Characterization of cysteinylation of pharmaceutical-grade human serum albumin by electrospray ionization mass spectrometry and low-energy collision-induced dissociation tandem mass spectrometry

Three samples of albumin derived from human plasma (pharmaceutical grade, HSA) obtained from different commercial sources were investigated for their micro-heterogeneities by means of electrospray ionization (ESI) ion trap mass spectrometry (ITMS). The study covered MS analyses of the intact proteins as well as on the tryptic peptide level. The intact protein samples were analyzed without any separation step except for simple desalting. With these samples we observed in the positive ion ESI mass spectra that the multiply charged ion signals of HSA consisted of a number of fully or partly resolved peaks with relative intensities depending on the analyzed sample. The non-modified form of HSA was detected in the three HSA preparations at m/z values of 66448 +/- 3.6, 66450 +/- 0.6 and 66451 +/- 3.2 ([MH]+), respectively. The value calculated from the amino acid sequence was 66439. The second compound present with high intensity (in two cases the base peak in the deconvoluted mass spectrum) is interpreted as a modified HSA, and the molecular mass increase in relation to the unmodified HAS was between 116 and 118 Da (m/z of 66 564, 66 567 and 66 569), suggesting the presence of a covalently bound cysteine residue. A further peak in the deconvoluted ESI spectra was found in all three samples with rather low signal/noise ratio at m/z 66 619, 66 621 and 66 613, respectively, which may correspond to a non-enzymatic glycation described in the literature. The verification of the proposed covalent HSA modifications was subsequently done on the peptide level using high-performance liquid chromatography (HPLC)/ESI-MS and HPLC/ESI-MS/MS including low-energy collision-induced dissociation (CID). Prior to the tryptic digestion, the HSA samples were alkylated without a prior reduction step. Following this procedure we detected peptides of the sequence T21-41 that included the Cys-34 residue in both forms: cysteinylated (m/z 639.15 [M+4H]4+) as well as vinylpyridine-alkylated (m/z 635.69 [M+4H]4+, which means in its previously native free SH form). In the next step on-line LC/ESI low-energy CID MS/MS experiments were performed to verify these two proposed structures. By means of MS/MS analysis of the mentioned ions the described modification (cysteinylation) at the Cys-34 residue could be proven. This abundant modification of HSA in pharmaceutical-grade preparations could be unambiguously identified as cysteinylation at the Cys-34 residue. On the other hand, the proposed non-enzymatic glycation was not detectable on the peptide level in the on-line HPLC/ESI-MS mode, maybe due to the low concentration in the three samples under investigation.     

Mass spectral fragmentation pathways in some dinitroaromatic compounds studied by collision-induced dissociation and tandem mass spectrometry

A collision-induced dissociation study of a series of dinitroaromatic compounds was carried out using a tandem BB mass spectrometer. Fragmentation pathways were determined in the electron impact mode. Loss of NO₂˙ from the molecular ion was observed In most of the investigated compounds. In some compounds loss of NO₂˙ occurred only after loss of OH˙. In other compounds it was not observed at all because of competitive processes, such as loss of NO˙, CO₂, CH₂O, C₂H₄ or H₂O. Loss of NO˙ was a major decomposition pathway, forming ‘dished peaks’ in some of the compounds having a nitro group ortho to a phenyl group, indicating a release of kinetic energy associated with the decomposition. Loss of OH˙ due to an ‘ortho effect’ occurred in compounds where a nitro group was ortho to a group containing a labile hydrogen, but was not observed when competitive processes such as loss of NO˙, NO₂˙ or H₂O occurred. ‘Nitro to nitrite’ isomerization was suggested to explain the decarboxylation process in 2,4- and 2,5-dinitrobenzoic acid and the loss of COH₂ in 2,4-dinitroanisole.     

Characterisation of anthocyanidins by electrospray ionisation and collision-induced dissociation tandem mass spectrometry.

The present study describes the use of electrospray ionisation mass spectrometry, in combination with collision-induced dissociation (CID) and tandem mass spectrometry, for the structural characterisation of anthocyanidins and their O-glycosides. The high-energy CID spectra of [M-Cl](+) ions of the free aglycones show characteristic fragmentation pathways, which provide useful information about the substitution pattern in the A- and B-rings of each compound. The major fragmentation observed in the high-energy CID spectra of [M-Cl](+) ions of anthocyanins involves loss of the mono- or disaccharide units resulting in ions containing only the aglycone moiety. From the spectral data, the identity of the aglycone can be established as well as the number and the class of monosaccharide units in the O-glycosides.     

