Precursor Ion Scan Mode Research Articles

Screening for the calstabin‐ryanodine receptor complex stabilizers JTV‐519 and S‐107 in doping control analysis

Recent studies outlined the influence of exercise on the stability of the skeletal muscle calstabin1-ryanodine receptor1-complex, which represents a major Ca(2+) release channel. The progressive modification of the type-1 skeletal muscle ryanodine receptor (RyR1) combined with reduced levels of calstabin1 and phosphodiesterase PDE4D3 resulted in a Ca(2+) leak that has been a suggested cause of muscle damage and impaired exercise capacity. The use of 1,4-benzothiazepine derivatives such as the drug candidates JTV-519 and S-107 enhanced rebinding of calstabin1 to RyR1 and resulted in significantly improved skeletal muscle function and exercise performance in rodents. Due to the fact that the mechanism of RyR1 remodelling under exercise conditions were proven to be similar in mice and humans, a comparable effect of JTV-519 and S-107 on trained athletes is expected, making the compounds relevant for doping controls. After synthesis of JTV-519, S-107, and a putative desmethylated metabolite of S-107, target compounds were characterized using nuclear magnetic resonance spectroscopy and electrospray ionization (ESI)-high-resolution/high-accuracy Orbitrap mass spectrometry. Collision-induced dissociation pathways were suggested based on the determination of elemental compositions of product ions and H/D-exchange experiments. The most diagnostic product ion of JTV-519 was found at m/z 188 (representing the 4-benzyl-1-methyl piperidine residue), and S-107 as well as its desmethylated analog yielded characteristic fragments at m/z 153 and 138 (accounting for 1-methoxy-4-methylsulfanyl-benzene and 4-methoxy-benzenethiol residues, respectively). The analytes were implemented in existing doping control screening procedures based on liquid chromatography, multiple reaction monitoring and simultaneous precursor ion scanning modes using a triple quadrupole mass spectrometer. Validation items such as specificity, recovery (68-92%), lower limit of detection (0.1-0.2 ng/mL), intraday (5.2-18.5%) and interday (8.7-18.8%) precision as well as ion suppression/enhancement effects were determined.

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.

A tandem MS precursor‐ion scan approach to identify variable covalent modification of albumin Cys34: a new tool for studying vascular carbonylation

We developed a liquid chromatography electrospray ionisation multi-stage mass spectrometry (LC-ESI-MS/MS) approach based on precursor-ion scanning and evaluated it to characterize the covalent modifications of Cys34 human serum albumin (HSA) caused by oxidative stress and reactive carbonyl species (RCS) adduction. HSA was isolated and digested enzymatically to generate a suitable-length peptide (LQQCPF) containing the modified tag residue. The resulting LQQCPF peptides were identified by LC-ESI-MS/MS in precursor-ion scan mode and further characterized in product-ion scan mode. The product ions for precursor-ion scanning were selected by studying the MS/MS fragmentation of a series of LQQCPF derivatives containing Cys34 modified with different alpha,beta-unsaturated aldehydes and di and ketoaldehydes. We used a Boolean logic to enhance the specificity of the method: this reconstitutes a virtual current trace (vCT) showing the peaks in the three precursor-ion scans, marked by the same parent ion. The method was first evaluated to identify and characterize the Cys34 covalent adducts of HSA incubated with 4-hydroxy-hexenal, 4-hydroxy-trans-2-nonenal (HNE) and acrolein (ACR). Then we studied the Cys34 modification of human plasma incubated with mildly oxidized low-density lipoproteins (LDL), and the method easily identified the LQQCPF adducts with HNE and ACR. In other experiments, plasma was oxidized by 2,2'-azobis(2-amidinopropane) HCl (AAPH) or by Fe2+/H2O2. In both conditions, the sulfinic derivative of LQQCPF was identified and characterized, indicating that the method is suitable not only for studying RCS-modified albumin, but also to check the oxidative state of Cys34 as a marker of oxidative damage.

