Electrochemiluminescent detection of the hybridization of oligonucleotides using an electrode modified with nanocomposite of carbon nanotubes and gold nanoparticles

We report on an electrochemiluminescent (ECL) sensing technique for the detection of the hybridization between oligonucleotides. A glassy carbon electrode was first functionalized with a composite prepared from gold nanoparticles and carbon nanotubes, and a sensor was then constructed by immobilizing the probing oligonucleotide. The ECL of luminol acts as the sensing signal. It is quenched, to a different degree, by the hybridized double strands of the oligonucleotide depending on the match status. The slope of the ECL response as a function of the status of hybridization drops with increasingly matched hybridization. The response is attributed to the interaction between luminol and the strands of oligomers, and also related to the reduction of reactive oxygen species.

Surface Induced Dissociation Yields Quaternary Substructure of Refractory Noncovalent Phosphorylase B and Glutamate Dehydrogenase Complexes

Ion mobility (IM) and tandem mass spectrometry (MS/MS) coupled with native MS are useful for studying noncovalent protein complexes. Collision induced dissociation (CID) is the most common MS/MS dissociation method. However, some protein complexes, including glycogen phosphorylase B kinase (PHB) and L-glutamate dehydrogenase (GDH) examined in this study, are resistant to dissociation by CID at the maximum collision energy available in the instrument. Surface induced dissociation (SID) was applied to dissociate the two refractory protein complexes. Different charge state precursor ions of the two complexes were examined by CID and SID. The PHB dimer was successfully dissociated to monomers and the GDH hexamer formed trimeric subcomplexes that are informative of its quaternary structure. The unfolding of the precursor and the percentages of the distinct products suggest that the dissociation pathways vary for different charge states. The precursors at lower charge states (+21 for PHB dimer and +27 for GDH hexamer) produce a higher percentage of folded fragments and dissociate more symmetrically than the precusors at higher charge states (+29 for PHB dimer and +39 for GDH hexamer). The precursors at lower charge state may be more native-like than the higher charge state because a higher percentage of folded fragments and a lower percentage of highly charged unfolded fragments are detected. The combination of SID and charge reduction is shown to be a powerful tool for quaternary structure analysis of refractory noncovalent protein complexes, as illustrated by the data for PHB dimer and GDH hexamer.

Gas-phase Dissociation of homo-DNA Oligonucleotides

Synthetic modified oligonucleotides are of interest for diagnostic and therapeutic applications, as their biological stability, pairing selectivity, and binding strength can be considerably increased by the incorporation of unnatural structural elements. Homo-DNA is an oligonucleotide homologue based on dideoxy-hexopyranosyl sugar moieties, which follows the Watson-Crick A-T and G-C base pairing system, but does not hybridize with complementary natural DNA and RNA. Homo-DNA has found application as a bioorthogonal element in templated chemistry applications. The gas-phase dissociation of homo-DNA has been investigated by ESI-MS/MS and MALDI-MS/MS, and mechanistic aspects of its gas-phase dissociation are discussed. Experiments revealed a charge state dependent preference for the loss of nucleobases, which are released either as neutrals or as anions. In contrast to DNA, nucleobase loss from homo-DNA was found to be decoupled from backbone cleavage, thus resulting in stable products. This renders an additional stage of ion activation necessary in order to generate sequence-defining fragment ions. Upon MS³ of the primary base-loss ion, homo-DNA was found to exhibit unspecific backbone dissociation resulting in a balanced distribution of all fragment ion series.

Cyanide–Arene Meisenheimer Complex Generated in Electrospray Ionization Mass Spectrometry Using Acetonitrile as a Solvent

The C – C bond formation activated under negative electrospray ionization of an acetonitrile solution of 1,3,5-trinitrobenzene is reported. The solvent function is to provide a source of cyanide ion, a highly problematic reagent, which is found to attack the electron-deficient aromatic ring to form a covalently bound anionic complex (Meisenheimer complex). The structure of the complex is elucidated by means of collision induced dissociation mass spectrometry and IR multiple photon dissociation spectroscopy in the ‘fingerprint’ region.

