Simultaneous genotyping of indels and SNPs by mass spectroscopy

Nucleotide insertion/deletion polymorphisms (indels) in ApoE gene were precisely genotyped using artificial ribonucleases and MALDI-TOF MS. The RNA fragments for MS analysis were prepared by treating RNA specimens with our artificial ribonucleases, which consist of LuCl(3) (molecular scissors) and oligonucleotides bearing two acridine groups (RNA-activator for site-selective scission). RNA scission by Lu(III) ion always occurred at the phosphodiester linkages in front of the two acridines, even when the RNA specimens involved consecutive cytidine sequences of different lengths. Thus, even complicated mixtures of these indel specimens were completely genotyped by using only one acridine-bearing oligonucleotide and by subjecting the reaction mixture to single MS measurement. Moreover, single nucleotide polymorphism (SNP) in the consecutive sequences could be genotyped simultaneously with the indels.     

Resonance ionization mass spectrometry for precise measurements of isotope ratios

Resonance ionization mass spectrometry offers extremely high sensitivity and elemental selectivity in microanalysis, but the isotopic precision attainable by this technique has been limited. Measured isotope ratios are sensitive to small fluctuations in the pointing, pulse timing, and wavelength of the resonance lasers. We show that, by minimizing these fluctuations using feedback controls and by power-broadening the optical transitions, we are able to measure chromium isotope ratios with statistics-limited precision better than 1%. Small additional improvements in reproducibility come from careful shaping of the electric field in the region where atoms are photoionized and from minimizing pulse-to-pulse variations in the time-of-flight mass spectrometer through which the photoions travel. The increased reproducibility of isotopic measurements on standard materials has enabled us to detect anomalous chromium isotopic abundances in presolar SiC grains extracted from primitive meteorites.     

CYP2D6 genotyping by liquid chromatography-electrospray ionization mass spectrometry

Genetic polymorphisms can significantly affect the enzyme activity of the drug metabolizing enzyme Cytochrome P450 2D6 (CYP2D6; OMIM 124030). Accordingly, CYP2D6 genotyping is considered as a valid approach to predict the individual CYP2D6 metabolizing status. We introduce ion-pair reversed-phase high-performance liquid chromatography-electrospray ionization mass spectrometry (ICEMS) as method for the characterization of single base variants, small deletions, and insertions in the CYP2D6 gene. A two-step polymerase chain reaction (PCR) was developed for the simultaneous amplification of nine polymorphic regions within the CYP2D6 gene. Cleanup, separation, and denaturation of PCR amplicons were achieved by high-performance liquid chromatography. High-performance molecular mass measurements provided nucleotide composition profiles that principally enable the resolution of 37 reported CYP2D6 alleles. The developed assay was applied to the genotyping of 93 unrelated Austrian individuals. For validation, a selected number of samples and polymorphic sites were retyped by alternative genotyping technologies. The PCR-ICEMS assay turned out to be an accurate, robust, and cost-effective CYP2D6 genotyping strategy.     

Rapid screening by MALDI-TOF mass spectrometry to probe binding specificity at enzyme active sites

The binding affinity of aspartate decarboxylase has been probed using MALDI-TOF spectrometry; adducts formed covalently in the active site were detected by MALDI-TOF mass spectrometry after incubation of the enzyme with a range of potential ligands in the presence of NaCNBH3; this has highighted key structural features which will aid design of potential inhibitors.     

Mass spectrometry characterization of the glycation sites of bovine insulin by tandem mass spectrometry

Bovine insulin was glycated under hyperglycemic reducing conditions and in nonreducing conditions. Purification through HPLC allowed isolating glycated forms of insulin and a novel triglycated form (6224.5 Da) was purified. Endoproteinase Glu-C digestion combined with mass spectrometry (MALDI-TOF/TOF) allowed determining the exact location of the glycation sites in each of the isolated glycated insulins. For the first time, a triglycated form of insulin was isolated and characterized accordingly to its glycation sites. These glucose binding sites were identified as the N-terminals of both chains (Gly1 and Phe1) and residue Lys29 of B-chain. Moreover, in diglycated insulin we found the coexistence of one specie glycated at the N-terminals of both chains (Gly1 and Phe1) and another specie containing the two glucitol adducts in B-chain (Phe1 and Lys29). Also, in monoglycated insulin generated in reducing and nonreducing conditions, one specie glycated at Phe1 and another specie glycated at Lys29, both B-chain residues coexist.     

