Improved adapter-ligation-mediated allele-specific amplification for multiplex genotyping by using software

Adapter-ligation-mediated allele-specific amplification (ALM-ASA) is a potential method for multiplex SNPs typing at an ultra low cost. Here, we describe a kind of software, which designs allele-specific primers for ALM-ASA assay on multiplex SNPs. DNA sequences containing SNPs of interest are submitted into the software which contains various endonucleases for options. Based on the SNP sequence information and the selected endonucleases, the software is capable of automatically generating sets of information needed to perform genotyping experiments. Each set contains a suitable endonuclease, qualified allele-specific primers with orientations and melting temperatures, sizes of allele-specific amplicons, and gel electropherograms simulated according to the sizes of the allele-specific amplicons and the mobility of DNA fragments in 2% agarose gel. Seven SNPs in the arylamines N-acetyltransferase 2 (NAT2) gene, five SNPs in the BRCA1 gene, five SNPs in the COMT gene, six SNPs in the CYP2E1 gene, five SNPs in the MPO gene, and six SNPs in the NRG1 gene were selected for evaluating the software. Without extra optimization, seven SNPs in the NAT2 gene were successfully genotyped for genomic DNA samples from 127 individuals by using the first set of allele-specific primers yielded by the software. Although several steps are used in the ALM-ASA assay, the whole genotyping process can be completed within 3 h by optimizing each step. Profiting from the software, the ALM-ASA assay is easy-to-perform, labor-saving, and accurate.     

A novel technique for detecting single nucleotide polymorphisms by analyzing consumed allele-specific primers

We present a simple and rapid polymerase chain reaction (PCR)-based technique, termed consumed allele-specific primer analysis (CASPA), as a new strategy for single nucleotide polymorphism (SNP) analysis. The method involves the use of labeled allele-specific primers, differing in length, with several noncomplementary nucleotides added in the 5'-terminal region. After PCR amplification, the amounts of the remaining primers not incorporated into the PCR products are determined. Thus, nucleotide substitutions are identified by measuring the consumption of primers. In this study, the CASPA method was successfully applied to ABO genotyping. In the present method, the allele-specific primer only anneals with the target polymorphic site on the DNA, so it is not necessary to analyze the PCR products. Therefore, this method is only little affected by modification of the PCR products. The CASPA method is expected to be a useful tool for typing of SNPs.     

Matrix-assisted laser desorption/ionization detection of polymerase chain reaction products by utilizing the 5'-3' exonuclease activity of Thermus aquaticus DNA polymerase

The 5'-3' exonuclease activity of DNA polymerase was utilized in the polymerase chain reaction system to generate a specific signal concomitant with amplification. These signals were detected by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). This method obviates the need to perform extensive DNA purification of reaction products that is often necessary for detecting larger DNA molecules by mass spectrometry. Oligonucleotides complementary to the internal region of the amplicon are degraded by the 5'-3' exonuclease activity and the degradation products are analyzed by MALDI mass spectrometry. We refer to this assay as the Exo-taq assay or probe degradation assay. This method should be amenable to automation.     

Analysis of carrageenans by matrix-assisted laser desorption/ionization and electrospray ionization mass spectrometry. I. kappa-Carrageenans.

Matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) mass spectra of small kappa-carrageenans are reported and discussed. MALDI spectra can be obtained in both positive and negative ion mode. In the absence of extraneous metal ions, positive ions are formed by the attachment of one Na(+) ion to the carrageenan, whereas for negative ions one Na(+) ion is detached from the sulfate group. Multiply charged species are not observed in MALDI. Intense ESI spectra can be obtained in negative ion mode and now multiply charged species are seen. Alkali exchange experiments show that in these small carrageenan anions one, but only one, alkali metal ion is bound in a bidentate coordination with two ionic sulfate groups. G2-type ab initio calculations on model ions HO(-) [M(+)] (-)OH (M = Li, Na, K, Cs), as well as arguments based on a simple Coulombic interaction model, show that the bidentate stabilization energy drops rapidly as the size of the alkali cation increases. Exchange of Na(+) with Li(+) leads to expulsion of the Na(+) ion generating, in ESI, intense multiply charged anions. An attempt is made to rationalize this behavior in terms of hydration effects.     

