Carboxylate-substituted superhalogens of the type RCOOMX
(M=Mg, Ca, Sr, Ba, Mn, Co, Ni, Zn; X=Cl, Br) are easily accessible in the gas phase by electrospray ionisation. Their collision-induced ...dissociation (CID) characteristics have been probed by using ion-trap and triple-quadrupole mass analysers with particular emphasis on the behaviour of RCOOCaCl
-type ions. In the ion trap these appear to react readily with residual water to yield HOCaCl
as the hydrolysis product. In the absence of water, a collision-induced McLafferty-type rearrangement takes over to produce HCaCl
with the expulsion of an olefin and CO
. A brief computational analysis using the CBS-QB3 model chemistry provides a satisfactory rationale for these observations. If complexed with MX
(M=Mg, Ca, Sr, Ba), long-chain unsaturated aliphatic carboxylate anions undergo various backbone cleavages upon collision. These lead to structure-diagnostic olefin losses because the position of the double bonds remains intact. Such cleavages are absent in the bare ion RCOO
. The long-chain ions RCOOMX
also produce the intriguing species CO
MX
. These have been characterised by CID experiments, and theory indicates that they may be viewed as a CO
molecule captured by the salt anion MX
. Finally, it is shown that the CID spectra of RCOOCaCl
ions derived from all-trans retinoic acid, a compound of current interest in biochemistry and medicine, show a unique structure-diagnostic dissociation that may greatly aid its qualitative and quantitative analysis.
Early combined computational and experimental studies by J.K. Terlouw and colleagues propose that low-energy methyl carbamate ions, NH2COOCH3•+ (MC-1), rearrange into distonic ions NH2C(OH)OCH2•+ and ...hydrogen-bridged radical cations NH2C=O–H–OCH2•+ (MC-5) en route to the observed losses of HCO• and CO. In this study, we report on the generation of ionsMC-5 by decarbonylation of ionized methyl oxamate NH2COCOOCH3•+. Theory and experiment agree that ionMC-5 is a key intermediate in the dissociation of low-energy ionsMC-1. The subsequent HCO• loss, however, may not proceed via the route proposed by Terlouw et al., but rather by an entirely different mechanism involving proton–transport catalysis (PTC) in ionMC-5. This view is further supported by the dissociation behaviour of theMC-5 isotopologue ND2C=O–D–OCH2•+, which is conveniently generated from the d3-labelled glycolamide ion DOCH2C(=O)ND2•+
In a previous report J.W. de Beukelaar, J.W. Gratama, P.A. Sillevis Smitt, G.M. Verjans, J. Kraan, Th.M. Luider, P.C. Burgers, Rapid Commun. Mass Spectrom. 21 (2007) 1282 on the quality assessment of ...synthetic peptides used in protein-spanning peptide pools by matrix-assisted laser desorption/ionization Fourier transform mass spectrometry (MALDI-FTMS) we noted that certain peptides showed remarkably intense signals for their calcium-containing analogues. Here we report on a detailed mass spectrometric study of the unimolecular chemistry of these calcium-containing peptides. By integration of the experimental findings with computational results derived from DFT and the CBS-QB3 model chemistry, we have traced the processes induced by Ca
2+ attachment in the peptide ions.
Key to our analysis is the observation that all of the studied calcium-bound peptides containing a threonine or serine residue show prominent losses of CH
3CH
O (from threonine) and/or CH
2
O (from serine) in both the positive and the negative ion mode. In the first step, Ca
2+ attaches itself to a negatively charged in-chain carboxylate group. Next, electrophilic attack of the calcium ion on the
CH(R)OH group of threonine (R
CH
3) or serine (R
H) releases the hydroxyl proton which can then move to a suitable acceptor site,
viz. a peptide bond. This leads to the formation of a very stable ionic bidentate structure. Upon collisional activation (MS/MS), this bidentate opens up leading to the loss of the exposed acetaldehyde or formaldehyde molecule, to yield another bidentate structure.
MS/MS spectra of selected peptides interacting with other metal ions have also been investigated and it is found that only divalent ions follow the Ca
2+-induced transformations.
Up until today, no proteomics approaches have been described for heart muscle development. We describe a proteomics method to study the proteome of different heart structures at three stages of ...chicken embryonic development. For this purpose, a combination of gel separation, nanoLC separation and mass spectrometry was used. With this method, we identified in total 267 proteins in different tissue structures of chicken heart. We observed differences in protein abundance for a number of proteins between the different tissue structures and time points of development using spectral counting as a semiquantitative measure of protein abundance. For myosin-heavy chain 6, myosin-heavy chain 7, titin, connectin, collagen alpha-1, and xin, differences in protein levels for the different stages and structures (great arteries, outflow tract and ventricles) have been observed. A pathway analysis is performed in which the identified proteins are related to theoretical protein networks. Most prominent was the ‘cardiovascular system development and function’ network with the abundantly present proteins myosin 6 and myosin 7. We showed that myosin 6 is highly regulated in a stage and heart tissue specific manner. In conclusion, this method can be used to study changes in protein levels of chicken heart tissue in a spatiotemporal manner.