A strategy for direct identification of protein S-nitrosylation sites by quadrupole time-of-flight mass spectrometry

S-nitrosylation of proteins serves an important role in regulating diverse cellular processes including signal transduction, DNA repair, and neurotransmission. Identification of S-nitrosylation sites is crucial for understanding the significance of this post-translational modification (PTM) in modulating the function of a protein. However, it is challenging to identify S-nitrosylation sites directly by mass spectrometric (MS) methods due to the labile nature of the S-NO bond. Here we describe a strategy for direct identification of protein S-nitrosylation sites in an electrospray ionization (ESI) quadrupole time-of-flight (QTOF) mass spectrometer without prior chemical derivatization of S-nitrosylated peptides. Both sample buffer composition and MS hardware parameters were carefully adjusted to ensure that S-nitrosylated peptide ions could be analyzed by the QTOF MS with optimal signal/noise ratios. It was crucial that the proteins were preserved in a sample solution containing 1 mM EDTA and 0.1 mM neocuproine at neutral pH. Proteins dissolved in this solution are amenable to in-solution tryptic digestion, which is important for the analysis of biological samples. S-nitrosylated peptides were effectively analyzed by LC/MS/MS on QTOF MS, with an optimized cone voltage of 20 V and collision energy of 4 V. We have successfully applied this method to thioredoxin, a key antioxidant protein, and identified within it an S-nitrosylation site at Cys73.     

Analysis of class I phosphoinositide 3-kinase autophosphorylation sites by mass spectrometry

This article describes the identification of the autophosphorylation sites of the G protein-sensitive class I phosphoinositide 3-kinase isoforms beta and gamma by mass spectrometry. Since discrimination and suppression effects prevented the immediate detection and sequencing of phosphopeptides in complex mixtures, a strategy was applied that involved (32)P-radiolabeling of the phosphoproteins, cleavage of the phosphoproteins with several proteases and/or cyanogen bromide, separation of the resulting peptide mixtures by micro-reversed-phase liquid chromatography, and mass spectrometric analysis of fractions containing phosphopeptides. As a result the primary autophosphorylation sites of phosphoinositide 3-kinase p110beta and p110gamma subunits could be unambiguously assigned to the C-terminal Ser 1070 and Ser 1101, respectively.     