微流控芯片电泳-多重聚合酶链反应法同时测定多个单碱基多态性位点

以微流控芯片电泳为检测平台,建立了多重PCR扩增法同时测定多个单碱基多态性(SNP)位点的方法。先通过PCR扩增得一段含所有待测SNP位点的长片段;用限制性内切酶消化成短片段,再将酶切反应产物与脱氧核糖核酸适配器(DNAadapter)相连;以连接产物为模板,分成两管,分别用n条等位基因特异性引物和一条通用引物进行n重PCR扩增;最后用微流控芯片电泳法分离PCR扩增产物,根据两管扩增产物的芯片电泳图谱中扩增片段的大小判断SNP的类型。以细胞色素P4502D6(CYP2D6)基因中的5个SNP位点(100C>T、1661G>C、1758G>T、2470T>C和2850C>T)为检测对象,考察了各等位基因特异性引物之间的相互影响和扩增反应的特异性,采用微流控芯片电泳法成功测定了20名健康中国人的CYP2D6基因中5个SNP位点的基因多态性,与聚合酶链反应-限制性片段长度多态性法(PCR-RFLP)测定结果完全一致。     

Negative ion production from peptides and proteins by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry

Negative ion production from peptides and proteins was investigated by matrix‐assisted laser desorption/ionization time‐of‐flight (MALDI‐TOF) mass spectrometry. Although most research on peptide and protein identification with ionization by MALDI has involved the detection of positive ions, for some acidic peptides protonated molecules are not easily formed because the side chains of acidic residues are more likely to lose a proton and form a deprotonated species. After investigating more than 30 peptides and proteins in both positive and negative ion modes, [M–H]⁻ ions were detected in the negative ion mode for all peptides and proteins although the matrix used was 2,5‐dihydroxybenzoic acid (DHB), which is a good proton donor and favors the positive ion mode production of [M+H]⁺ ions. Even for highly basic peptides without an acidic site, such as myosin kinase inhibiting peptide and substance P, good negative ion signals were observed. Conversely, gastrin I (1‐14), a peptide without a highly basic site, will form positive ions. In addition, spectra obtained in the negative ion mode are usually cleaner due to absence of alkali metal adducts. This can be useful during precursor ion isolation for MS/MS studies. Copyright © 2008 John Wiley & Sons, Ltd.     

Method development for the analysis of resinous materials with MALDI‐FT‐ICR‐MS: novel internal standards and a new matrix material for negative ion mode

Matrix‐assisted laser desorption/ionization (MALDI) is a mass spectrometry (MS) ionization technique suitable for a wide variety of sample types including highly complex ones such as natural resinous materials. Coupled with Fourier transform ion cyclotron resonance (FT‐ICR) mass analyser, which provides mass spectra with high resolution and accuracy, the method gives a wealth of information about the composition of the sample. One of the key aspects in MALDI‐MS is the right choice of matrix compound. We have previously demonstrated that 2,5‐dihydroxybenzoic acid is suitable for the positive ion mode analysis of resinous samples. However, 2,5‐dihydroxybenzoic acid was found to be unsuitable for the analysis of these samples in the negative ion mode. The second problem addressed was the limited choice of calibration standards offering a flexible selection of m/z values under m/z 1000. This study presents a modified MALDI‐FT‐ICR‐MS method for the analysis of resinous materials, which incorporates a novel matrix compound, 2‐aminoacridine for the negative ion mode analysis and extends the selection of internal standards with m/z <1000 for both positive (15 different phosphazenium cations) and negative (anions of four fluorine‐rich sulpho‐compounds) ion mode. The novel internal calibration compounds and matrix material were tested for the analysis of various natural resins and real‐life varnish samples taken from cultural heritage objects. Copyright © 2017 John Wiley & Sons, Ltd.     

