Comparison of different mass spectrometry techniques in the measurement of L‐[ring‐13C6]phenylalanine incorporation into mixed muscle proteins

Precise measurement of low enrichment of stable isotope labeled amino‐acid tracers in tissue samples is a prerequisite in measuring tissue protein synthesis rates. The challenge of this analysis is augmented when small sample size is a critical factor. Muscle samples from human participants following an 8 h intravenous infusion of L‐[ring‐¹³C₆]phenylalanine and a bolus dose of L‐[ring‐¹³C₆]phenylalanine in a mouse were utilized. Liquid chromatography tandem mass spectrometry (LC/MS/MS), gas chromatography (GC) MS/MS and GC/MS were compared to the GC‐combustion‐isotope ratio MS (GC/C/IRMS), to measure mixed muscle protein enrichment of [ring‐¹³C₆]phenylalanine enrichment. The sample isotope enrichment ranged from 0.0091 to 0.1312 molar percent excess. As compared with GC/C/IRMS, LC/MS/MS, GC/MS/MS and GC/MS showed coefficients of determination of R² = 0.9962 and R² = 0.9942, and 0.9217 respectively. However, the precision of measurements (coefficients of variation) for intra‐assay are 13.0%, 1.7%, 6.3% and 13.5% and for inter‐assay are 9.2%, 3.2%, 10.2% and 25% for GC/C/IRMS, LC/MS/MS, GC/MS/MS and GC/MS, respectively. The muscle sample sizes required to obtain these results were 8 µg, 0.8 µg, 3 µg and 3 µg for GC/C/IRMS, LC/MS/MS, GC/MS/MS and GC/MS, respectively. We conclude that LC/MS/MS is optimally suited for precise measurements of L‐[ring‐¹³C₆]phenylalanine tracer enrichment in low abundance and in small quantity samples. Copyright © 2013 John Wiley & Sons, Ltd.     

Structure and dynamics of silk fibroin studied with 13C, 15N and 2H solid state NMR

[1-¹³C]Gly, L-[1-¹³C]Ala, [¹⁵N]Gly, L-[¹⁵N]Ala, [2,2-²H₂]Gly, L-[3,3-²H₂]Ser and [3,3,3-²H₃]Ala labeled silk fibroin fibers from Bombyx mori and Samia cynthia ricini silkworms were prepared in order to analyze structure of backbone and dynamics of side chain. The torsion angles ϕ and Ψ were determined from the angular dependent ¹³C and ¹⁵N solid state NMR spectra for uniaxially oriented fiber samples. In addition, the characteristic side chain dynamics of Ser residue determined from solid state ²H NMR measurements was compared with those of Ala and Gly residues.     

Synthesis of Deuterium Labeled Tryptamine Derivatives

The synthesis of deuterium labeled tryptamine derivatives, [2‐(1H‐indol‐3‐yl)‐[²H₄]‐ethyl]‐dimethylamine (DMT), [²H₁₀]‐diethyl‐[2‐(1H‐indol‐3‐yl)‐ethyl]‐amine (DET), [2‐(1H‐indol‐3‐yl)‐ethyl]‐[²H₆]‐dipropyl‐amine (DPT) and [²H₂]‐alpha‐methyltryptamine (AMT) is described. The isotopically labeled compounds are used as internal standards in gas chromatography‐mass spectrometry (GC‐MS) assays.     

Enzymatic synthesis of dopamine ring labeled with hydrogen isotopes

The synthesis of two isotopomers of L-DOPA, the precursors for labeled dopamine is reported. The ring labeled with deuterium or tritium [2′,5′,6-²H₃]-L-DOPA, and [2′,5′,6-³H₃]-L-DOPA were obtained using acid catalyzed isotopic exchange between L-DOPA and heavy or tritiated water, respectively. Their derivatives, i.e., [2′,5′,6-²H₃], and [2′,5′,6-³H₃]-dopamine were obtained by enzymatic decarboxylation of deuterated or tritiated isotopomers of L-DOPA using enzyme tyrosine decarboxylase (EC 4.1.1.25).     