High throughput screening of bradykinin-potentiating peptides in Bothrops moojeni snake venom using precursor ion mass spectrometry

Snake venoms are known to be an extensive source of bioactive peptides. Bradykinin-potentiating peptides (BPPs) are inhibitors of the angiotensin-converting enzyme that have already been identified in the venom of many snake, scorpion, spider and batrachian species. Their most characteristic structural features are an invariable N-terminal pyroglutamate residue (pGlu or Z) and two consecutive proline residues at the C-terminus. Fragmentation of BPPs by collision-induced dissociation during electrospray tandem mass spectrometry analysis (ESI-MS/MS) generates a predominant signal at m/ z 213.1 corresponding to the y-ion of the terminal Pro–Pro fragment. In addition, signals at m/ z 226.1 and 240.1 that correspond to the b ions of the N-terminus pGlu–Asn and pGlu–Lys, respectively, can often be observed. Based on these structural determinants, the present work describes an original methodology for the discovery of BPPs in natural extracts using liquid chromatography coupled to ESI-MS/MS operated in precursor ion-scan mode. The venom of the Bothrops moojeni snake was used as a model and the methodology was applied for subsequent structural analysis of the identified precursors by tandem mass spectrometry on quadrupole-time-of-flight (Q-TOF) and matrix-assisted laser-desorption/ionization time-of-flight tandem mass spectrometry (MALDI-TOF-MS/MS) instruments. More than 40 peptides below 2500 Da could be detected, among them 20 were shown to belong to the BPP-like family including the related tripeptides pGlu–Lys–Trp and pGlu–Asn–Trp. A total of 15 new sequences have been identified using this approach.

Systematic and comprehensive strategy for reducing matrix effects in LC/MS/MS analyses

A systematic, comprehensive strategy that optimizes sample preparation and chromatography to minimize matrix effects in bioanalytical LC/MS/MS assays was developed. Comparisons were made among several sample preparation methods, including protein precipitation (PPT), liquid–liquid extraction (LLE), pure cation exchange solid-phase extraction (SPE), reversed-phase SPE and mixed-mode SPE. The influence of mobile phase pH and gradient duration on the selectivity and sensitivity for both matrix components and basic analytes was investigated. Matrix effects and overall sensitivity and resolution between UPLC ® technology and HPLC were compared. The amount of specific matrix components, or class of matrix components, was measured in the sample preparation extracts by LC/MS/MS with electrospray ionization (ESI) using both precursor ion scanning mode and multiple reaction monitoring (MRM). PPT is the least effective sample preparation technique, often resulting in significant matrix effects due to the presence of many residual matrix components. Reversed-phase and pure cation exchange SPE methods resulted in cleaner extracts and reduced matrix effects compared to PPT. The cleanest extracts, however, were produced with polymeric mixed-mode SPE (both reversed-phase and ion exchange retention mechanisms). These mixed-mode sorbents dramatically reduced the levels of residual matrix components from biological samples, leading to significant reduction in matrix effects. LLE also provided clean final extracts. However, analyte recovery, particularly for polar analytes, was very low. Mobile phase pH was manipulated to alter the retention of basic compounds relative to phospholipids, whose retention tends to be relatively independent of pH. In addition to the expected resolution, speed and sensitivity benefits of UPLC ® technology, a paired t-test demonstrated a statistically significant improvement with respect to matrix effects when this technology was chosen over traditional HPLC. The combination of polymeric mixed-mode SPE, the appropriate mobile phase pH and UPLC ® technology provides significant advantages for reducing matrix effects resulting from plasma matrix components and in improving the ruggedness and sensitivity of bioanalytical methods.

Mapping of arsenic species and identification of a novel arsenosugar in giant clams Tridacna maxima and Tridacna derasa using advanced mass spectrometric techniques

Environmental context. Arsenic is known to accumulate in various marine organisms. The high acute toxicity of inorganic arsenic species and the potential chronic toxicity of some organoarsenic species require detailed knowledge about the occurrence and metabolism of arsenic compounds in marine organisms. The application of advanced analytical techniques still allows, even after decades of arsenic speciation, the identification of novel species. In addition, comprehensive mapping of all arsenic species present in marine organisms may allow for a more detailed understanding of arsenic metabolism. Abstract. Because of their symbiotic microalgae, giant clams (Tridacna species) exhibit a unique arsenic metabolism, which has been shown in previous studies to involve a large number of arsenic species. This study demonstrates the application of liquid chromatography (HPLC) online with electrospray tandem mass spectrometry (ES-MS/MS) as well as inductively coupled plasma mass spectrometry (ICP-MS) for arsenic speciation analysis in giant clam extracts. Selected reaction monitoring (SRM) was used for sensitive and selective detection of a large number of arsenic species in a single chromatographic run. Novel aspects are the analysis of 10 tissue fractions from one clam and the analysis of kidney extracts both from T. maxima and T. derasa with the same method thus offering the possibility for direct comparisons. Moreover, HPLC-ES-MS/MS in the precursor ion scan mode and product ion scan mode allowed the identification of a novel sulfonated dimethylarsenosugar and the partial characterisation of another unknown arsenic species. The results indicate that most arsenic species are accumulated in the kidneys. However, arsenobetaine was found at similar contents in all analysed tissue fractions of one T. maxima clam.

