Gordon R. Nicol

Signal suppression in electrospray ionization Fourier transform mass spectrometry of multi-component samples

The high resolution, mass range and sensitivity of Fourier transform mass spectrometry (FTMS) suggest that it could be a valuable tool for the quantitative analysis of biomolecules. To determine the applicability of electrospray ionization combined with FTMS to the quantitation of biomolecules in multi-component samples, mixtures of varying compositions and concentrations of cytochrome c, angiotensin II, insulin and chicken egg white lysozyme were examined. The instrument used has an electrospray source with a hexapole trap to accumulate ions for injection into an ion cyclotron resonance mass analyzer. Linear responses for single component samples of angiotensin II and insulin were in the range 0.031-3 microM and those of both cytochrome c and lysozyme were between 0.031 and 1 microM. In examining various mixtures of the proteins with angiotensin II, it was found that the presence of the large molecules suppresses the signal of the smaller molecules. This is suggested to be a result of ion-ion interactions producing selective ion loss from either the hexapole trap or the ion cyclotron resonance mass analyzer trap. More massive, more highly charged ions can collisionally transfer large amounts of translational energy to smaller, less highly charged ions, ejecting the smaller ions from the trap. Mass discrimination effects resulting from the trapping voltage were also examined. It was found that relative signal intensities of ions of different masses depend on trapping voltage for externally produced ions. The effect is most significant for spectra including masses that differ by 30% or more. This suggests that for quantitation all samples and standards be run at a constant trapping potential.

Improved Mass Spectrometric Characterization of Protein Glycosylation Reveals Unusual Glycosylation of Maize-Derived Bovine Trypsin

Although bottom-up proteomics using tryptic digests is widely used to locate post-translational modifications (PTM) in proteins, there are cases where the protein has several potential modification sites within a tryptic fragment and MS(2) strategies fail to pinpoint the location. We report here a method using two proteolytic enzymes, trypsin and pepsin, in combination followed by tandem mass spectrometric analysis to provide fragments that allow one to locate the modification sites. We used this strategy to find a glycosylation site on bovine trypsin expressed in maize (TrypZean). Several glycans are present, and all are attached to a nonconsensus N-glycosylation site on the protein.

Proton affinities of saturated aliphatic methyl esters

The kinetic method was used to determine the proton affinities of methyl esters of several saturated fatty acids. Decompositions of the proton-bound dimers of the methyl esters, AHB+, were observed under different conditions with two instruments. The proton affinities (PAs) of the methyl esters increase continually with increasing carbon number in the acid. Equilibrium and initial rate experiments were performed with a Fourier transform ion cyclotron resonance mass spectrometer on the methyl ester of the C22 saturated acid (methyl behenate). These experiments give values for PA (methyl behenate) that are perhaps slightly lower than those obtained with the kinetic method. The PAs of the methyl esters of the fatty acids could be correlated with the equation: PA (ester) = (40.0 ± 2.5)*log(n) + (784.7 ± 3.9) kJ/mol or PA (ester) = (864 ± 2) − (479 ± 41)/n, wheren = number of atoms in the molecule. Proton affinities of smaller sets of 1-alkylamines and 1-alkanols can be fit to similar equations.

Hydrothermal reactions of 1-nitrobutane in high-temperature water

Abstract Development of a rational basis for the design of safe and reliable methods for the destruction of high-energy materials and explosives motivated the study of the reactions of model explosive compounds in high-temperature water (HTW). Specifically, the reaction of 1-nitrobutane in HTW occurred with a pseudo-first-order-rate constant almost three times greater than that for neat pyrolysis. Water also influenced the product spectrum. The preliminary reaction network included the formation of nitriles and aldehydes as primary products, amides and acids as secondary products, and esters and acids as higher-rank products. In contrast, pyrolysis, under otherwise identical conditions, yielded nitriles, pyrolidine derivatives, and char as products. Free radical and hydrolysis mechanisms explain 1-nitrobutane product formation pathways well.