Synthesis of L-tryptophan labeled with hydrogen isotopes in the indole ring

The isotopomers of L-tryptophan (L-Trp) labeled with hydrogen isotopes in the indole ring were obtained by isotope exchange method. Heavy and tritiated water were used as the sources of stable and radioactive label. Three isotopomers, i.e., [4′-²H]-L-Trp, [4′-³H]-L-Trp, and doubly labeled [4′-²H/³H]-L-Trp were synthesized by isotope exchange enhanced by irradiation with a mercury discharge lamp. The indole whole ring labeled isotopomers, i.e., [2′,4′,5′,6′,7′-²H₅]-L-, [2′,4′,5′,6′,7′-³H₅]-L-, and doubly labeled [2′,4′,5′,6′,7′-²H/³H]-L-Trp were synthesized by isotope exchange between L-Trp and D₂O or DTO in the presence of deuteriated trifluoroacetic acid.     

Kinetics of anation of chromium(III) by L-phenylalanine

Summary The kinetics of anation of chromium(III) species, [Cr(H₂O)₆]⁴⁺ and [Cr(H₂O)₅OH]²⁺, by L-phenylalanine in aqueous acid has been studied spectrophotometrically. Effects of varying [substrate], [ligand], [H⁺], , % ethanol and temperature were investigated. The kinetic data suggest a mechanism where outersphere-associations [between chromium(III) species and phenylalanine in the zwitterionic form] precede anation. Comparison of the results with published data suggest an I_a path for the [Cr(H₂O)₆]³⁺ reaction and I_d path for the [Cr(H₂O)₅OH]²⁺ reaction.     

Characterization of [M–H] cations,radicals and anions of glycine in the gas phase: a combined experimental and ab initio study

The gas phase structures of the [M–H] cations and anions of glycine have been studied by using a combination of ab initio calculations (at the MP2(FC)/6–31+G1 level of theory) and tandem mass spectrometry (MS/MS). It was found that the ab initio stability order for the anions is [H₂NCH₂CO₂]⁻ > [H₂NCHCO₂H]⁻ > [HNCH₂CO₂H]⁻. In contrast, the cations exhibit different behaviour, whereas [H₂NCHCO₂H]⁺ is predicted to be a stable structure, [H₂NCH₂CO₂]⁺ spontaneously fragments to the ion–molecule complex [H₂NCH₂⁺ ⋯ (OCO)] and the singlet [HNCH₂CO₂H]⁺ isomer is predicted to undergo a skeletal rearrangement to form [CH₂NHCO₂H]⁺. MS/MS spectra of [M–H]⁺ cations of various glycine isotopomers were obtained via: (i) collisional activation of electron impact generated cations and (ii) charge reversal of anions formed via HO⁻ negative ion chemical ionization. The resulting spectra were significantly different, suggesting different structures were involved. Neutralization–reionization experiments were performed on [M–H]⁻ anions in order to gain insights into the structures of the intermediate radicals.     

Gamma-radiation combined with tricycloazole to protect tempera paintings in ancient Egyptian tombs (Nile Delta,Lower Egypt)

In order to comprehensively understanding the metabolism process of fluvastatin, the stable isotope-labeled (3R, 5S)-fluvastatin and (3S, 5R)-fluvastatin were required. Both of (3R, 5S)-fluvastatin and (3S, 5R)-fluvastatin were synthesized via a seven steps procedure starting from aniline and [²H₆] 2-bromopropane. Two versions of deuterium labeled compound revealed over 98% deuterium enrichment, using as an internal standard.