Binding of DNA purine sites to dirhodium compounds probed by mass spectrometry

The adducts formed between the antitumor active compounds [Rh(2)(O(2)CCH(3))(2)(CH(3)CN)(6)](BF(4))(2), Rh(2)(O(2)CCH(3))(4), and Rh(2)(O(2)CCF(3))(4) with DNA oligonucleotides have been assessed by matrix-assisted laser desorption ionization (MALDI) and nanoelectrospray (nanoESI) coupled to time-of-flight mass spectrometry (TOF MS). A series of MALDI studies performed on dipurine (AA, AG, GA, and GG)-containing single-stranded oligonucleotides of different lengths (tetra- to dodecamers) led to the establishment of the relative reactivity cis-[Pt(NH(3))(2)(OH(2))(2)](2+) (activated cisplatin) approximately Rh(2)(O(2)CCF(3))(4) > cis-[Pt(NH(3))(2)Cl(2)] (cisplatin) > [Rh(2)(O(2)CCH(3))(2)(CH(3)CN)(6)](BF(4))(2) > Rh(2)(O(2)CCH(3))(4) approximately Pt(C(6)H(6)O(4))(NH(3))(2) (carboplatin). The relative reactivity of the complexes is associated with the lability of the leaving groups. The general trend is that an increase in the length of the oligonucleotide leads to enhanced reactivity for Rh(2)(O(2)CCH(3))(2)(CH(3)CN)(6)](BF(4))(2) and Rh(2)(O(2)CCH(3))(4) (except for the case of [Rh(2)(O(2)CCH(3))(2)(CH(3)CN)(6)](2+), which reacts faster with the GG octamers than with the dodecamers), whereas the reactivity of Rh(2)(O(2)CCF(3))(4) is independent of the oligonucleotide length. When monitored by ESI, the dodecamers containing GG react faster than the respectiveAA oligonucleotides in reactions with Rh(2)(O(2)CCF(3))(4) and Rh(2)(O(2)CCH(3))(2)(CH(3)CN)(6)](BF(4))(2), whereas AA oligonucleotides react faster with Rh(2)(O(2)CCH(3))(4). The mixed (AG, GA) purine sequences exhibit comparable rates of reactivity with the homopurine (AA, GG) dodecamers in reactions with Rh(2)(O(2)CCH(3))(4). The observation of initial dirhodium-DNA adducts with weak axial (ax) interactions, followed by rearrangement to more stable equatorial (eq) adducts, was achieved by electrospray ionization; the Rh-Rh bond as well as coordinated acetate or acetonitrile ligands remain intact in these dirhodium-DNA adducts. MALDI in-source decay (ISD), collision-induced dissociation (CID) MS-MS, and enzymatic digestion studies followed by MALDI and ESI MS reveal that, in the dirhodium compounds studied, the purine sites of the DNA oligonucleotides interact with the dirhodium core. Ultimately, both MALDI and ESI MS proved to be complementary, valuable tools for probing the identity and stability of dinuclear metal-DNA adducts.     

Simultaneous ion-beam collection for precise measurement of nuclidic ion-current ratios in spark-source mass spectrometry

Simultaneous ion-beam collection techniques make significant improvement possible in the precision of measurements when electrical ion-detection is used with the spark ion-source. The advantage of reference of specific ion-current signals only to those from desired regions of the mass spectrum is that reproducible measurements may be made on heterogeneous samples such as oxide-graphite pellets. For example, isotopic ratio measurements from separate samplings of an oxide (pelleted with graphite) are reproducible to better than +/-0.5%. The technique is also advantageous when sparking dissimilar electrodes, such as a gold probe and a metal bar.     

Extension of the GOOD assay for genotyping single nucleotide polymorphisms by matrix-assisted laser desorption/ionization mass spectrometry

Over the past years several methods using mass spectrometry for high-throughput genotyping of single nucleotide polymorphisms (SNPs) have been developed. Most of these procedures require stringent purification. Only the GOOD assay does not need any sample purification. Here, several new implementations of this assay are presented. The molecular biological procedure of the GOOD assays is based on the principle that the analysis of DNA by matrix-assisted laser desorption/ionization (MALDI) is strongly dependent on the charge state. A 100-fold increase in sensitivity can be achieved if the analyzed DNA product is conditioned by a chemical procedure termed 'charge-tagging'. The GOOD assay starts with a PCR; allele-specific DNA molecules are generated by extension of modified primers. These contain up to three phosphorothioates and optionally a quaternary ammonium charged group with ddNTPs or alpha-S-ddNTPs. Then the unmodified part of the primers is digested by phosphodiesterase II and the negative charges of the phosphorothioates are neutralized by an alkylation reaction resulting in charge-tagged DNA products. Through the use of a novel DNA polymerase for the primer extension, which preferably incorporates ddNTPs over dNTPs, an enzymatic degradation of residual dNTPs from the PCR is not required. Additionally, the unique property of charge-tag technology is demonstrated to detect specifically on the same sample allele-specific DNA products carrying a positive charge-tag in the positive ion mode while products carrying a negative charge-tag are analyzed in the negative ion mode. We also generated zwitterionic allele-specific products that were detectable with high sensitivity in positive ion mode. The findings of this study raise interesting questions about the ionization process of nucleic acids in MALDI. The new variations of the GOOD assay were applied to genotype SNPs of a candidate gene for cardiovascular disease.     