Analysis of various organic and organometallic compounds using nanostructure-assisted laser desorption/ionization time-of-flight mass spectrometry (NALDI-TOFMS)

Nanostructure-assisted laser desorption/ionization time-of-flight mass spectrometry (NALDI-TOFMS) has been developed recently as a matrix-free/surface-assisted alternative to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). The NALDI surface of silicon nanowires is already very effective for the analysis of small to medium sized, polar organic molecules in positive ion mode. The current study examined this technology for the analysis of several nonpolar organic, organometallic, and ionic compounds in positive ion mode, as well as a fluorinated compound and various acids in negative ion mode. NALDI data are compared and contrasted with MALDI data for the same compounds, and the higher sensitivity of NALDI is highlighted by the successful characterization of two porphyrins for a sample amount of 10 amol per spot.     

MALDI-MS/MS with Traveling Wave Ion Mobility for the Structural Analysis of N-Linked Glycans

The preference for singly charged ion formation by MALDI makes it a better choice than electrospray ionization for profiling mixtures of N-glycans. For structural analysis, fragmentation of negative ions often yields more informative spectra than fragmentation of positive ones but such ions are more difficult to produce from neutral glycans under MALDI conditions. This work investigates conditions for the formation of both positive and negative ions by MALDI from N-linked glycans released from glycoproteins and their subsequent MS/MS and ion mobility behaviour. 2,4,6-Trihydroxyacetophenone (THAP) doped with ammonium nitrate was found to give optimal ion yields in negative ion mode. Ammonium chloride or phosphate also yielded prominent adducts but anionic carbohydrates such as sulfated N-glycans tended to ionize preferentially. Carbohydrates adducted with all three adducts (phosphate, chloride, and nitrate) produced good negative ion CID spectra but those adducted with iodide and sulfate did not yield fragment ions although they gave stronger signals. Fragmentation paralleled that seen following electrospray ionization providing superior spectra than could be obtained by PSD on MALDI-TOF instruments or with ion traps. In addition, ion mobility drift times of the adducted glycans and the ability of this technique to separate isomers also mirrored those obtained following ESI sample introduction. Ion mobility also allowed profiles to be obtained from samples whose MALDI spectra showed no evidence of such ions allowing the technique to be used in conditions where sample amounts were limiting. The method was applied to N-glycans released from the recombinant human immunodeficiency virus glycoprotein, gp120.     

Evaluation of pyridoindoles, pyridylindoles and pyridylpyridoindoles as matrices for ultraviolet matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

In an effort to gain an understanding of the processes governing ultraviolet matrix-assisted laser desorption/ionization (UV-MALDI), direct comparison was made of the mass spectra of proteins, carbohydrates and synthetic polymers (polyethylene glycol, polyester and polyamide) by using pyridylindoles, pyridoindoles and pyridylpyridoindoles as UV (337 nm)-MALDI-TOFMS matrices in positive and negative ion mode. In order to study the combined effect of the indole N-H and the pyridine nitrogen of the MALDI matrix on the desorption/ionization process in MALDI, compounds were selected that include either or both of these functions in their structure. Within the compounds studied only those that possess simultaneously both functions in a 1,4-relation behave as very good matrices for proteins. These compounds also work as matrices for some carbohydrates and synthetic polymers used as analytes in the present study. Some of the compounds were also found to be useful for the post-source decay (PSD) analysis of cyclodextrins in positive and negative ion mode. In several cases we also examined the matrix behavior of the corresponding N-methylindole derivatives.     