A duplex–triplex nucleic acid nanomachine that probes pH changes inside living cells during apoptosis

A duplex–triplex switchable DNA nanomachine was fabricated and has been applied for the demonstration of intracellular acidification and apoptosis of Ramos cells, with graphene oxide (GO) not only as transporter but also as fluorescence quencher. The machine constructed with triplex-forming oligonucleotide exhibited duplex–triplex transition at different pH conditions. By virtue of the remarkable difference in affinity of GO with single-stranded DNA and triplex DNA, and the super fluorescence quenching efficiency of GO, the nanomachine functions as a pH sensor based on fluorescence resonance energy transfer. Moreover, taking advantage of the excellent transporter property of GO, the duplex–triplex/GO nanomachine was used to sense pH changes inside Ramos cells during apoptosis. Fluorescence images showed different results between living and apoptotic cells, illustrating the potential of DNA scaffolds responsive to more complex pH triggers in living systems.

Electron Capture Dissociation and Collision Induced Dissociation of S-Dipalmitoylated Peptides

Here we investigate the effect of S-dipalmitoylation on the electron capture dissociation (ECD) behavior of peptides. The ECD and collision induced dissociation (CID) of peptides modified by covalent attachment of [(RS)-2,3-di(palmitoyloxy)-propyl] (PAM2) group to cysteine residues [C(PAM2)LEYDTGFK and RPPGC(PAM2)SPFK] were examined. The results suggest that ECD of S-dipalmitoylated peptides can provide both primary sequence information and structural information regarding the modification. The structural information provided by CID is complementary to that provided by ECD.

Use of Doubly Charged Precursors to Validate Dissociation Mechanisms of Singly Charged Poly(Dimethylsiloxane) Oligomers

Collision-induced dissociation of doubly charged poly(dimethylsiloxane) (PDMS) molecules was investigated to provide experimental evidence for fragmentation reactions proposed to occur upon activation of singly charged oligomers. This study focuses on two PDMS species holding trimethylsilyl or methoxy end-groups and cationized with ammonium. In both cases, introduction of the additional charge did not induce significant differences in dissociation behavior, and the use of doubly charged precursors enabled the occurrence of charge-separation reactions, allowing molecules always eliminated as neutrals upon activation of singly charged oligomers to be detected as cationized species. In the case of trimethylsilyl-terminated oligomers, random location of the adducted charge combined with rapid consecutive reactions proposed to occur from singly charged precursors could be validated based on MS/MS data of doubly charged oligomers. In the case of methoxy-terminated PDMS, favored interaction of the adducted ammonium with both end-groups, proposed to rationalize the dissociation behavior of singly charged molecules, was also supported by MS/MS data obtained for molecules adducted with two ammonium cations.

Study of an Unusual Advanced Glycation End-Product (AGE) Derived from Glyoxal Using Mass Spectrometry

Glycation is a post-translational modification (PTM) that affects the physiological properties of peptides and proteins. In particular, during hyperglycaemia, glycation by α-dicarbonyl compounds generate α-dicarbonyl-derived glycation products also called α-dicarbonyl-derived advanced glycation end products. Glycation by the α-dicarbonyl compound known as glyoxal was studied in model peptides by MS/MS using a Fourier transform ion cyclotron resonance mass spectrometer. An unusual type of glyoxal-derived AGE with a mass addition of 21.98436 Da is reported in peptides containing combinations of two arginine-two lysine, and one arginine-three lysine amino acid residues. Electron capture dissociation and collisionally activated dissociation results supported that the unusual glyoxal-derived AGE is formed at the guanidino group of arginine, and a possible structure is proposed to illustrate the 21.9843 Da mass addition.

