A Computational Analysis of the Collision-Induced Dissociation Mechanisms of the Dipeptide Glycine-Serine Under Mass Spectrometry

Collision-induced dissociation (CID) of peptides using tandem mass spectrometry (MS) has been used to determine the identity of peptides and other large biological molecules. Mass spectrometry (MS) is a useful tool for determining the identity of molecules based on their interaction with electromagnetic fields. If coupled with another method like infrared (IR) vibrational spectroscopy, MS can provide structural information, but in its own right, MS can only provide the mass-to-charge (m/z) ratio of the fragments produced, which may not be enough information to determine the mechanism of the collision-induced dissociation (CID) of the molecule. In this case, theoretical calculations provide a useful companion for MS data and yield clues about the energetics of the dissociation. In this study, negative ion electrospray tandem MS was used to study the CID of the deprotonated dipeptide glycine-serine (Gly-Ser). Though negative ion MS is not as popular a choice as positive ion MS, studies by Bowie et al. show that it yields unique clues about molecular structure which complement positive ion spectroscopy, such as characteristic fragmentations like the loss of formaldehyde from the serine residue.2 The increase in the collision energy in the mass spectrometer alters the flexibility of the dipeptide backbone, enabling isomerizations (reactions not resulting in a fragment loss) and dissociations to take place. The mechanism of the CID of Gly-Ser was studied using two computational methods, B3LYP/6-311+G* and M06-2X/6-311++G**. The main pathway for molecular dissociation was analyzed in 5 conformers in an attempt to verify the initial mechanism proposed by Dr. James Swan after examination of the MS data. The results suggest that the loss of formaldehyde from serine, which Bowie et al. indicates is a characteristic of the presence of serine in a protein residue, is an endothermic reaction that is made possible by the conversion of the translational energy of the ion into internal energy as the ion collides with the inert collision gas. It has also been determined that the M06-2X functional¿s improved description of medium and long-range correlation makes it more effective than the B3LYP functional at finding elusive transition states. M06-2X also more accurately predicts the energy of those transition states than does B3LYP. A second CID mechanism, which passes through intermediates with the same m/z ratio as the main pathway for molecular dissociation, but different structures, including a diketopiperazine intermediate, was also studied. This pathway for molecular dissociation was analyzed with 3 conformers and the M06-2X functional, due to its previously determined effectiveness. The results suggest that the latter pathway, which meets the same intermediate masses as the first mechanism, is lower in overall energy and therefore a more likely pathway of dissociation than the first mechanism.

(Table 1) Sr isotopes and chemical composition of DSDP Hole 61-462A basalts

Sixteen elemental abundances and 87Sr/86Sr ratio of the Nauru Basin basalt (Cores 75 to 90: sub-bottom depths 950 m to 1050 m) from Hole 462A have been determined by inductively coupled plasma-optical emission spectroscopy and mass spectrometry. The result indicates that the basalt is a new type of oceanic tholeiite, elementally similar to normal mid-oceanic ridge basalts and isotopically similar to oceanic island-type basalts.

Optimization of InGaAsN(Sb)/GaAs quantum dots for optical emission at 1.55 µm with low optical degradation

Low optical degradation in GaInAsN(Sb)/GaAs quantum dots (QDs) p–i–n structures emitting up to 1.55 μm is presented in this paper. We obtain emission at different energies by means of varying N content from 1 to 4%. The samples show a low photoluminescence (PL) intensity degradation of only 1 order of magnitude when they are compared with pure InGaAs QD structures, even for an emission wavelength as large as 1.55 μm. The optimization studies of these structures for emission at 1.55 μm are reported in this work. High surface density and homogeneity in the QD layers are achieved for 50% In content by rapid decrease in the growth temperature after the formation of the nanostructures. Besides, the effect of N and Sb incorporation in the redshift and PL intensity of the samples is studied by post-growth rapid thermal annealing treatments. As a general conclusion, we observe that the addition of Sb to QD with low N mole fraction is more efficient to reach 1.55 μm and high PL intensity than using high N incorporation in the QD. Also, the growth temperature is determined to be an important parameter to obtain good emission characteristics. Finally, we report room temperature PL emission of InGaAsN(Sb)/GaAs at 1.4 μm.

DNA-bound transcription factor complexes analysed by mass-spectrometry: binding of novel proteins to the human c-fos SRE and related sequences

Transcription factors control eukaryotic polymerase II function by influencing the recruitment of multiprotein complexes to promoters and their subsequent integrated function. The complexity of the functional ‘transcriptosome’ has necessitated biochemical fractionation and subsequent protein sequencing on a grand scale to identify individual components. As a consequence, much is now known of the basal transcription complex. In contrast, less is known about the complexes formed at distal promoter elements. The c-fos SRE, for example, is known to bind Serum Response Factor (SRF) and ternary complex factors such as Elk-1. Their interaction with other factors at the SRE is implied but, to date, none have been identified. Here we describe the use of mass-spectrometric sequencing to identify six proteins, SRF, Elk-1 and four novel proteins, captured on SRE duplexes linked to magnetic beads. This approach is generally applicable to the characterisation of nucleic acid-bound protein complexes and the post-translational modification of their components.