Negative ion MALDI‐TOF MS,ISD and PSD of neutral underivatized oligosaccharides without anionic dopant strategies,using 2,5‐DHAP as a matrix

Oligosaccharides represent complex class of analytes for mass spectrometric analysis due to the high variety of structural isomers concerning glycosidic linkages and possible branching. A systematic study of the negative ion mode matrix‐assisted laser desorption/ionization (MALDI) mass spectrometry of various neutral oligosaccharides under selection of an appropriate matrix, like 2,5‐dihydroxyacetophenone (2,5‐DHAP) is reported here, without commonly used anion dopant strategies. Nevertheless, we were able to generate relevant in‐source decay (ISD) cross‐ring fragment ions, typically obtained in the negative ion mode. Data observed indicate that the intrinsic property of the terminal non‐reduced aldose is crucial for this behavior. A systematic study of the post source decay (PSD) of molecular, pseudomolecular and ISD cross‐ring cleavage precursor ions is reported here. A direct comparison of the positive and negative ion mode MALDI MS1 and PSD behavior of neutral oligosaccharides could also be performed under the use of the same matrix preparation, because 2,5‐DHAP is fully compatible with positive ion mode acquisition. We found that PSD spectra of deprotonated neutral oligosaccharides obtained in the negative ion mode are richer, because they contained both glycosidic and cross‐ring fragment ions. However, we also found that cross‐ring fragment ions are readily produced in the positive ion mode when potassiated precursor ions were selected. In addition, we show evidence that non‐anionic dopants and specific instrumental parameters can also significantly influence the ISD fragmentation. Taken together, our results should increase our understanding of oligosaccharide behavior in the negative ion mode as well as increase our knowledge regarding many aspects of in‐source MALDI chemistry. Copyright © 2016 John Wiley & Sons, Ltd.     

Generation of CsI cluster ions for mass calibration in matrix-assisted laser desorption/ionization mass spectrometry

A simple method was developed for the generation of cesium iodide (CsI) cluster ions up to m/z over 20,000 in matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS). Calibration ions in both positive and negative ion modes can readily be generated from a single MALDI spot of CsI₃ with 2-[(2E)-3-(4-tert-butylphenyl)-2-methylprop-2-enylidene] malononitrile (DCTB) matrix. The major cluster ion series observed in the positive ion mode is [(CsI)_nCs]⁺, and in the negative ion mode is [(CsI)_nI]⁻. In both cluster series, ions spread evenly every 259.81 units. The easy method described here for the production of CsI cluster ions should be useful for MALDI MS calibrations.     

Influence of chain end groups on the matrix-assisted laser desorption/ionization spectra of polymer blends

The positive ion matrix-assisted laser desorption/ionization (MALDI) spectrum of an equimolar blend of Nylon 6 (Ny6) and hydroxyl-terminated polybutyleneterephthalate (PBT) shows a surprisingly strong imbalance between the two components. Since the average molar masses and the polydispersions of the two polymers are comparable, it follows that the efficiency of MALDI is quite different for the two components of the blend. This finding prompted us to a more detailed study, and to synthesize Ny6 and PBT samples terminated with different end groups, in order to analyze their blends by MALDI. The negative ion MALDI spectra of the mixtures investigated show that PBT samples do not yield signals, so that only Ny6 peaks appear in these spectra. By comparing positive and negative ion MALDI spectra of mixtures of Ny6 terminated with various end groups, it was found that the peak intensity depends on the nature of the end groups. The results reported in the present study may help to clarify some fundamental aspects of the mechanisms of ion formation, when MALDI mass spectrometry is applied to macromolecules. End group ionization efficiency appears to be the most important parameter in determining the relative intensity of peaks in the MALDI spectra of the polymer blends investigated. End-group-dependent ionization is the key to the rationalization of the relative peak intensities in MALDI spectra of polymer mixtures.     

Production and fragmentation of negative ions from neutral N-linked carbohydrates ionized by matrix-assisted laser desorption/ionization