Metabolite identification of small interfering RNA duplex by high-resolution accurate mass spectrometry

On-line liquid chromatography/electrospray ionization high-resolution mass spectrometry (LC/ESI-HRMS) using an LTQ-Orbitrap mass spectrometer was employed to investigate the metabolite profiles of a model siRNA duplex designated HBV263. The HBV263 duplex was incubated in rat and human serum and liver microsomes in vitro. The siRNA drug and its metabolites were then extracted using a liquid-liquid extraction followed by solid-phase extraction (LLE-SPE), and analyzed by LC/ESI-MS. High-resolution accurate mass data enabled differentiation between two possible metabolite sequences with a monoisotopic molecular mass difference of less than 1 Da. ProMass deconvolution software was used to provide semi-automated data processing. In vitro serum and liver microsome incubation samples afforded different metabolite patterns: the antisense strand of the duplex was degraded preferentially in rat and human serum, while the sense strand of the duplex was less stable in rat and human liver microsomes.     

Identifying sites of protein modification by using matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry and on-line membrane preconcentration-capillary electrophoresis-tandem mass spectrometry

A strategy for rapidly identifying the number and sites of chemical or posttranslational modification of proteins is described. The use of matrix-assisted laser desorption/ionization-time-of-flight-mass spectrometry to determine the molecular weight of the adducted protein as well as map the proteolytic digest of peptides offers a rapid method to screen for the possible site of adduction. To unequivocally determine the amino acid sequence of the peptide bearing the adduct as well as structurally characteize the covalent modification, the peptide mixture is subjected to membrane preconcentration-capillary electrophoresis-mass spectrometry and tandem mass spectrometry (mPC-CE-MS/MS). The high resolving separation capability of capillary electrophoresis (CE) afford a chromatograhic step that lends itself to separation of complex mixtures of peptides with minimal sample loss. The membrane preconcentration-CE cartridge allows sample loading volumes 10,000-fold greater than conventional CE. In this work the binding site of the fluorescent label acrylodan to the intestinal fatty binding protein is characterized and shown to be covalently bound at lysine-27, by using mPC-CE-MS/MS.     

Characterization of O-glycosylation sites in MUC2 glycopeptides by nanoelectrospray QTOF mass spectrometry

Sequencing of eight O-glycosylated peptides by nanoESI-QTOF-MS/MS was carried out to provide a sensitive general characterization method for determination of glycosylation site(s) and of the type of the attached carbohydrate moiety in a single experiment. The glycopeptide structures were chosen to demonstrate the feasibility of this sensitive and accurate approach, where isobaric peptide structures either (i) with the same number of attachment sites in different position in the peptide backbone, and (ii) with the same number of sugar moieties distributed on different attachment sites in the peptide backbone, can be clearly distinguished. Beside the B-type carbohydrate sequence ions of high abundance, it is possible to register diagnostic b- and y-type glycosylated peptide ions of lower abundance due to high dynamic range of the QTOF analyser. The applicability of this approach for detailed analysis of highly clustered O-glycan structures as found in biological mucin samples is discussed.     

Determination of calcium binding sites in gas-phase small peptides by tandem mass spectrometry

Low-energy (LE) and high-energy (HE) collisionally activated decompositions (CAD) of calcium/peptide complexes of the form [M-H+Ca]⁺ and [M+Ca]²⁺ reflect the site of calcium binding in various gas-phase peptides that are models of the calcium binding site III of rabbit skeletal troponin C. The Ca²⁺ binding sites involve an aspartic acid, glutamic acid, and asparagine, which are in the metal-binding loops of calcium-binding proteins. Both fast atom bombardment (FAB) and electrospray ionization (ESI) were used to generate the metal/peptide complexes. When submitted to LE CAD, ESI-produced Ca²⁺/peptide complexes undergo fragmentations that are controlled by Ca²⁺ binding and provide information on the Ca²⁺ binding site. The LE CAD spectra are simple, indicating that Ca²⁺ binding involves specific oxygen ligands including acidic side chains and that only a few low-energy fragmentation channels exist. The HE CAD spectra of FAB-produced Ca²⁺/peptide complexes are more complex, owing to the introduction of high internal energy into the precursor ion. Interactions of the other alkaline-earth metal ions Mg²⁺ and Ba²⁺ with these peptides reveal that the ligand preferences of these metal ions are slightly different than those of Ca²⁺.     