Atmospheric pressure chemical ionization of fluorinated phenols in atmospheric pressure chemical ionization mass spectrometry, tandem mass spectrometry, and ion mobility spectrometry

Atmospheric pressure chemical ionization (APCI)-mass spectrometry (MS) for fluorinated phenols (C6H5-xFxOH Where x = 0-5) in nitrogen with Cl- as the reagent ion yielded product ions of M Cl- through ion associations or (M-H)- through proton abstractions. Proton abstraction was controllable by potentials on the orifice and first lens, suggesting that some proton abstraction occurs through collision induced dissociation (CID) in the interface region. This was proven using CID of adduct ions (M Cl-) with Q2 studies where adduct ions were dissociated to Cl- or proton abstracted to (M-H)-. The extent of proton abstraction depended upon ion energy and structure in order of calculated acidities: pentafluorophenol > tetrafluorophenol > trifluorophenol > difluorophenol. Little or no proton abstraction occurred for fluorophenol, phenol, or benzyl alcohol analogs. Ion mobility spectrometry was used to determine if proton abstraction reactions passed through an adduct intermediate with thermalized ions and mobility spectra for all chemicals were obtained from 25 to 200 degrees C. Proton abstraction from M Cl- was not observed at any temperature for phenol, monofluorophenol, or difluorophenol. Mobility spectra for trifluorophenol revealed the kinetic transformations to (M-H)- either from M Cl- or from M2 Cl- directly. Proton abstraction was the predominant reaction for tetra- and penta-fluorophenols. Consequently, the evidence suggests that proton abstraction occurs from an adduct ion where the reaction barrier is reduced with increasing acidity of the O-H bond in C6H5-xFxOH.     

Fragmentation study of protonated chalcones by atmospheric pressure chemical ionization and tandem mass spectrometry

Fragmentation mechanisms of protonated chalcone and its derivatives with different functional groups were investigated by atmospheric pressure chemical ionization with tandem mass spectrometry (MS/MS). The major fragmentation pathways were loss of the phenyl group from the A or B ring, combined with loss of CO. Losses of H(2)O and CO from the precursor ions of [M+H](+) are proposed to occur via rearrangements. Elimination of water from protonated chalcones was observed in all the title compounds to yield a stable ion but it was difficult to obtain skeletal fragmentation of a precursor ion. Loss of CO was found in the MS/MS spectra of all the compounds except the nitro-substituted chalcones. When the [M+H--CO](+) ion was fragmented in the MS/MS experiments, there were distinctive losses of 15 and 28 Da, as the methyl radical and ethylene, respectively. The ion at m/z 130, found only in the nitro-substituted chalcones, was assigned as C(9)H(6)O by Fourier transform ion cyclotron resonance (FTICR)-MS/MS; m/z 130 is a common fragment ion in the electron ionization (EI) spectra of chalcones. In order to more easily distinguish the constitutional isomers of these chalcones, breakdown curves were produced and these provided strong support in this study.     

Investigations of taxoids by electrospray and atmospheric pressure chemical ionization with tandem mass spectrometry

The fragmentation behavior of taxoids was studied using electrospray (ESI) and atmospheric pressure chemical ionization (APCI) sources with multi-stage tandem mass spectrometry. In the positive ion mode taxoids gave prominent [M+Na]+ and [M+K]+ ions with the ESI source, and [M+NH4]+ or [M+H]+ ions with the APCI source. The MS/MS fragmentations of ions produced by APCI and ESI sources were very similar. For both sources, the presence of cinnamoyl or benzoyl groups could be characterized by initial losses of 148 or 122 u, respectively, from molecular adduct ions. However, the elimination of cinnamic acid was relatively difficult for the molecular adduct ions formed by APCI, and was comparable in importance to the loss of acetic acid. The other fragments involved losses of CH2CO, CO, and H2O. The 5/7/6 type taxoids underwent characteristic losses of 58 or 118 u from ions produced by both APCI and ESI sources. The fragmentation behavior was remarkably influenced by substitution locations. The elimination of the C-10 benzoyl group was usually the first fragmentation step, while that of the C-2 benzoyl group was relatively difficult. The acetoxyl group at C-7 was more active than those at C-2, C-9, and C-10, which in turn were more active than that at C-4. These fragmentation rules could facilitate the rapid screening and structural characterization of taxoids in plant extracts by high-performance liquid chromatography/mass spectrometry (HPLC/MS).     

