphic nucleotides. The application of MALDI MS identification of contiguously stacked 5mers to increase the length of DNA for sequence analysis is discussed.     

Mineral oil-, glycerol-, and Vaseline-coated plates as matrix-assisted laser desorption/ionization sample supports for high-throughput peptide analysis

A novel protocol for rapid and high-quality sample preparation prior to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been developed by coating bare stainless steel plates with one of three adhesives: mineral oil, glycerol, or Vaseline. The advantages of these three adhesive coats are that they take little time to both prepare and wipe away, hold the matrices to prevent them from flying from the support, reduce the background matrix, and affect neither the resolution of the peptide peaks nor the accuracy of their determined molecular masses. Consequently, the signal intensity, detection limit, and tolerance of the analytes to contaminants on the three adhesive-coated plates are improved. In the two strategies of on-plate desalting and concentration of the peptide mixture, all three adhesives reduced the loss of peptides, especially in the case of larger molecular mass peptides. The microscope and stereomicroscope images of the deposited droplets showed that after dropping onto the adhesive coats, the droplets formed a reduced spot size, were more homogeneous, and showed sticky crystallization. Therefore, this is an easy-to-use, reproducible, highly sensitive, tolerant (to salts), and high-throughput method of peptide sample preparation for MALDI-TOF MS 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.     

A MALDI TOF MS-based minisequencing method for rapid detection of TEM-type extended-spectrum beta-lactamases in clinical strains of Enterobacteriaceae

A minisequencing method based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS) was developed for rapid identification of single nucleotide polymorphisms at bla(TEM) gene codons 104, 164 and 238 associated with extended-spectrum activity on TEM-type beta-lactamases. The method was validated by testing the Escherichia coli and Klebsiella pneumoniae strains possessing the known bla(TEM) gene sequences.     

An improved method for the analysis of Cryptosporidium parvum oocysts by matrix-assisted laser desorption/ionization time of flight mass spectrometry

Cryptosporidium parvum oocysts were analyzed using matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). Sample preparation proved to be a crucial step in the acquisition of acceptable mass spectra. Oocysts of C. parvum and the matrix were mixed and held for at least 45 min to produce reproducible, representative mass spectra. Sporozoites were also excysted from oocysts, purified, and analyzed using MALDI-TOF MS. The mass spectra of the intact oocysts contained many of the same peaks found in the mass spectra of the sporozoites, suggesting that during analysis, the internal constituents, not just the oocyst wall, are ablated by the laser.     

The use of mass spectrometry in genomics

Recent advances in the development of electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) now permit the near routine analysis of oligonucleotides and intact nucleic acids. These developments have led to the use of mass spectrometry (MS) as a detection platform for genomics studies. Among the various uses of mass spectrometry in genomics, applications focused on the characterization of single nucleotide polymorphisms (SNPs) and short tandem repeats (STRs) are particularly well-suited to MALDI or ESI-based analysis. It is predicted that continued developments in methodology and instrumentation will further improve the capabilities of mass spectrometry for nucleic acid analysis.     

Facile method for automated genotyping of single nucleotide polymorphisms by mass spectrometry

In the future, analysis of single nucleotide polymorphisms (SNPs) should become a powerful tool for many genetic applications in areas such as association studies, pharmacogenetics and traceability in the agro-alimentary sector. A number of technologies have been developed for high-throughput genotyping of SNPs. Here we present the simplified GOOD assay for SNP genotyping by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI). The simplified GOOD assay is a single-tube, purification-free, three-step procedure consisting of PCR, primer extension and phosphodiesterase II digestion followed by mass spectrometric analysis. Due to the application of charge-tag technology, no sample purification is required prior to the otherwise very impurity-sensitive MALDI analysis. The use of methylphosphonate containing primers and ddNTPs or α-S-ddNTPs together with a novel DNA polymerase derived from Thermotoga maritima for primer extension allow the fluent preparation of negatively charge-tagged, allele-specific products. A key feature of this polymerase is its preference for ddNTPs and α-S-ddNTPs over dNTPs. The simplified GOOD assay was run with automatic liquid handling at the lowest manageable volumes, automatic data acquisition and interpretation. We applied this novel procedure to genotyping SNPs of candidate genes for hypertension and cardiovascular disease.     