     

Evaluation of AM1-calculated radical cation ion-neutral complexes

AM1 semiempirical molecular orbital calculations are reported for 20 ion-neutral complexes, including hydrogen-bonded complexes, presumably involved in the gas-phase unimolecular decomposition of simple organic radical cations. The systems investigated are [C₂H₄O₂]^˙+, [C₂H₅NO]^˙+, [C₂H₆O]^˙+, [C₂H₆O₂]^˙+, [C₃H₆O]^˙+, [C₃H₆O₂]^˙+, [C₃H₈O]^˙+, and [C₃H₈O₂]^˙+. The AM1 results are compared with ab initio molecular orbital calculations at different levels of theory up to MP3/6-31G(d, p)//SCF/6-31G(d) + ZPVE and the available experimental data. AM1 fails to predict some local minima and the equilibrium geometries calculated for several complexes are found to be qualitatively different from those predicted by the ab initio calculations. However, reasonable agreement is generally found for the stabilization energies of the complexes toward dissociation into their loosely bound components. © John Wiley & Sons, Inc.     

Isomeric [C,H3,N,O]+˙ ions and their neutral counterparts

The isomeric ions [H₂NC(H)O]⁺˙, [H₂NCOH]⁺˙, [H₃CNO]⁺˙ and [H₂CNOH]⁺˙ were examined in the gas phase by mass spectrometry. Ab initio molecular orbital theory was used to calculate the relative stabilities of [H₂NC(H)O]⁺˙, [H₂NCOH]⁺˙, [H₃NCO]⁺˙ and their neutral counterparts. Theory predicted [H₂NC(H)O]⁺˙ to be the most stable ion. [H₂NCOH]⁺˙ ions were generated via a 1,4-hydrogen transfer in [H₂NC(O)OCH₃]⁺˙, [H₂NC(O)C(O)OH]⁺˙ and [H₂NC(O)CH₂CH₃]⁺˙. Its metastable dissociation takes place via [H₃NCO]⁺˙ with the isomerization as the rate-determining step. [H₂CNOH]⁺˙ undergoes a rate-determining isomerization into [H₃CNO]⁺˙ prior to metastable fragmentation. Neutralization-reionization mass spectrometry was used to identify the neutral counterparts of these [H₃,C,N,O]⁺˙ ions as stable species in the gas phase. The ion [H₃NCO]⁺˙ was not independently generated in these experiments; its neutral counterpart was predicted by theory to be only weakly bound.     

Characterization of isomeric [CH5P]+˙ and [C2H7P]+˙ radical cations and some [C2H6P]+ phosphonium ions in the gas phase by their collisional activation spectra

Collisional activation spectra were used to characterize isomeric ion structures for [CH₅P]^+˙ and [C₂H₇P]^+˙ radical cations and [C₂H₆P]⁺ even-electron ions. Apart from ionized methylphosphane, [CH₃PH₂]^+˙, ions of structure [CH₂PH₃]^+˙ appear to be stable in the gas phase. Among the isomeric [C₂H₇P]^+˙ ions stable ion structures [CH₂PH₂CH₃]^+˙ and [CH₂CH₂PH₃]^+˙/[CH₃CHPH₃]^+˙ are proposed as being generated by appropriate dissociative ionization reactions of alkyl phosphanes. At least three isomeric [C₂H₆]⁺ ions appear to exist, of which \documentclass{article}\pagestyle{empty}\begin{document}$ {\rm CH}_{\rm 3} - \mathop {\rm P}\limits^{\rm + } {\rm H = CH}_{\rm 2} $\end{document} could be identified positively.     

Alkali Metal/Ammonia Reductions of Ketones Should Be Run in the Presence of Ammonium Ion