Porcine P2 myelin protein primary structure and bound fatty acids determined by mass spectrometry

Complementary collision-induced/electron capture dissociation Fourier-transform ion cyclotron resonance mass spectrometry was used to fully sequence the protein P2 myelin basic protein. It is an antigenic fatty-acid-binding protein that can induce experimental autoimmune neuritis: an animal model of Guillain–Barré syndrome, a disorder similar in etiology to multiple sclerosis. Neither the primary structure of the porcine variant, nor the fatty acids bound by the protein have been well established to date. A 1.8-Å crystal structure shows but a bound ligand could not be unequivocally identified. A protocol for ligand extraction from protein crystals has been developed with subsequent gas chromatography MS analysis allowing determination that oleic, stearic, and palmitic fatty acids are associated with the protein. The results provide unique and general evidence of the utility of mass spectrometry for characterizing proteins from natural sources and generating biochemical information that may facilitate attempts to elucidate the causes for disorders such as demyelination.

Repeat MALDI MS imaging of a single tissue section using multiple matrices and tissue washes

Matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI MSI) techniques are continually being assessed with a view to improving the quality of information obtained from a given sample. A single tissue section will typically only be analyzed once by MALDI MSI and is then either used for histological staining or discarded. In this study, we explore the idea of repeat analysis of a single tissue section by MALDI MSI as a route toward improving sensitivity, structural characterization, and diversity of detected analyte classes. Repeat analysis of a single tissue section from a fresh frozen mouse brain is investigated with both α-cyano-4-hydroxycinnamic acid (CHCA) and para-nitroaniline (PNA). Repeat analysis is then applied to the acquisition of MALDI MSI and MALDI tandem mass spectrometry imaging employing collision induced dissociation (MS/MS imaging employing CID) from a formalin-fixed mouse brain section. Finally, both lipid and protein data are acquired from the same tissue section via repeat analysis utilizing CHCA, sinapinic acid (SA), and a tissue wash step. PNA was found to outperform CHCA as a matrix for repeat analysis; multiple lipids were identified using MS/MS imaging; both lipid and protein images were successfully acquired from a single tissue section.

Activated Ion ETD Performed in a Modified Collision Cell on a Hybrid QLT-Oribtrap Mass Spectrometer

We describe the implementation and characterization of activated ion electron transfer dissociation (AI-ETD) on a hybrid QLT-Orbitrap mass spectrometer. AI-ETD was performed using a collision cell that was modified to enable ETD reactions, in addition to normal collisional activation. The instrument manifold was modified to enable irradiation of ions along the axis of this modified cell with IR photons from a CO₂ laser. Laser power settings were optimized for both charge (z) and mass to charge (m/z) and the instrument control firmware was updated to allow for automated adjustments to the level of irradiation. This implementation of AI-ETD yielded 1.6-fold more unique identifications than ETD in an nLC-MS/MS analysis of tryptic yeast peptides. Furthermore, we investigated the application of AI-ETD on large scale analysis of phosphopeptides, where laser power aids ETD, but can produce b- and y-type ions because of the phosphoryl moiety’s high IR adsorption. nLC-MS/MS analysis of phosphopeptides derived from human embryonic stem cells using AI-ETD yielded 2.4-fold more unique identifications than ETD alone, demonstrating a promising advance in ETD sequencing of PTM containing peptides.

Implementation of Dipolar Resonant Excitation for Collision Induced Dissociation with Ion Mobility/Time-of-Flight MS

An ion mobility/time-of-flight mass spectrometer (IMS/TOF MS) platform that allows for resonant excitation collision induced dissociation (CID) is presented. Highly efficient, mass-resolved fragmentation without additional excitation of product ions was accomplished and over-fragmentation common in beam-type CID experiments was alleviated. A quadrupole ion guide was modified to apply a dipolar AC signal across a pair of rods for resonant excitation. The method was characterized with singly protonated methionine enkephalin and triply protonated peptide angiotensin I, yielding maximum CID efficiencies of 44 % and 84 %, respectively. The Mathieu q_x,y parameter was set at 0.707 for these experiments to maximize pseudopotential well depths and CID efficiencies. Resonant excitation CID was compared with beam-type CID for the peptide mixture. The ability to apply resonant waveforms in mobility-resolved windows is demonstrated with a peptide mixture yielding fragmentation over a range of mass-to-charge (m/z) ratios within a single IMS-MS analysis.