A homologous series of eunicellin-based diterpenes from Acalycigorgia sp. characterised by tandem mass spectrometry

The discovery and structure determination of a homologous series of eunicellin-based diterpenes from the gorgonian Acalycigorgia sp. is described. Extensive use was made of 1D and 2D NMR data to determine the structure of the diterpene skeleton. The relative stereochemistry was confirmed via the use of NOE data in conjunction with molecular modelling. A series of homologues were identified using a combination of product and precursor ion scanning modes in tandem mass spectrometry. This powerful technique afforded excellent clarification to aid the analysis of the complex mass spectral data.

LC-MS-based method for the qualitative and quantitative analysis of complex lipid mixtures

A simple and robust LC-MS-based methodology for the investigation of lipid mixtures is described, and its application to the analysis of human lipoprotein-associated lipids is demonstrated. After an optional initial fractionation on Silica 60, normal-phase HPLC-MS on a YMC PVA-Sil column is used first for class separation, followed by reversed-phase LC-MS or LC-tandem mass spectrometry using an Atlantis dC18 capillary column, and/or nanospray MS, to fully characterize the individual lipids. The methodology is applied here for the analysis of human apolipoprotein B-associated lipids. This approach allows for the determination of even low percentages of lipids of each molecular species and showed clear differences between lipids associated with apolipoprotein B-100-LDL isolated from a normal individual and those associated with a truncated version, apolipoprotein B-67-containing lipoproteins, isolated from a homozygote patient with familial hypobetalipoproteinemia. The methods described should be easily adaptable to most modern MS instrumentation.

A general precursor ion‐like scanning mode on quadrupole‐TOF instruments compatible with chromatographic separation

MS protein identification and quantitation are key proteomic techniques in biological research. Besides identification of proteins, MS is used increasingly to characterize secondary protein modifications. This often requires trimming the analytical strategy to a specific type of modification. Direct analysis of protein modifications in proteomic samples is often hampered by the limited dynamic range of current analytical tools. Here we present a fast, sensitive, multiplexed precursor ion scanning mode--implemented on a quadrupole-TOF instrument--that allows the specific detection of any modified peptide or molecule that reveals itself by a specific fragment ion or pattern of fragment ions within a complex proteomic sample. The high mass accuracy of the TOF mass spectrometer is available for the marker ion specificity and the precursor ion mass determination. The method is compatible with chromatographic separation. Fragment ions and intact molecular ions are acquired quasi-simultaneously by continuously switching the collision energy between elevated and low levels. Using this technique many secondary modifications can be analyzed in parallel; however, the number of peptides carrying a specific modification that can be analyzed successfully is limited by the chromatographic resolution or, more generally, by the depth of the resolved time domain.

N-Terminal Adducts of Bovine Hemoglobin with Glutaraldehyde in a Hemoglobin-Based Oxygen Carrier

Hemoglobin-based oxygen carriers (HBOCs) are being developed for the medical field, but because they could increase an athlete's performance, they are misapplied for doping purposes. We previously presented a screening method to detect Oxyglobin (Biopure Corp.) and PolyHeme (Northfield Laboratories Inc.) in serum samples using total acid hydrolysis followed by electrospray mass spectrometry analyses. An alternative mass spectrometric method involving enzymatic hydrolysis is here presented. Digestion of Oxyglobin by endoproteinase Glu-C and LC/MS analyses of the mixture allowed the detection of unique peptidic fragments in comparison with a bovine hemoglobin digest. Tandem mass spectrometry experiments of these peptide ions were performed, and two specific species were actually identified as the N-terminal enzymatic fragment of the beta chain carrying two different modifications. Sequential MS3 experiments using an ion trap mass spectrometer permitted us to locate the chemical modification by the glutaraldehyde on the NH2-terminal group and to propose a structure for the modified peptides. In another set of experiments, screening of these two diagnostic ions into Oxyglobin-spiked serums using precursor ion scan mode in a triple quadrupole instrument allowed the detection of this HBOC with a detection limit of 2 g L(-1).