Although negative ion fragmentation mass spectra of neutral N-linked carbohydrates (those attached to Asn in glycoproteins) provide much more structural information than spectra recorded in positive ion mode, neutral carbohydrates are reluctant to form negative ions by matrix-assisted laser desorption/ionization (MALDI) unless ionized from specific matrices such as nor-harmane or adducted with anions such as chloride. This paper reports the results of experiments to optimize negative ion formation from adducts of N-linked glycans with respect to ion abundance and fragment ion production. The best results were obtained with 2,4,6-trihydroxyacetophenone (THAP) as the matrix with added ammonium nitrate as the salt providing the anion. This approach is demonstrated to be applicable for a wide range of N-linked glycan structures. Phosphate adducts, analogous to those that are usually encountered in electrospray spectra from N-glycans released by protein N-glycosidase F, were produced by addition of ammonium phosphate to the matrix but in relatively low yield allowing competitive ionization of endogenous anionic compounds leading to complex spectra. Fragmentation of the nitrate adducts, which were formed in higher yield, generally paralleled that seen by collision-induced dissociation following ionization by electrospray, with the first stage of the dissociation being the elimination of the nitrate with a proton from one of the hydroxyl groups of the sugar. The spectra of the resulting [M-H](-) species displayed very specific fragment ions, mainly cross-ring and C-type glycosidic cleavage products, that revealed more structural (linkage and branching) information of the compounds than the mainly glycosidic cleavage products that dominated the positive ion spectra.     

Comparative study of organic dyes with time-of-flight static secondary ion mass spectrometry and related techniques

A series of cationic, zwitterionic and anionic fluorinated carbocyanine dyes, spin-coated on Si substrates, were measured with time-of-flight static secondary ion mass spectrometry (TOF-S-SIMS) under Ga(+) primary ion bombardment. Detailed fragmentation patterns were developed for all dyes measured. In the positive mode, the resulting spectra showed very intense signals for the precursor ions of the cationic dyes, whereas the protonated signals of the anionic dyes were hardly detected. Differences of three orders of magnitude were repeatedly observed for the secondary ion signal intensities of cationic and anionic dyes, respectively. All measured dyes yielded mass spectra containing several characteristic fragment ions. Although the secondary ion yields were still higher for the cationic than the anionic dye fragments, the difference was reduced to a factor of < or =10. This result and the fact that M(+), [M + H](+) or [M + 2H](+) are even-electron species make it very likely that the recorded fragments were not formed directly out of the (protonated) parent ions M(+), [M + H](+) or [M + 2H](+). In the negative mode, none of the recorded spectra contained molecular information. Only signals originating from some characteristic elements of the molecules (F, Cl), the anionic counter ion signal and some low-mass organic ions were detected. A comparative study was made between TOF-S-SIMS, using Ga(+) primary ions, and other mass spectrometric techniques, namely fast atom bombardment (FAB), electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI). The measurements showed that MALDI, ESI and FAB all give rise to spectra containing molecular ion signals. ESI and FAB produced M(+) and [M + H](+) signals, originating from the cationic and zwitterionic dyes, in the positive mode and M(-) and [M - H](-) signals of the anionic and zwitterionic dyes in the negative mode. With MALDI, molecular ion signals were measured in both modes for all the dyes. Structural fragment ions were detected for FAB, ESI and MALDI in both the positive and negative modes. Compared with the other techniques, TOF-S-SIMS induced a higher degree of fragmentation.     

Tandem mass spectrometry characteristics of polyester anions and cations formed by electrospray ionization

Electrospray ionization of polyesters composed of isophthalic acid and neopentyl glycol produces carboxylate anions in negative mode and mainly sodium ion adducts in positive mode. A tandem mass spectrometry (MS/MS) study of these ions in a quadrupole ion trap shows that the collisionally activated dissociation pathways of the anions are simpler than those of the corresponding cations. Charge-remote fragmentations predominate in both cases, but the spectra obtained in negative mode are devoid of the complicating cation exchange observed in positive mode. MS/MS of the Na(+) adducts gives rise to a greater number of fragments but not necessarily more structural information. In either positive or negative mode, polyester oligomers with different end groups fragment by similar mechanisms. The observed fragments are consistent with rearrangements initiated by the end groups. Single-stage ESI mass spectra also are more complex in positive mode because of extensive H/Na substitutions; this is also true for matrix-assisted laser desorption ionization (MALDI) mass spectra. Hence, formation and analysis of anions might be the method of choice for determining block length, end group structure and copolymer sequence, provided the polyester contains at least one carboxylic acid end group that is ionizable to anions.     