Determination of protein phosphorylation sites by mass spectrometry: a novel electrospray-based method

Several methods are used to identify protein phosphorylation sites. We report a novel electrospray-based method for the determination of phosphorylation sites by mass spectrometry, using two different declustering potential values. This method allows one to obtain, with a single liquid chromatography/mass spectrometry (LC/MS) run, the pattern with either the phosphorylated or the unphosphorylated species of a protein tryptic digest, that can be further analyzed by tracing back the origin of each HPO3-deprived form using the capabilities of tandem mass spectrometers.     

Identification of phosphorylation sites in proteins by nanospray quadrupole ion trap mass spectrometry

A method is described for identifying serine phosphorylation sites in proteins, based on conventional (32)P labeling followed by electrophoretic separation, 'in-gel' digestion with a protease, peptide extraction, reversed-phase high-performance liquid chromatographic separation and collection and off-line analysis of the radioactive fractions by nanospray ion trap mass spectrometry. The method was successfully applied to the identification of three phosphorylation sites in two proteins which were subjected to in vitro phosphorylation under physiological conditions. Different combinations of the various scanning modes of the ion trap, including high-resolution, multiple subfragmentation (or MS(n)) and fast scan analysis, were employed to identify the phosphopeptides, determine their sequence and localize the exact site of phosphorylation. 'Blind' fragmentation using fast scans was used to analyze a phosphopeptide which was undetectable in other scanning modes. The sequence, phosphorylation site and double cysteine modification of the potassium adduct of a peptide containing 35 residues were also determined by multiple fragmentation. The results not only support the validity of the proposed method for routine identification of phosphorylation sites, but also demonstrate the exceptional capability of off-line ion trap mass spectrometry in combination with nanospray ionization for performing very detailed studies on the structure of peptides.     

Determination of binding sites in carboplatin-bound cytochrome c using electrospray ionization mass spectrometry and tandem mass spectrometry

Interaction of carboplatin with cytochrome c (Cyt. c) has been investigated by electrospray ionization mass spectrometry (ESI-MS) and tandem mass spectrometry (MS/MS). ESI-MS studies revealed that the ring-opened adducts of carboplatin with Cyt. c were formed in the stoichiometric ratio of 1:1 and 2:1 at pH 5.0 and 37 degrees C and in the stoichiometric ratio of 1:1 only at pH 7.0 and 37 degrees C. It was also found that Cyt. c could be cleaved by carboplatin at pH 2.5 and 50 degrees C. The cleaved fragments of Cyt. c were determined by ESI-MS and MS/MS analysis to be Glu66 approximately Met80, Ac-Gly01 approximately Met65, Glu66 approximately Glu104, Ac-Gly01 approximately Met80 and Ile81 approximately Glu104. The carboplatin prefers to anchor to Met65 first, then to Met80. To further confirm the binding site of Met, AcMet-Gly was used as the model molecule to investigate its interaction with carboplatin and its hydrolysis reaction. On the basis of species detected during the reaction monitored by ESI-MS, a possible pathway of the cleavage reaction was proposed.     

Tandem mass spectrometry for the determination of the sites of DNA interstrand cross-link

Formation of DNA interstrand cross-link is implicated in the mechanism of anticancer activity of some drugs. Here we examined the fragmentation of deprotonated ions of double-stranded oligodeoxynucleotides (ODNs) that are covalently held together with either a mitomycin C or a 4,5',8-trimethylpsoralen. Our results showed that, upon collisional activation, the covalently-bound duplex ODNs cleaved to give a series of wn and [an-base] ions; the sites of interstrand cross-linking could be determined from the mass shifts of some product ions. In addition, compared with the product-ion spectra acquired on an ion trap, those obtained from sustained off-resonance irradiation-collisionally activated dissociation (SORI-CAD) on a Fourier transform mass spectrometer offered high mass-resolving power, which facilitated unambiguous assignment of product ions and made it an effective method for locating the cross-linking sites.