Determination of glucosinolate profiles in Chinese vegetables by precursor ion scan and multiple reaction monitoring scan mode (LC-MS/MS)

Liquid chromatography-electrospray ionization (ESI) mass spectrometry was used to describe the glucosinolate (GSL) profiles in three Chinese vegetables. The strategy was based on first screening for possible glucosinolates via precursor ion scan mode (PreIS). Further validating was done using the multiple reaction monitoring scan mode (MRM). The obtained fragment ions [S=C=NOH]− for m/z 75 and [SO4H]− for m/z 97 were used in both scan modes to reveal the masses of the GSL precursor ions [M-H]− which were further validated by the mostly known chromatographic behavior of the resulting intact GSL. The feasibility of the strategy was first demonstrated using crude extracts of broccoli. The tandem mass spectrometric experiments in negative ion ESI proved to be sensitive and selective enough to rapidly examine the GSL profiles even of crude plant extracts. The results adequately characterize the target differences between various GSL distributions in vegetables induced by treatment of methyl jasmonate.

Glycoprotein profiling by electrospray mass spectrometry

This work compares several different methods of site-specific analysis of glycoproteins using electrospray mass spectrometry. The glycoprotein, oLHα (ovine luteinizing hormone, α-subunit) was chosen as an appropriate example protein for these studies because of its biological relevance and extreme microheterogeneity. More than 20 unique glycoforms were detected for this glycoprotein at the Asn 56 site of oLHα. The carbohydrates present at this site affect receptor binding affinity, so understanding the great variety in the composition of these carbohydrates is important in studying ligand binding interactions. MS data was acquired on a quadrupole ion trap, a triple quadrupole, and a quadrupole time of flight mass spectrometer, and carbohydrate composition at the Asn 56 site of oLHα was determined using these instruments. Additionally, neutral loss and precursor ion scanning modes were also used to identify the glycoforms present, and these techniques were compared to the standard MS data. Of the three instruments compared in the study, the qTOF mass spectrometer achieved the lowest sample consumption, but all three instruments were useful in profiling the glycopeptide composition.

Screening for transformation products of pesticides using tandem mass spectrometric scan modes

The applicability of tandem mass spectrometric (MS–MS) scan modes such as constant neutral-loss and precursor-ion scanning to screen for unknown transformation products (TPs) of pesticides at environmentally relevant concentrations (low-μg/l level) is studied. The selection of the MS–MS scan modes is based on the product-ion scan of the parent pesticide, and TPs are detected which are unaltered in the part of the structure concerned. The screening approach is applied to a surface water sample spiked with atrazine and three known TPs at a level of 3 μg/l to study the possibility to extract the TPs from the total ion chromatogram. Next, the approach is used to identify unknown TPs formed after (bio)degradation of two test compounds, fenchlorazole-ethyl (FCE) and furathiocarb (FTC). By using the precursor-ion scan mode, two TPs were detected after biodegradation of FCE, fenchlorazole-methyl and fenchlorazole; in surface water only fenchlorazole was found. The constant neutral-loss scan mode was used to identify carbofuran as TP of FTC. The added value of the proposed procedure is the increased selectivity at the cost of sensitivity. Best results are, therefore, obtained for samples which contain large amounts of matrix constituents.

The impact of accurate mass measurements using quadrupole/time-of-flight mass spectrometry on the characterisation and screening

Atmospheric pressure ionisation quadrupole/ time-of-flight mass spectrometry was used to perform accurate mass measurements on the product ion fragments of moclobemide and remikiren. The low energy collision induced dissociation fragmentation pathways of the two compounds could be established using accurate mass, MSn data and MS interpretation software (Mass Frontier). An atypical fragmentation corresponding to the loss of neutral dimethylvinyl could be clearly identified. The knowledge gained from these experiments was used to demonstrate the application of data dependent experiments on a triple quadrupole instrument for the screening of metabolites. Data dependent experiments were performed using either Q3 single scan or precursor ion scan mode as the survey scan, and product ion scan as the dependent scan.

Duty Cycle Improvement for a Quadrupole—Time-of-Flight Mass Spectrometer and its Use for Precursor Ion Scans

Normally, quadrupole–time-of-flight (QqTOF) instruments benefit from a simultaneous recording of all masses, and their sensitivity is higher than that of a triple quadrupole when a full scan is recorded. This is not the case when only a single type of ion has to be monitored, as in precursor ion scanning or multiple-reaction monitoring (MRM) mode. This is because ion losses in TOF are higher than in a quadrupole. A significant part of these losses is due to the low duty cycle of an orthogonal TOF instrument, typically 5 to 30%. Trapping ions in the collision cell and then gating them out in short bursts synchronized with TOF extraction can restore the duty cycle at the expense of mass range. The mechanism of ion trapping, gating and bunching is studied under various conditions. By varying trapping and gating parameters, different modes of operation are achievable: from a 100% duty cycle restoration for a narrow mass range to <50% duty cycle for a wider mass range. The former mode is best for a single-fragment precursor ion scan, whereas the latter is better when a full product-ion spectrum needs to be recorded, or when a multiple-fragment precursor ion scan is performed. Several examples of precursor ion scans obtained with peptides and lipids are demonstrated. Sensitivity improvements range from ×15 for the immonium fragment ion ( m/z 86) to ×5 for simultaneous recording of lipid fragments with m/z = 184 and 264.