Apparent proton affinities of highly charged peptide ions

The apparent proton affinities (PA) of various charge states of three highly basic peptides [(KAP)10, (KAP)8, (KAA)8] were measured by the “bracketing” method. For all three peptides the apparent PA decreases as the charge state increases and the magnitude of the decrease is consistent with an increase in coulombic repulsion in the highly protonated species. Based on a simple electrostatic model, theoretical PAs were predicted for each charge state and the values for (KAP)10 and (KAP)8 were within 10 kcal/mol of the experimental values. The maximum charge state of these peptides was observed in all cases even when the most volatile solvent was sufficiently basic to deprotonate that charge state in the gas phase. In solution (KAP)8 exhibits a random coil secondary structure while (KAA)8 exhibits an α-helix structure. Comparison of measured and calculated apparent PAs suggests that (KAP)8 retains its solution random coil structure in the gas phase and (KAA)8 retains the solution compact α-helix structure in the lower charge states but opens up to a β structure in the gas phase to minimize electrostatic repulsions in higher charge states.

Gas chromatography/mass spectrometry of polyethylene glycol oligomers

Polyethylene glycol (PEG) oligomers, H(OC2H4O) n H, were analyzed by gas chromatography/mass spectrometry (GC/MS) for oligomers, n = 2–11, without indications of pyrolysis of the higher oligomers at oven temperatures of 330°C. The electron ionization (EI) mass spectra (70 eV and 12–14 eV) of these PEG oligomers contain only low molecular weight fragment ions and, essentially, no M+ ions. The low-voltage EI spectra contain more abundant high mass ions and allow easier identification of the PEG oligomers. The major fragment ions in all EI spectra are (C2H4O) x H+ at m/z 45, 89, 133 and 177. The base peak in the low-voltage spectra varies with molecular weight and suggests preferential cleavage at the second and third ether linkages. The relative molar sensitivities (70 eV EI) of PEG oligomers (n = 1–6) are linear functions of molecular weight or polarizability ratios. The CH4 chemical ionization mass spectra of the PEG oligomers contain, predominantly, (C2H4O) x H+ ions at m/z 45, 89, 133 and 177. The relative abundances of MH+ ions vary markedly across the chromatographic peaks because these ions are formed predominantly by sample ion / sample molecule reactions. Proton transfer reactions from CH5+ and C2H5+ to the PEG oligomers are almost entirely dissociative. The i-C4H10 CI mass spectra of PEG oligomers contain MH+ as the base peaks. The relative abundances of MH+ ions do not vary significantly across the chromatographic peaks but do increase with increasing pressure of i-C4H10. The extent of decomposition of MH+ ions in i-C4H10 CI mass spectra increases with increasing molecular weight of the PEG oligomers. Relative molar sensitivities (CH4 CI and i-C4H10 CI) of PEG oligomers (n = 1–6) are essentially the same linear functions of molecular weight ratio.

A method of rate constant measurement in Fourier transform ion cyclotron resonance pulsed valve experiments

Rate constant measurements in Fourier-transform ion cyclotron resonance (FT-ICR) pulsed valve experiments are complicated by the pressure changes which occur rapidly after the opening of the pulsed valve. A method is described here to overcome that complication. The time-dependent number density n(t) is sampled by determining the total ion signal I(tn) in spectra resulting from pulsing the electron beam on at various delay times tn after opening the valve. The I(tn) are fit to an analytic function to give I(t) which can be integrated to give a kinetic variable comparable to pressure at fixed time or time at fixed pressure. A reactant ion isolated in the cell should disappear exponentially with ∫ 0 t n I(t) dt where the valve opens at time 0 admitting reactant neutral and the reactant ion concentration is sampled at time tn. To obtain the rate constant from such an exponential decay the ionization cross section of the neutral must be known or estimated and the overall sensitivity of the instrument must be known. The latter can be determined by examining a reaction of known rate constant. The procedure is illustrated for the reaction of Re2+ with cyclohexane. The implications of this treatment for other types of pulsed valve experiments are discussed.