Isolation and sequence analysis of peptides from the skin secretion of the Middle East tree frog Hyla savignyi

Novel peptides were identified in the skin secretion of the tree frog Hyla savignyi. Skin secretions were collected by mild electrical stimulation. Peptides were separated by reversed-phase high-performance liquid chromatography. Mass spectra were acquired by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS), and fragment ion spectra were obtained after collision-induced dissociation and electron capture dissociation. Peptides were analyzed by manual de novo sequencing and composition-based sequencing (CBS). Sequence analyses of three so far undescribed, structurally unrelated peptides are presented in this paper, having the sequences DDSEEEEVE-OH, P*EEVEEERJK-OH, and GJJDPJTGJVGGJJ-NH₂. The glutamate-rich sequences are assumed to be acidic spacer peptides of the prepropeptide. One of these peptides contains the modified amino acid hydroxyproline, as identified and localized by high-accuracy FTICR-MS. Combination of CBS and of experience-based manual sequence analysis as complementary and database-independent sequencing strategies resulted in peptide identification with high reliability.

Multipurpose Dissociation Cell for Enhanced ETD of Intact Protein Species

We describe and characterize an improved implementation of ETD on a modified hybrid linear ion trap-Orbitrap instrument. Instead of performing ETD in the mass-analyzing quadrupole linear ion trap (A-QLT), the instrument collision cell was modified to enable ETD. We partitioned the collision cell into a multi-section rf ion storage and transfer device to enable injection and simultaneous separate storage of precursor and reagent ions. Application of a secondary (axial) confinement voltage to the cell end lens electrodes enables charge-sign independent trapping for ion–ion reactions. The approximately 2-fold higher quadrupole field frequency of this cell relative to that of the A-QLT enables higher reagent ion densities and correspondingly faster ETD reactions, and, with the collision cell’s longer axial dimensions, larger populations of precursor ions may be reacted. The higher ion capacity of the collision cell permits the accumulation and reaction of multiple full loads of precursor ions from the A-QLT followed by FT Orbitrap m/z analysis of the ETD product ions. This extends the intra-scan dynamic range by increasing the maximum number of product ions in a single MS/MS event. For analyses of large peptide/small protein precursor cations, this reduces or eliminates the need for spectral averaging to achieve acceptable ETD product ion signal-to-noise levels. Using larger ion populations, we demonstrate improvements in protein sequence coverage and aggregate protein identifications in LC-MS/MS analysis of intact protein species as compared to the standard ETD implementation.

An innovative strategy for direct electrochemical detection of microRNA biomarkers

We report an electrochemical method for direct, reagentless, and label-free detection of microRNA, based on a conjugated copolymer, poly(5-hydroxy-1,4-naphthoquinone-co-5-hydroxy-2-carboxyethyl-1,4-naphthoquinone), acting as hybridization transducer. Hybridization between the oligonucleotide capture probe and a microRNA target of 22 base pairs generates an increase in the redox current (“signal-on”), which is evidenced by square wave voltammetry. Selectivity is good, with little hybridization for non-complementary targets, and the limit of detection reaches 650 fM. It is also evidenced that this sensitivity benefits from the high affinity of DNA for RNA.

Activation Energies of Fragmentations of Disaccharides by Tandem Mass Spectrometry

A simple multiple collision model for collision induced dissociation (CID) in quadrupole was applied for the estimation of the activation energy (E_o) of the fragmentation processes for lithiated and trifluoroacetated disaccharides, such as maltose, cellobiose, isomaltose, gentiobiose, and trehalose. The internal energy-dependent rate constants k(E_int) were calculated using the Rice-Ramsperger-Kassel-Marcus (RRKM) or the Rice-Ramsperger-Kassel (RRK) theory. The E_o values were estimated by fitting the calculated survival yield (SY) curves to the experimental ones. The calculated E_o values of the fragmentation processes for lithiated disaccharides were in the range of 1.4–1.7 eV, and were found to increase in the order trehalose < maltose < isomaltose < cellobiose < gentiobiose.