Reduction of (+)-[3,3?²H₂]camphor ([3,3?²H₂] 1 ) with lithium, sodium or potassium in ammonia and a co-solvent gave; 1) the enolate of [3,3?²H₂] 1 and the alcoholates of (?)-[2,3,3?²H₃]isoborneol ([2,3,3?²H₃] and (+)-[2,3,3?²H₃]borneol ([2,3,3?²H₃] 3 ); 2) the alcoholates of [3,3?²H₂] 2 and [3,3?²H₂] 3 ; 3) the dialcoholates of the pinacols [3,3,3′,3′?²H₄] 4 and [3,3,3′,3′?²H₄] 5 . It is proposed that these are formed from the ketyls [3,3?²H₂] 1 - M⁺, by: 1) disproportionation; 2) H-atom abstraction from the medium; 3) dimerization. Protonation upon work-up afforded [endo?3²H] 1 , [2,3,3?²H₃] 2 , [2,3,3?²H₃] 3 ,[3,3?²H₂] 2 , [3,3?²H₂] 3 , [3,3,3′,3′?²H₄] 4 and [3,3,3′,3′?²H₄] 5 . Pinacol [3,3,3′,3′?²H₄] 5 was the main and pinacol [3,3,3′,3′?²H₄] 4 a minor product in the reductions with lithium and both were minor products in the reductions with sodium; pinacols were not formed in the reductions with potassium. Parallel reductions of 1 , unlabeled, analogously led to 2 , 3 , 4 and 5 , and the ratios 2/3 differed from the ratios ([2,3,3?²H₃] 2 +[3,3?²H₂] 2 /([2,3,3?²H₃]+[3,3?²H₂] 3 ) under certain conditions. Different values for these ratios were found in the reductions with each metal, all of which corresponded to low overall diastereoselectivities. Reactions 1 and 3 persisted when the reductions were carried out in ammonia/water/co-solvent mixtures and the enolate formed via reaction 1 was protonated and the resulting [endo-3-²H] 1 recycled. Reaction 2 cannot be monitored under these conditions. Reactions 1 and 3, and by inference also reaction 2, were almost completely suppressed when analogous reductions were carried out in the presence of ammonium chloride, [3,3?²H₂] 2 and [3,3?²H₂] 3 being obtained almost exclusively, in a 6: 94 ratio. It is proposed that the mechanism outlined in House [1] was dominant when, and only when, ammonium ion was the proton source; it may have competed when water was the proton source.     

Characterization and application of the fac-[188Re(CO)3(H2O)3]+ core

Summary Fac-[¹⁸⁸Re(CO)₃(H₂O)₃]⁺ was synthesized with an overall radiochemical yield of 80±5%, and more than 95% radiochemical purity after a QMA Sep-Pak column separation. Fac-[Re(CO)₃(H₂O)₃]⁺ was also synthesized as a reference sample. The structure of the precursor, fac-[¹⁸⁸Re(CO)₃(H₂O)₃]⁺, was confirmed by high performance of liquid chromatography (HPLC). MN-His (magnetic nanoparticles coated with silica and modified with an amino silane coupling agent, N-[3-(trimethyoxysilyl)propyl]-ethylenediamine (SG-Si900) and immobilized with histidine) was labeled with fac-[¹⁸⁸Re(CO)₃(H₂O)₃]⁺ and an initial animal test of MN-His was conducted for a magnetic targeting study.     

Fast-atom bombardment tandem mass spectrometry of [13C]arachidonic acid labeled phospholipid molecular species

A method employing stable isotope labeling and fast-atom bombardment (FAB) tandem mass spectrometry has been developed to directly assess events of biosynthesis and metabolism of arachidonic acid containing phospholipid molecular species by cells carried in culture. Mast cells, cultured with [¹³C]linoleic acid, converted this precursor into arachidonic acid which was then incorporated into cellular phospholipids. Over a 24 hour period, the extent of label enrichment in each arachidonate-containing phospholipid molecular species was monitored by using negative FAB ionization with selected reaction monitoring. Specific incorporation of [¹³C₁₇] labeled arachidonate was determined from the ratio of the carboxylate anions at m/z 320 and 303, which correspond to [¹³C₁₇]arachidonate and unlabeled arachidonate, respectively, produced by collision-induced dissociation of each specific molecular anion. The use of [¹³C]linoleic acid as a precursor of arachidonic acid avoids the problem of changing the endogenous pool size by directly adding labeled arachidonic acid. Measurement of the [¹³C₁₇]label also avoids interferences from endogenous isobaric fatty acids that are naturally present at low levels.     