Probing the oligomeric structure of an enzyme by electrospray ionization time-of-flight mass spectrometry.

Electrospray ionization time-of-flight (ESI-TOF) mass spectrometry was used to study the quaternary structure of 4-oxalocrotonate tautomerase (EC 5.3.2; 4OT), and four analogues prepared by total chemical synthesis. Wild-type 4OT is a hexamer of 62 amino acid subunits and contains no cysteine residues. The analogues were: (desPro1)4OT, a truncated construct in which Pro1 was deleted; (Cpc1)4OT in which Pro1 was replaced with cyclopentane carboxylate; a derivative [Met(O)45]4OT in which Met45 was oxidized to the sulfoxide; and an analogue (Nle45)4OT in which Met45 was replaced with norleucine. ESI of (Nle45)4OT, (Cpc1)4OT, and 4OT from solution conditions under which the native enzyme was fully active (5 mM ammonium bicarbonate buffer, pH 7.5) gave the intact hexamer as the major species detected by TOF mass spectrometry. In contrast, analysis of [Met(O)45]4OT and (desPro1)4OT under similar conditions yielded predominantly monomer ions. The ESI-TOF measurements were consistent with structural data obtained from circular dichroism spectroscopy. In the context of kinetic data collected for 4OT and these analogues, ESI-TOF mass spectrometry also provided important evidence for the structural and mechanistic significance of the catalytically important Pro1 residue in 4OT.

Non-spectral interferences due to the presence of sulfuric acid in inductively coupled plasma mass spectrometry

Results of a systematic study concerning non-spectral interferences from sulfuric acid containing matrices on a large number of elements in inductively coupled plasma–mass spectrometry (ICP-MS) are presented in this work. The signals obtained with sulfuric acid solutions of different concentrations (up to 5% w w− 1) have been compared with the corresponding signals for a 1% w w− 1− nitric acid solution at different experimental conditions (i.e., sample uptake rates, nebulizer gas flows and r.f. powers). The signals observed for 128Te+, 78Se+ and 75As+ were significantly higher when using sulfuric acid matrices (up to 2.2-fold for 128Te+ and 78Se+ and 1.8-fold for 75As+ in the presence of 5 w w-1 sulfuric acid) for the whole range of experimental conditions tested. This is in agreement with previously reported observations. The signal for 31P+ is also higher (1.1-fold) in the presence of sulfuric acid. The signal enhancements for 128Te+, 78Se+, 75As+ and 31P+ are explained in relation to an increase in the analyte ion population as a result of charge transfer reactions involving S+ species in the plasma. Theoretical data suggest that Os, Sb, Pt, Ir, Zn and Hg could also be involved in sulfur-based charge transfer reactions, but no experimental evidence has been found. The presence of sulfuric acid gives rise to lower ion signals (about 10–20% lower) for the other nuclides tested, thus indicating the negative matrix effect caused by changes in the amount of analyte loading of the plasma. The elemental composition of a certified low-density polyethylene sample (ERM-EC681K) was determined by ICP-MS after two different sample digestion procedures, one of them including sulfuric acid. Element concentrations were in agreement with the certified values, irrespective of the acids used for the digestion. These results demonstrate that the use of matrix-matched standards allows the accurate determination of the tested elements in a sulfuric acid matrix.

A rapid HPLC-mass spectrometry cyclosporin method suitable for current monitoring practices

Objectives: Cyclosporin is an immunosuppressant drug with a narrow therapeutic window. Trough and 2-h post-dose blood samples are currently used for therapeutic drug monitoring in solid organ transplant recipients. The aim of the current study was to develop a rapid HPLC-tandem mass spectrometry (HPLC-MS) method for the measurement of cyclosporin in whole blood that was not only suitable for the clinical setting but also considered a reference method. Methods: Blood samples (50 mu L) were prepared by protein precipitation followed by C-18 solid-phase extraction while using d(12) cyclosporin as the internal standard. Mass spectrometric detection was by selected reaction monitoring with an electrospray interface in positive ionization mode. Results: The assay was linear from 10 to 2000 mu g/L (r(2) > 0.996, n = 9). Inter-day,analytical recovery and imprecision using whole blood quality control samples at 10, 30, 400, 1500, and 2000 mu g/L were 94.9-103.5% and

Matrix-free mass spectrometric imaging using laser desorption ionisation Fourier transform ion cyclotron resonance mass spectrometry

Mass spectrometry imaging (MSI) is a powerful tool in metabolomics and proteomics for the spatial localization and identification of pharmaceuticals, metabolites, lipids, peptides and proteins in biological tissues. However, sample preparation remains a crucial variable in obtaining the most accurate distributions. Common washing steps used to remove salts, and solvent-based matrix application, allow analyte spreading to occur. Solvent-free matrix applications can reduce this risk, but increase the possibility of ionisation bias due to matrix adhesion to tissue sections. We report here the use of matrix-free MSI using laser desorption ionisation performed on a 12 T Fourier transform ion cyclotron resonance (FTICR) mass spectrometer. We used unprocessed tissue with no post-processing following thaw-mounting on matrix-assisted laser desorption ionisation (MALDI) indium-tin oxide (ITO) target plates. The identification and distribution of a range of phospholipids in mouse brain and kidney sections are presented and compared with previously published MALDI time-of-flight (TOF) MSI distributions.