Comparison between GC–MS and GC–ICPMS using isotope dilution for the simultaneous monitoring of inorganic and methyl mercury,butyl and phenyl tin compounds in biological tissues

The aim of this work is to compare simultaneous isotope dilution analysis of organotin and organomercury compounds by gas chromatography–mass spectrometry (GC–MS) and gas chromatography–inductively coupled plasma mass spectrometry (GC–ICP/MS) on certified bivalve samples. These samples were extracted by microwave with tetramethylammonium hydroxide (TMAH). Derivatization with both NaBEt₄ and NaBPr₄ was evaluated, and analytical performances were compared. Two CRM materials, BCR-710 and CRM-477, were analyzed by both techniques to verify accuracy. A mixed spike containing ²⁰¹Hg-enriched methylmercury (MeHg), ¹⁹⁹Hg-enriched inorganic mercury (iHg), ¹¹⁹Sn-enriched monobutyltin (MBT), dibutyltin (DBT), and tributyltin (TBT) as well as homemade ¹¹⁶Sn-enriched monophenyltin (MPT), diphenyltin (DPT), and triphenyltin (TPT) was used for the isotope dilution analysis of samples. The two techniques studied were compared in terms of classic analytical parameters: linearity, precision or repeatability (i.e., percent relative standard deviation, RSD%), limit of detection (LOD), and limit of quantification (LOQ), showing excellent linearity, precision below 12 % for all analytes, and LOQs of 0.06–1.45 pg for GC–MS and 0.02–0.27 pg for GC–ICP/MS.

Energy Dependence of HCD on Peptide Fragmentation: Stepped Collisional Energy Finds the Sweet Spot

An understanding of the process of peptide fragmentation and what parameters are best to obtain the most useful information is important. This is especially true for large-scale proteomics where data collection and data analysis are most often automated, and manual interpretation of spectra is rare because of the vast amounts of data generated. We show herein that collisional cell peptide fragmentation, in this case higher collisional dissociation (HCD) in the Q Exactive, is significantly affected by the normalized energy applied. Both peptide sequence and energy applied determine what ion fragments are observed. However, by applying a stepped normalized collisional energy scheme and combining ions from low, medium, and high collision energies, we are able to increase the diversity of fragmentation ions generated. Application of stepped collision energy to HEK293T lysate demonstrated a minimal effect on peptide and protein identification in a large-scale proteomics dataset, but improved phospho site localization through increased sequence coverage. Stepped HCD is also beneficial for tandem mass tagged (TMT) experiments, increasing intensity of TMT reporters used for quantitation without adversely effecting peptide identification.

Neutron-Encoded Protein Quantification by Peptide Carbamylation

We describe a chemical tag for duplex proteome quantification using neutron encoding (NeuCode). The method utilizes the straightforward, efficient, and inexpensive carbamylation reaction. We demonstrate the utility of NeuCode carbamylation by accurately measuring quantitative ratios from tagged yeast lysates mixed in known ratios and by applying this method to quantify differential protein expression in mice fed a either control or high-fat diet.

Analysis of flame retardants and elements of concern in printed wiring boards with respect to origin and year of construction

Thirty-one populated printed wiring boards, covering a range of 30 years of construction, and originating from various electronic devices, were investigated using different analytical procedures. Noble, precious and rare metals, as well as environmentally relevant elements were identified by EDXRF, and lead and the flame retardant (FR) indicator bromine were localised by means of microbeam EDXRF. A GC/MS procedure was developed to identify and quantify FR substances. Several sample preparation techniques were applied, optimised and compared. The method of first choice was ultrasonic extraction because it provided the best compromise between effort, cost and quality of the analytical results. Altogether, a wide variety of elements of concern, and halogenated and phosphate-based FRs were found in the investigated boards. Their occurrence is partially related to the origin and/or year of construction.