Quantitative aspects of and ionization mechanisms in positive-ion atmospheric pressure chemical ionization mass spectrometry

The behavior in atmospheric pressure chemical ionization of selected model polycyclic aromatic compounds, pyrene, dibenzothiophene, carbazole, and fluorenone, was studied in the solvents acetonitrile, methanol, and toluene. Relative ionization efficiency and sensitivity were highest in toluene and lowest in methanol, a mixture of molecular ions and protonated molecules was observed in most instances, and interferences between analytes were detected at higher concentrations. Such interferences were assumed to be caused by a competition among analyte molecules for a limited number of reagent ions in the plasma. The presence of both molecular ions and protonated analyte molecules can be attributed to charge-transfer from solvent radical cations and proton transfer from protonated solvent molecules, respectively. The order of ionization efficiency could be explained by incorporating the effect of solvation in the ionization reactions. Thermodynamic data, both experimental and calculated theoretically, are presented to support the proposed ionization mechanisms. The analytical implications of the results are that using acetonitrile (compared with methanol) as solvent will provide better sensitivity with fewer interferences (at low concentrations), except for analytes having high gas-phase basicities.     

Determination of selected sulfonamide antibiotics and trimethoprim in manure by electrospray and atmospheric pressure chemical ionization tandem mass spectrometry

A method for the determination of sulfonamides and trimethoprim in the complex matrix liquid manure has been developed using reversed-phase liquid chromatography and atmospheric pressure chemical ionization (APCI) tandem mass spectrometry. A comparison was made between electrospray and atmospheric pressure chemical ionization. APCI proved to be more robust and less sensitive to matrix effects. High-performance liquid chromatographic (HPLC) separation of the analytes was achieved in less than 7 min. The compounds were extracted with ethyl acetate and the extracts were cleaned up by solid-phase extraction on an aminopropyl column. Recoveries were not dependent on the concentration level. The mean recoveries were as follows: trimethoprim 79.0%, sulfadiazine 80.5%, N(4)-acetylsulfadiazine 91.0%, sulfamerazine 78.6%, sulfadimidine 77.2% and sulfamethoxazole 82.8%. Linearity was established over a concentration range of 5 to 5000 microg/kg with correlation coefficients greater than 0.99. The method had a limit of quantitation (LOQ) of 5 microg/kg manure.     

Low-mass ions produced from peptides by high-energy collision-induced dissociation in tandem mass spectrometry

High-energy collision-induced dissociation (CID) mass spectrometry provides a rapid and sensitive means for determining the primary sequence of peptides. The low-mass region (below mass 300) of a large number of tandem CID spectra of peptides has been analyzed. This mass region contains several types of informative fragment ions, including dipeptide ions, immonium ions, and other related ions. Useful low-mass ions are also present in negative-ion CID spectra. Immonium ions (general structure [H₂N=CH-R]⁺, where R is the amino acid side chain) and related ions characteristic of specific amino acid residues give information as to the presence or absence of these residues in the peptide being analyzed. Tables of observed immonium and reiated ions for the 20 standard amino acids and for a number of modified amino acids are presented. A database consisting of 228 high-energy CID spectra of peptides has been established, and the frequency of occurrence of various ions indicative of specific ammo acid residues has been determined. Two model computer-aided schemes for analysis of the ammo-acid content of unknown peptides have been developed and tested against the database.     

Sequencing of oligosaccharides by collision-induced dissociation matrix-assisted laser desorption/ionization mass spectrometry

A study of the collision-induced dissociation post-source decay (PSD) spectra of free oligosaccharides is presented. These spectra, when obtained with helium as collision gas, show (1,5)X fragments containing the reducing end sugar. The presence of these fragments permits Y ions and, consequently, B and C peaks to be identified. This is a common behaviour from which it has been possible to delineate a general method for the easy assignment of the peaks in PSD spectra of underivatized neutral sugars, allowing the sequence of a real unknown to be obtained.     

Study of atmospheric pressure chemical ionization of PETN by ion mobility spectrometry/tandem mass spectrometry

We present the results of studying the effects of temperature and humidity of the reaction medium and the intensity of ultraviolet radiation on the atmospheric pressure chemical ionization of Penthrite. The peculiarities of the ion mobility spectra of this compound obtained by ion mobility spectrometry-tandem mass spectrometry are analyzed.     