Characterization of novel oxidation products of cysteine in an active site motif peptide of PTP1B

We investigated the formation of hydroxyl radical (OH^·) and H₂O₂ mediated oxidation products of a synthetic peptide, HCSAGIGRS, which is an active site sequence motif of protein tyrosine phosphatase 1B (PTP1B). We determined that a novel cysteine sulfinamide HC[S(O)N]SAGIGRS is produced in the oxidation reaction by Fenton reagents (Fe⁺²/H₂O₂) as well as by H₂O_2. These products were characterized by tandem mass spectrometry experiments on both singly and doubly charged precursor ions. MS³ experiments using an ion trap instrument as well as LC-MS/MS experiments using a quadrupole time-of-flight (Q-TOF) instrument demonstrated that HC[S(O)N]SAGIGRS is not a water loss product of cysteine sulfinic acid [HC(SO₂H)SAGIGRS]. We also obtained data from tandem mass spectrometry experiments that provided evidence for the existence of stable cysteine sulfenic acid [HC(SOH)SAGIGRS] in solution. A mechanism for the formation of the cysteine sulfinamide product is proposed based on the above experimental results. The preparation and identification of cysteine sulfinamide in this study may provide insight into the mechanism of both OH^· and H₂O₂ induced oxidation reactions of protein tyrosine phosphatases.     

The heats of formation of some protonated olefinic carbonyl compounds: [C5H9O]+ and [C4H7O2]+ ions

The proton transfer equilibrium reactions involving 3-penten-2-one, 3-methyl-3-buten-2-one, crotonic acid and methacrylic acid were carried out in an ion cyclotron resonance (ICR) spectrometer. The semiempirical method MNDO, used to estimate the heats of formation for 14 protonated [C₅H₉O]⁺ and [C₄H₇O₂]⁺ ions and the energetic aspect of the fragmentations of metastable [C₆H₁₂O]^+. and [C₆H₁₂O₂]^+. ions, leads to the conclusion that the ions corresponding to protonation at the carbonyl oxygen are the most stable. Thus the experimentally determined heats of formation of protonated olefinic carbonyl compounds can be attributed to the following structures: [CH₃COHCHCHCH₃]⁺ (δH_f = 490 KJ mol^?1), [CH₃COHC(CH₃)CH₂]⁺ (δH_f = 502 KJ mol^?1), [HOCOHCHCHCH₃]⁺ (δH_f = 330 KJ mol^?1) and [HOCOHC(CH₃)CH₂]⁺ (δH_f = 336 KJ mol^?1).     

Structure and mechanism of fragmentation of [C2H5O]+

The decomposition reactions of [C₂H₅O]⁺ ions produced by dissociative electron-impact ionization of 2-propanol have been studied, using ¹³C and deuterium labeling coupled with metastable intensity studies. In addition, the fragmentation reactions following protonation of appropriately labeled acetaldehydes and ethylene oxides with [H₃]⁺ or [D₃]⁺ have been investigated. In both studies particular attention has been paid to the reactions leading to [CHO]⁺, [C₂H₃]⁺ and [H₃O]⁺. In both the electron-impact-induced reactions and the chemical ionization systems the fragmentation of [C₂H₅O]⁺ to both [H₃O]⁺ and [C₂H₃]⁺ proceeds by a single mechanism. For each case the reaction involves a mechanism in which the hydrogen originally bonded to oxygen is retained in the oxygen containing fragment while the four hydrogens originally bonded to carbon become indistinguishable. The fragmentation of [C₂H₅O]⁺ to produce [CHO]⁺ proceeds by a number of mechanisms. The lowest energy route involves complete retention of the α carbon and hydrogen while a higher energy route proceeds by a mechanism in which the carbons and the attached hydrogens become indistinguishable. A third distinct mechanism, observed in the electron-impact spectra only, proceeds with retention of the hydroxylic hydrogen in the product ion. Detailed fragmentation mechanisms are proposed to explain the results. It is suggested that the [C₂H₅O]⁺ ions formed by protonation of acetaldehyde or ionization of 2-propanol are produced initially with the structure [CH₃CH?\documentclass{article}\pagestyle{empty}\begin{document}$ \mathop {\rm O}\limits^ + $\end{document}H] (a), but isomerize to [CH₂?CH? \documentclass{article}\pagestyle{empty}\begin{document}$ \mathop {\rm O}\limits^ + $\end{document}H₂] (e) prior to decomposition to [C₂H₃]⁺ or [H₃O]⁺. The results indicate that the isomerization a → e does not proceed directly, possibly because it is symmetry forbidden, but by two consecutive [1,2] hydrogen shifts. A more general study of the electron-impact mass spectrum of 2-propanol has been made and the fragmentation reactions proceeding from the molecular ion have been identified.     