Protein oxidation:role in signalling and detection by mass spectrometry

Proteins can undergo a wide variety of oxidative post-translational modifications (oxPTM); while reversible modifications are thought to be relevant in physiological processes, non-reversible oxPTM may contribute to pathological situations and disease. The oxidant is also important in determining the type of oxPTM, such as oxidation, chlorination or nitration. The best characterized oxPTMs involved in signalling modulation are partial oxidations of cysteine to disulfide, glutathionylated or sulfenic acid forms that can be reversed by thiol reductants. Proline hydroxylation in HIF signalling is also quite well characterized, and there is increasing evidence that specific oxidations of methionine and tyrosine may have some biological roles. For some proteins regulated by cysteine oxidation, the residues and molecular mechanism involved have been extensively studied and are well understood, such as the protein tyrosine phosphatase PTP1B and MAP3 kinase ASK1, as well as transcription factor complex Keap1-Nrf2. The advances in understanding of the role oxPTMs in signalling have been facilitated by advances in analytical technology, in particular tandem mass spectrometry techniques. Combinations of peptide sequencing by collisionally induced dissociation and precursor ion scanning or neutral loss to select for specific oxPTMs have proved very useful for identifying oxidatively modified proteins and mapping the sites of oxidation. The development of specific labelling and enrichment procedures for S-nitrosylation or disulfide formation has proved invaluable, and there is ongoing work to establish analogous methods for detection of nitrotyrosine and other modifications.

Redox signalling and detection of protein oxidation by mass spectrometry

It is now recognised that redox control of proteins plays an important role in many signalling pathways both in health and disease. Proteins can undergo a wide variety of oxidative post-translational modifications (oxPTM); while the reversible modifications are thought to be most important in physiological processes, non-reversible oxPTM may contribute to pathological situations and disease. The oxidant is also important in determining the type of oxPTM (chlorination, nitration, etc.), and the susceptibilities of residues vary depending on their structural location. The best characterized oxPTMs involved in signalling modulation are partial oxidations of cysteine to the disulfide, glutathionylated or sulfenic acid forms, but there is increasing evidence that specific oxidations of methionine and tyrosine may have some biological roles. Well understood examples of oxidative regulation include protein tyrosine phosphatases, e.g. PTP1B/C, and members of the MAPK pathways such as MEKK1 and ASK1. Transcription factors such as NFkB and Nrf-2 are also regulated by redox-active cysteines. Improved methods for analysing specific oxPTMs in biological samples are critical for understanding the physiological and pathological roles of these changes, and tandem or MS3 mass spectrometry techniques interfaced with nano-LC separation are being now used. MS3 fragmentation markers for a variety of oxidized residues including tyrosine, tryptophan and proline have been identified, and a precursor ion scanning method that allows the selective identification of these oxPTMs in complex samples has been developed. Such advances in technology offer potential for biomarker development, disease diagnosis and understanding pathology.

Protein oxidative modifications:new mass spectrometry approaches to detection in biological samples

In inflammatory diseases, release of oxidants leads to oxidative damage to proteins. The precise nature of oxidative damage to individual proteins depends on the oxidant involved. Chlorination and nitration are markers of modification by the myeloperoxidase-H2O2-Cl- system and nitric oxide-derived oxidants, respectively. Although these modifications can be detected by western blotting, currently no reliable method exists to identify the specific sites damage to individual proteins in complex mixtures such as clinical samples. We are developing novel LCMS2 and precursor ion scanning methods to address this. LC-MS2 allows separation of peptides and detection of mass changes in oxidized residues on fragmentation of the peptides. We have identified indicative fragment ions for chlorotyrosine, nitrotyrosine, hydroxytyrosine and hydroxytryptophan. A nano-LC/MS3 method involving the dissociation of immonium ions to give specific fragments for the oxidized residues has been developed to overcome the problem of false positives from ions isobaric to these immonium ions that exist in unmodified peptides. The approach has proved able to identify precise protein modifications in individual proteins and mixtures of proteins. An alternative methodology involves multiple reaction monitoring for precursors and fragment ions are specific to oxidized and chlorinated proteins, and this has been tested with human serum albumin. Our ultimate aim is to apply this methodology to the detection of oxidative post-translational modifications in clinical samples for disease diagnosis, monitoring the outcomes of therapy, and improved understanding of disease biochemistry.