Top-down tandem mass spectrometry of tRNA via ion trap collision-induced dissociation

Transfer RNA is a class of highly modified and structured non-coding RNA molecules generally comprised of 74–95 nucleotides. In this study, tandem mass spectrometry of intact multiply charged tRNA anions of roughly 25 kDa in mass has been demonstrated using a quadrupole/time-of-flight tandem mass spectrometer adapted for ion/ion reaction studies. The sample proved to be a mixture of tRNA molecules. The mass of the most abundant component of the mixture was not consistent with that of the nominal identity of the tRNA from the supplier, viz., tRNA^phe; rather, the mass was consistent with tRNA^Phe bearing an incomplete 3′-terminus. Multiply-charged anions from the major components were isolated in the gas phase and subjected to ion trap collision-induced dissociation without subsequent ion/ion reactions. Abundant fragments from the 5′- and 3′-termini of the molecule could be used to identify the major component as tRNA^phe-3′adenosine (without 3′-phosphorylation). Roughly 15% of the primary sequence of the intact tRNA was unambiguously reflected in the product ion spectrum. The existence of a possible tRNA^Phe variant and the intact tRNA^Phe was also supported by ion trap CID data. The multiply-charged fragment ions derived from tRNA^Phe-3′adenosine were further charge-reduced to mostly singly- and doubly-charged species via proton transfer ion/ion reactions with benzoquinoline cations. The resulting reduction in spectral overlap and charge state ambiguity simplified interpretation of the product ion spectrum and allowed for the identification of product ions from roughly 60% of the sequence.     

Fragmentation study of iridoid glucosides through positive and negative electrospray ionization, collision-induced dissociation and tandem mass spectrometry

Mass spectrometric methodology based on the combined use of positive and negative electrospray ionization, collision-induced dissociation (CID) and tandem mass spectrometry (MS/MS) has been applied to the mass spectral study of a series of six naturally occurring iridoids through in-source fragmentation of the protonated [M+H]+, deprotonated [M--H]- and sodiated [M+Na]+ ions. This led to the unambiguous determination of the molecular masses of the studied compounds and allowed CID spectra of the molecular ions to be obtained. Valuable structural information regarding the nature of both the glycoside and the aglycone moiety was thus obtained. Glycosidic cleavage and ring cleavages of both aglycone and sugar moieties were the major fragmentation pathways observed during CID, where the losses of small molecules, the cinnamoyl and the cinnamate parts were also observed. The formation of the ionized aglycones, sugars and their product ions was thus obtained giving information on their basic skeleton. The protonated, i.e. [M+H]+ and deprotonated [M--H]-, ions were found to fragment mainly by glycosidic cleavages. MS/MS spectra of the [M+Na]+ ions gave complementary information for the structural characterization of the studied compounds. Unlike the dissociation of protonated molecular ions, that of sodiated molecules also provided sodiated sugar fragments where the C0+ fragment corresponding to the glucose ion was obtained as base peak for all the studied compounds.     

Determination of the fragmentation mechanisms of organophosphorus ions by H2O and D2O atmospheric-pressure ionization tandem mass spectrometry

Dimethylmethyl phosphonate (DMMP), dimethyl phosphite (DMPI), trimethyl phosphite (TMPI) and trimethyl phosphate (TMP) were investigated using H₂O and D₂O atmospheric-pressure ionization (API) tandem mass Spectrometry. All daughter ions could be explained by losses of one or a successive number of stable molecules as opposed to losses of radicals such as the hydride, methyl and methoxy species. Losses of neutral methanol and dimethyl ether and of protonated methanol and formaldehyde ions from all four organophosphorus pseudo-molecular ions were observed. The DMMP and DMPI MH⁺ pseudomolecular ions produced the losses of neutral C₂H₆ and water, respectively. Formaldehyde loss was not observed for the MH⁺ ions, but it was well represented in the decomposition pathways of daughter ions. The D₂O reagent gas highlighted the role of the ionizing proton/ deuteron in the various daughter ions, including m/z 95, 79, 65, 49, 33, 31 and 47. The last ion was found to be isobaric in that m/z 47 and 48 both appeared with similar abundances in the D₂O-API daughter ion mass spectra of TMPI and TMP.     

Detection mass bias in atmospheric pressure ionization mass spectrometry

A previously uncharacterized source of detection mass bias is shown to be associated with atmospheric pressure ionization mass spectrometry (APIMS), and is attributed to a mass dependence in the sampling of ions from the supersonic free jet expansion of gas emerging from the ion source. The halide ions Cl ^?, Br^?, and I^? are shown to be transported from the ion source aperture to a quadrupole mass filter with efficiencies that increase linearly with increasing mass of the ion. While the polyatomic anions SF ₆ ^- and C₇F ₁₄ ^- are detected with even greater efficiencies than would be expected for monatomic anions of the same mass, this additional sensitivity to the polyatomic anions is thought to be related to ion loss processes occurring within the ion source. The experimental conditions under which these mass bias effects can be minimized or enhanced in APIMS are described.     