Doubly charged ion mass spectra: IV—chlorinated and brominated alkanes

Doubly charged ion mass spectra have been obtained for 42 chlorinated and brominated n-alkane (methyl through octyl) hydrocarbons. A double focusing Hitachi RMU-7L mass spectrometer, operated at 1.6kV accelerating voltage, has been used to measure the spectra. Molecular doubly charged ions have not been observed. Intense fragment ions have been produced from extensive H and halogen loss as well as C? C bond rupture of the parent molecule. The most abundant ions in the doubly charged ion spectra observed in this investigation resulted from reactions of [Cl]²⁺˙, [Br]²⁺˙, [CCL₂]²⁺, [C₂H₂Cl]²⁺˙, [C₃H₂]²⁺, [C₃HCl]²⁺, [C₃HBr]²⁺, [C₄H₃]²⁺˙, [C₄H₄]²⁺, [C₄H₆Br]²⁺˙, [C₄H₈Br]²⁺˙, [C₅H₂]²⁺, [C₆H₆]²⁺, [C₆H₈]²⁺ and [C₇H₈]²⁺. The prominent doubly charged fragment ions formed by electron impact of the smaller halogenated alkanes generally contained halogen, whereas ions of the type [C_nH_x]²⁺ were dominant in the spectra of higher molecular weight mono- and dihalogenated alkanes. Appearance energies of several ions have been measured. A geometry optimized quantum mechanical SCF treatment has been used to compute energies, charge densities and structures of doubly charged halogenated alkane ions.     

Structures of decomposing and non-decomposing gas-phase [C4H6O]+· radical cations,and their [C2H2O]+· and [C3H6]+· product ions

The structures of gas-phase [C₄H₆O]^+· radical cations and their daughter ions of composition [C₂H₂O]^+· and [C₃H₆]^+· were investigated by using collisionally activated dissociation, metastable ion measurement, kinetic energy release and collisional ionization tandem mass spectrometric techniques. Electron ionization (70 eV) of ethoxyacetylene, methyl vinyl ketone, crotonaldehyde and 1-methoxyallene yields stable [C₄H₆O]^+· ions, whereas the cyclic C₄H₆O compounds undergo ring opening to stable distonic ions. The structures of [C₂H₃O]^+· ions produced by 70-eV ionization of several C₄H₆O compounds are identical with that of the ketene radical cation. The [C₃H₆]^+· ions generated from crotonaldehyde, methacrylaldehyde, and cyclopropanecarboxaldehyde have structures similar to that of the propene radical cations, whereas those ions generated from the remainder of the [C₄H₆O]^+· ions studied here produced a mixed population of cyclopropane and propene radical cations.     

Carbon skeletal rearrangements in 2-phenylthiophene and 2,5-diphenylthiophene under conditions of electron-impact

The mass spectra of ¹³C-labelled 2-phenylthiophenes and 2,5-diphenylthiophenes were studied. The label distributions for the [HCS]⁺, [C₂H₂S]^+·, [C₈H₆]^+·, [C₉H₇]⁺ and [C₇H₅]S⁺ ions from 2-phenylthiophene and the [HCS]⁺, [C₉H₇]⁺, [C₇H₅S]^+·, and [C₁₅H₁₁]⁺ ions from 2,5-diphenylthiophene were interpreted in terms of both carbon skeletal rearrangements in the thiophene ring and migration of the phenyl substituent. The degree of carbon scrambling in the thiophene ring appeared to be almost independent of the electron beam energy. The formation of some of the fragment ions studied seems to be so fast that no carbon scrambling could be detected at all; in neither case was complete scrambling of the carbon atoms of the thiophene ring observed.