We investigated calcium-binding motifs of peptides and their recognition of active functionalities for coordination. This investigation generates the fundamentals to design carrier material for ...calcium-bound peptide-peptide interactions. Interactions of different peptides with active calcium domains were investigated. Evaluation of selectivity was performed by electrospray ionization mass spectrometry by infusing solutions containing two different peptides (P1 and P2) in the presence of calcium ions. In addition to signals for monomer species, intense dimer signals are observed for the heterodimer ions ( P 1 ⋯ C a 2 + ⋯ P 2 ) ( ⋯ represents the noncovalent binding of calcium with the peptide) in the positive ion mode and for ions ( P 1 - 2 H 2 - ⋯ Ca 2 + ⋯ P 2 - 2 H 2 - ) in the negative ion mode. Monitoring of the dissociation from these mass selected dimer ions via the kinetic method provides information on the calcium affinity order of different peptide sequences.
In this report we explore the use of MALDI-FTICR mass spectrometry for the quantitative analysis of five HIV-1 protease inhibitors in cell lysates. 2,5-Dihydroxybenzoic acid (DHB) was used as the ...matrix. From a quantitative perspective, DHB is usually a poor matrix due to its poor shot-to-shot and poor spot-to-spot reproducibilities. We found that the quantitative precisions improved significantly when DMSO (dimethylsulfoxide) was added to the matrix solution. For lopinavir and ritonavir, currently the most frequently prescribed HIV-1 protease inhibitors, the signal-to-noise ratios improved significantly when potassium iodide was added to the matrix solution. The mean quantitative precisions, expressed as % relative standard deviation, were 6.4% for saquinavir, 7.3% for lopinavir, 8.5% for ritonavir, 11.1% for indinavir, and 7.2% for nelfinavir. The mean quantitative accuracies, expressed as % deviation, were 4.5% for saquinavir, 6.0% for lopinavir, 5.9% for ritonavir, 6.6% for indinavir, and 8.0% for nelfinavir. The concentrations measured for the individual quality control samples were all within 85–117% of the theoretical concentrations. The lower limits of quantification in cell lysates were 4 fmol/µL for saquinavir, 16 fmol/µL for lopinavir, 31 fmol/µL for ritonavir, and 100 fmol/µL for indinavir and nelfinavir. The mean mass accuracies for the protease inhibitors were ⩽0.28 ppm using external calibration. Our results show that MALDI-FTICR mass spectrometry can be successfully used for precise, accurate, and selective quantitative analyses of HIV-1 protease inhibitors in cell lysates. In addition, the lower limits of quantification obtained allow clinical applications of the technique.
Mass spectrometry is a powerful tool for studying the intracellular pharmacokinetics of antiretroviral drugs. However, the biohazard of HIV-1 calls for a safety protocol for such analyses. To this ...end, we extracted HIV-1 producing cells with methanol or ethanol at 4
°C. After extraction, no viral infectivity was detected, as shown by a reduction in infectious titers of more than 6
log. In addition, this protocol is compatible with the quantitative analysis of antiretroviral drugs in cell extracts using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS. Thus, using this protocol, infectious HIV-1 is inactivated and antiretroviral drugs are extracted from cells in a single step.
Cobby et al use a depth cut-off of 1.5 mm, which is three times the standard deviation above the norm. 1 Warren et al conclude that a torn anterior cruciate ligament is suggested by a depth of more ...than 2.0 mm. 2 An association between a lateral femoral notch sign and a torn anterior cruciate ligament is found in 0% to 26% of patients, depending on the definition used, the population, and the mechanism of injury. 3 4 5 6 Based on radiographic results from four (>2.0 mm) of 124 patients with a torn anterior cruciate ligament, Yu et al found a sensitivity of 3.2%, specificity of 100%, and positive predictive value of 100% for complete tears of the anterior cruciate ligament. 6 Gentili et al found a sensitivity of 19% and a specificity of 100% on magnetic resonance imaging (MRI) of a group of 89 patients (54 torn and 35 normal anterior cruciate ligaments), using a depth of >1.5 mm. 7 On MRI, depending on the location and depth, Grimberg et al found a sensitivity of 49% to 66% and a specificity of 70% to 89%. MRI findings confirmed the tear, together with a posterior root tear of the lateral meniscus, a grade 1 sprain of the medial collateral ligament, and extensive bone marrow oedema of the lateral femoral condyle and proximal tibia. The lateral femoral notch sign is indicative of anterolateral rotatory instability of the knee and warrants further evaluation of the soft tissue by MRI. 1 Cobby MJ, Schweitzer ME, Resnick D. The deep lateral femoral notch: an indirect sign of a torn anterior cruciate ligament.
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