In vivo metabolism of epothilone B in tumor-bearing nude mice: identification of three new epothilone B metabolites by capillary high-pressure liquid chromatography/mass spectrometry/tandem mass spectrometry.

Epothilone B is a 16-membered macrolide produced by the myxobacterium Sorangium cellulosum and is currently under clinical investigation. Experimentally, epothilone B demonstrates potent antiproliferative activity at the nanomolar level in vitro, and potent regression-producing antitumor activity in vivo, similar to paclitaxel (Taxol). In order to foster the design of improved derivatives, the potential biotransformation products of epothilone B formed in liver of tumor-bearing mice after intravenous administration of 10 mg/kg were characterized in an early stage of compound development. Solely on the basis of capillary high-performance liquid chromatography, combined either with electrospray tandem mass spectrometry (in precursor and product ion scan mode) and single analyzing time-of-flight mass spectrometry (H/D exchange and accurate mass measurement), three main metabolites could be detected. The three metabolites, formed by the liver, have in common that the epoxide ring was hydrolyzed and that the macrocyclic lactone ring was opened to the acid. In two cases it is assumed that open-chain intermediates re-cycled either to a lactone or, after conjugation with taurine, to the respective lactam. The proposed structures were additionally supported by the determination of the number of the exchangeable hydrogen atoms and by confirmation of the proposed elemental composition by exact mass measurement.

Fast, sensitive and selective liquid chromatographic–tandem mass spectrometric determination of tumor-promoting diterpene esters

A liquid chromatography–tandem mass spectrometry (LC–MS–MS) method was developed to detect tumor-promoting diterpene esters of the tigliane and ingenane types within plant extracts. Fractionation on a C 18 high-performance liquid chromatography (HPLC) column was followed by MS–MS–multiple reaction monitoring (MRM) using the precursor→product ion pairs of m/z 311→293 and 293→265 for phorbol esters. The ion pairs m/z 313→295 and 295→267 were used for ingenol and deoxyphorbol esters. In a second run, the characteristic ions at m/z 311 and 313 were followed in precursor ion scan mode. These quasi-molecular ions were utilized to obtain full scan spectra of the compounds in product ion scan mode. Due to its selectivity, the present on-line method can be applied for plant cultivar selection and plant product control without time-consuming extraction procedures and complex bioassays.

Structural characterisation of N-linked glycan mixtures by precursor ion scanning and tandem mass spectrometric analysis

2-Aminoacridone (2-AMAC) labelled N-linked glycan pools were analysed directly by a hybrid quadrupole orthogonal acceleration time-of-flight mass spectrometer (Q-Tof) in the precursor ion scanning and tandem mass spectrometry (MS/MS) modes. The use of a precursor ion scanning strategy on this instrument provides a rapid and sensitive method of screening glycan mixtures, without prior separation by chromatographic methods. It allows facile and preliminary characterisation of glycans into different classes, for example, high-mannose or complex glycans. Preliminary sequencing information for each glycan is obtained in the initial precursor ion scanning mode, but further sequencing information of selected glycans can be obtained using the MS/MS mode. Copyright 1999 John Wiley & Sons, Ltd.

Surface ionization mass spectroscopy of airbone particulates: W D Davis, Abstract. (Proc 19th Nat Symp Am Vac Soc) J Vac Sci Technol, 10 (1), Jan/Feb 1973, 278

A new non-targeted screening strategy was developed for the detection and relative quantification of known and potentially unknown drug-related impurities in the active pharmaceutical ingredient daptomycin using ultra-high performance liquid chromatography, coupled with hybrid triple quadrupole-linear ion trap electrospray ionization mass spectrometry in three different modes. The precursor ion scan mode was first applied using the m/z transitions of 159.2, 313.2, and 341.2 for rapid screening of all daptomycin-related impurities. Precursor ions detected at specific retention times were selected, fragmented, and focused in enhanced product ion scan mode to obtain secondary ion mass spectra and identify drug-related impurities. Finally, each potential compound was relatively quantified in multiple reaction monitoring mode. Repeatability experiments suggest that this impurity profiling method is a reliable strategy for identifying unknown impurities in daptomycin and other drugs.