Single-Base Extension and ELISA-Based Approach for Single-Nucleotide Polymorphisms Genotyping

Single-nucleotide polymorphisms (SNPs) emerge as a fundamental tool in personalized medicine due to their association with drug responses or disease predisposition. Single-base extension (SBE) is a common method for characterizing known SNPs, but involves complicated procedures or requires costly analytical instruments. Here, we describe a novel SNP genotyping based on SBE and enzyme-linked immunosorbent assay (ELISA). During the SBE, the 5′ end fluorescein isothiocyanate-labeled allele-specific primer will extend with biotinylated dideoxynucleotides which are complementary to the SNP sites. The extension product will then be captured by streptavidin-coated nanoparticle and develop blue color in the ELISA assay. We validated this method by detecting SNPs for TP53 gene codon 273 from 68 individuals and the data were 100% in concordant with DNA sequencing. Thus, SBE and ELISA-based SNPs assay is a simple and accurate method for SNP genotyping.     

Fragmentation pathway studies of oligonucleotides in matrix-assisted laser desorption/ionization mass spectrometry by charge tagging and H/D exchange

The desorption and decompositions of synthesized oligonucleotides bearing fixed charge sites have been investigated by linear, delayed-extraction, reflecting and post-source decay mode matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. In contrast to the conventional [M + H]⁺ forms of unmodified molecules where a proton is likely attached to a nucleobase, here the charge is fixed at one of the termini. In this case the observed fragment ions always incorporate the charge-tag. H/D exchange experiments provide no evidence for intramolecular migration of protons on the phosphate backbone to initiate the fragmentation event. New unique pathways of proton migration from the ribose have been elucidated and are rationalized by a charge-remote fragmentation pathway.     

5-Ethyl-2-mercaptothiazole as matrix for matrix-assisted laser desorption/ionization of a broad spectrum of analytes in positive and negative ion mode

A preliminary investigation of the use of 5-ethyl-2-mercaptothiazole as matrix in matrix-assisted laser desorption/ionization (MALDI) of a broad spectrum of analytes is reported. The analytes studied are substance P, insulin, beta-cyclodextrin, triacylglycerols of coconut oil and polypropylene glycol 2000 (PPG 2000). In the positive ion mass spectra of the matrix/analyte combinations, the formation of [M + H]+ and [M + cation]+ species were observed and compared with those obtained by using well-established matrices such as alpha-cyano-4-hydroxycinnamic acid, genticic acid, sinapinic acid and dithranol. In addition, the usefulness of this new matrix for MALDI in negative ion mode is also described using substance P and beta-cyclodextrin as examples.     

Negative matrix-assisted laser desorption/ionization time-of-flight/time-of-flight tandem mass spectrometry fragmentation of synthetic analogs of the O-specific polysaccharide of Vibrio cholerae O:1 in the presence of anionic dopants

Oligosaccharides (tri- to hexamers) that represent terminal epitopes of O-antigens of Vibrio cholerae O:1, serotypes Ogawa and Inaba, have been studied by negative matrix-assisted laser desorption/ionization time of flight/time-of-flight mass spectrometry (MALDI ToF/ToF MS). The [M - H(+)](-) ions are formed after expulsion of a proton from molecules studied under condition of MALDI MS analysis in the negative mode. Several ammonium salts (chloride, nitrate, hydrogencarbonate and hydrogensulfate) were used as additives to increase the formation of negative ions from saccharides. The most efficient was the addition of ammonium hydrogencarbonate, which increased the amount of [M - H(+)](-) ions more than six times. Between three fragmentation pathways, the new conjugated transfer of electrons within the second downstream unit of oligosaccharides was discovered. Production of these ions, which has not been observed in any other kinds of measurement, distinguishes substances belonging to Ogawa and Inaba serotypes. The negative MALDI ToF/ToF mass spectra are simpler and, at the same time, more informative, as compared with positive and negative electrospray ionization ion trap as well as with positive MALDI ToF/ToF analysis.