Probing the mechanism of the Petasis olefination reaction by atmospheric pressure chemical ionization mass and tandem mass spectrometry

Atmospheric pressure chemical ionization mass (APCI-MS) and tandem mass spectrometry (APCI-MS/MS) is used to probe the mechanism of the Petasis olefination reaction. Oxatitanacycle intermediates 4 were transferred from solution to the gas phase, detected as 4H+ by APCI-MS with characteristic Ti-isotopic patterns, and structurally characterized by APCI-MS/MS. Detection of 4H+, which upon collision activation dissociates to both 3H+ and Cp(2)TiOH+, fully supports the Hughes mechanism as depicted above. [reaction: see text]     

Comparison of charged derivatives for high energy collision-induced dissociation tandem mass spectrometry

Fixed-charge derivatives have been used to direct the fragmentation pattern of high energy collision-induced dissociation tandem mass spectra for several years. It has been noted that a fixed-charge placed at a terminus of a peptide will simplify the pattern of fragment ions that are produced in collision-induced dissociation. Trimethylammoniumacetyl, dimethyloctylammoniumacetyl, and triphenylphosphoniumethyl derivatives have been cited in the literature for this purpose and many other structures are possible. This work compares the cited derivatives as well as some new structures. The criteria used include the ease of synthesis and purification of the derivatized peptide and the effects of the derivative on the peptide sequence fragment ion yield and ionization efficiency. The trimethylammoniumacetyl derivative is concluded to be the most practical for general use, whereas the dimethyloctylammoniumacetyl derivative is found to be desirable for use with hydrophilic peptides.     

Collision-induced dissociation pathways of anabolic steroids by electrospray ionization tandem mass spectrometry

Anabolic steroids are structurally similar compounds, and their product-ion spectra obtained by tandem mass spectrometry under electrospray ionization conditions are quite difficult to interpret because of poly-ring structures and lack of a charge-retaining center in their chemical structures. In the present study, the fragmentation of nine anabolic steroids of interest to the racing industry was investigated by using triple quadrupole mass spectrometer, Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer, and a linear ion trap instrument. With the aid of an expert system software (Mass Frontier version 3.0), accurate mass measurements, and multiple stage tandem mass spectrometric (MS(n)) experiments, fragmentation pathways were elucidated for boldenone, methandrostenolone, tetrahydrogestrinone (THG), trenbolone, normethandrolone and mibolerone. Small differences in the chemical structures of the steroids, such as an additional double-bond or a methyl group, result in significantly different fragmentation pathways. The fragmentation pathways proposed in this paper allow interpretation of major product ions of other anabolic steroids reported by other researchers in a recent publication. The proposed fragmentation pathways are helpful for characterization of new steroids. The approach used in this study for elucidation of the fragmentation pathways is helpful in interpretation of complicated product-ion spectra of other compounds, drugs and their metabolites.     

Precursor ion scan profiles of acylcarnitines by atmospheric pressure thermal desorption chemical ionization tandem mass spectrometry

The fatty acyl esters of L-carnitine (acylcarnitines) are useful biomarkers for the diagnosis of some inborn errors of metabolism analyzed by liquid chromatography/tandem mass spectrometry. In this study the acylcarnitines were analyzed by atmospheric pressure thermal desorption chemical ionization using a commercial tandem mass spectrometer (APTDCI-MS/MS). The method is based on the precursor ion scan mode determination of underivatized acylcarnitines desorbed from samples by a hot desolvation gas flow and ionized by a corona pin discharge. During desorption/ionization step the temperature induces the degradation of acylcarnitines; nevertheless, the common fragment to all acylcarnitines [MH-59](+) is useful for analyzing their profile. APTDCI parameters, including angle of collection and incidence, gas flows and temperatures, were optimized for acylcarnitines. The experiments were performed drying 2 microL of an equimolar mixture of acylcarnitine standards on a glass slide. The specificity was evaluated by comparing product ion spectra and the precursor ion spectra of 85 m/z of acylcarnitines obtained by the APTDCI method and by electrospray ionization flow injection analysis (ESI-FIA). The method was also employed to analyze acylcarnitines extracted from a pathological dried blood spot and a control. The method enables analysis of biological samples and recognition of some acylcarnitines that are diagnostic markers of inherited metabolic diseases. The intrinsic high-throughput analysis of the ambient desorption ionization methods offers a new opportunity either for its potential application in clinical chemistry and for the expanded screening of some inborn errors of metabolism.