Straightforward detection of SNPs in double-stranded DNA by using exonuclease III/nuclease S1/PNA system

Single-nucleotide polymorphisms (SNPs) in double-stranded DNA (dsDNA) have been straightforwardly genotyped by matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry (MALDI-TOF MS). Peptide nucleic acid (PNA), a DNA analog, was used as a probe molecule. In its presence, genomic dsDNA was first treated with exonuclease III and then with nuclease S1. By these one-pot reactions, single-stranded DNA fragments including the SNP sites were formed in situ. These fragments were directly analyzed by MALDI-TOF MS, and the identity of the DNA base at the SNP site was determined in terms of mass number. By using two or more PNA probes simultaneously, multiplex analysis was also successful. Various genotypes of apolipoprotein E gene (ε2/ε2, ε3/ε3, ε4/ε4, ε2/ε3 and ε3/ε4) were identified from dsDNA obtained by PCR from corresponding patients.     

Identification of proteins from two-dimensional polyacrylamide gels using a novel acid-labile surfactant

Protein identification by peptide mass mapping usually involves digestion of gel-separated proteins with trypsin, followed by mass measurement of the resulting peptides by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Positive identification requires measurement of enough peptide masses to obtain a definitive match with sequence information recorded in protein or DNA sequence databases. However, competitive binding and ionization of residual surfactant introduced during polyacrylamide gel electrophoresis (PAGE) can inhibit solid-phase extraction and MS analysis of tryptic peptides. We have evaluated a novel, acid-labile surfactant (ALS) as an alternative to sodium dodecylsulfate (SDS) for two-dimensional (2-D) PAGE separation and MALDI-MS mapping of proteins. ALS was substituted for SDS at the same concentration in buffers and gels used for 2-D PAGE. Manual and automated procedures for spot cutting and in-gel digestion were used to process Coomassie stained proteins for MS analysis. Results indicate that substituting ALS for SDS during PAGE can significantly increase the number of peptides detected by MALDI-MS, especially for proteins of relatively low abundance. This effect is attributed to decomposition of ALS under acidic conditions during gel staining, destaining, peptide extraction and MS sample preparation. Automated excision and digestion procedures reduce contamination by keratin and other impurities, further enhancing MS identification of gel separated proteins.     

Analysis of the Proteinaceous Components of the Organic Matrix of Calcitic Sclerites from the Soft Coral Sinularia sp.

An organic matrix consisting of a protein-polysaccharide complex is generally accepted as an important medium for the calcification process. While the role this “calcified organic matrix” plays in the calcification process has long been appreciated, the complex mixture of proteins that is induced and assembled during the mineral phase of calcification remains uncharacterized in many organisms. Thus, we investigated organic matrices from the calcitic sclerites of a soft coral, Sinularia sp., and used a proteomic approach to identify the functional matrix proteins that might be involved in the biocalcification process. We purified eight organic matrix proteins and performed in-gel digestion using trypsin. The tryptic peptides were separated by nano-liquid chromatography (nano-LC) and analyzed by tandem mass spectrometry (MS/MS) using a matrix-assisted laser desorption/ionization (MALDI) – time-of-flight-time-of-flight (TOF-TOF) mass spectrometer. Periodic acid Schiff staining of an SDS-PAGE gel indicated that four proteins were glycosylated. We identified several proteins, including a form of actin, from which we identified a total of 183 potential peptides. Our findings suggest that many of those peptides may contribute to biocalcification in soft corals.     

Biomarker discovery for kidney diseases by mass spectrometry

By the development of soft ionization such as matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI), mass spectrometry (MS) has become an indispensable technique to analyze proteins. The combination of protein separation and identification such as two-dimensional gel electrophoresis and MS, surface-enhanced laser desorption/ionization-MS, liquid chromatography/MS, and capillary electrophoresis/MS has been successfully applied for proteome analysis of urine and plasma to discover biomarkers of kidney diseases. Some urinary proteins and their proteolytic fragments have been identified as biomarker candidates for kidney diseases. This article reviews recent advances in the application of proteomics using MS to discover biomarkers for kidney diseases.