Unbiased high-throughput screening of reactive metabolites on the linear ion trap mass spectrometer using polarity switch and mass tag triggered data-dependent acquisition

Constant neutral loss (CNL) and precursor ion (PI) scan have been widely used for the in vitro screening of glutathione conjugates derived from reactive metabolites, but these two methods are only applicable to triple quadrupole or hybrid triple quadrupole mass spectrometers. Additionally, the success of CNL and PI scanning largely depends on structure and CID fragmentation pathways of GSH conjugates. In the present study, a highly efficient methodology has been developed as an alternative approach for high-throughput screening and structural characterization of reactive metabolites using the linear ion trap mass spectrometer. In microsomal incubations, a mixture of glutathione [GSH, gamma-glutamyl-cystein-glycin] and the stable-isotope labeled compound [GSX, gamma-glutamyl-cystein-glycin-(13)C2-(15)N] was used to trap reactive metabolites, resulting in formation of both labeled and unlabeled conjugates at a given isotopic ratio. A mass difference of 3.0 Da between the natural and labeled GSH conjugate (mass tag) at a fixed isotopic ratio constitutes a unique mass pattern that can selectively trigger the data-dependent MS(2) scan of both isotopic partner ions, respectively. In order to eliminate the response bias of GSH adducts in the positive and negative mode, a polarity switch is executed between the mass tag-triggered data dependent MS(2) scan, and thus ESI- and ESI+ MS(2) spectra of both labeled and nonlabeled GSH conjugates are obtained in a single LC-MS run. Unambiguous identification of glutathione adducts was readily achieved with great confidence by MS(2) spectra of both labeled and unlabeled conjugates. Reliability of this method was vigorously validated using several model compounds that are known to form reactive metabolites. This approach is not based on the appearance of a particular product ion such as MH(+) - 129 and anion at m/z 272, whose formation can be structure-dependent and sensitive to the collision energy level; therefore, the present method can be suitable for unbiased screening of any reactive metabolites, regardless of their CID fragmentation pathways. Additionally, this methodology can potentially be applied to triple quadrupole or hybrid triple quadrupole mass spectrometers.     

Use of a trapping agent for simultaneous capturing and high-throughput screening of both "soft" and "hard" reactive metabolites

Glutathione (GSH) has been widely used for in vitro trapping and subsequently detecting reactive metabolites using liquid chromatography-mass spectrometry. A major drawback of GSH is its low trapping efficiency for "hard" reactive metabolites such as reactive aldehydes. In the present study, a bifunctional trapping agent (gamma GSK, gamma-glutamylcysteinlysine) is investigated as an alternative of GSH for simultaneous trapping both "hard" and "soft" reactive metabolites. In microsomal incubations, soft and hard reactive metabolites are captured by conjugation to the free thiol and the amine group of gamma GSK, respectively, resulting in formation of stable peptide adducts. Similar to GSH conjugates, all gamma GSK adducts derived from both soft and hard reactive metabolites contain a gamma-glutamyl moiety and, thus, undergo a neutral loss of 129 Da under collision-induced dissociation. As a result, an NL MS/MS scan can be utilized as a generic method for rapid detecting of both hard or soft reactive metabolites. As demonstrated by a number of model compounds, this approach, in combination with the isotope trapping technique, is reliable, sensitive, and efficient and can be potentially utilized as a high-throughput method for screening and rapid identification of both soft and hard reactive metabolites. In comparison with other methods, this approach is highly efficient and suitable in drug discovery for screening a wide variety of compounds for different reactive metabolites.     

High-throughput screening and characterization of reactive metabolites using polarity switching of hybrid triple quadrupole linear ion trap mass spectrometry

A highly sensitive and efficient method has been developed for detection and characterization of glutathione (gamma-glutamyl-cysteinylglycine, GSH)-trapped reactive metabolites using a negative precursor ion (PI) as the survey scan to trigger the acquisition of positive enhanced product ion (EPI) spectra on a triple quadrupole linear ion trap mass spectrometer. The negative precursor ion scan step was carried out monitoring the anion at m/z 272, corresponding to deprotonated gamma-glutamyl-dehydroalanyl-glycine originating from the glutathionyl moiety. Because of the uniqueness and abundance of the anion at m/z 272, this single survey scan exhibited broad utility in the detection of unknown GSH conjugates. Further structural characterization was achieved by analyzing positive MS2 spectra that featured rich fragments without mass cutoff and were acquired in the same liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis. The effectiveness and reliability of this approach was evaluated using a number of model compounds in human liver microsomal incubations, including acetaminophen, clozapine, diclofenac, imipramine, meclofenamic acid, and ticlopidine. As a result, the PI-EPI approach revealed the presence of known adducts and, in many instances, identified additional conjugates that had not been reported previously. In comparison to the widely used neutral loss (NL) scanning analysis, this approach provided superior sensitivity and selectivity for different types of GSH conjugates. More importantly, the PI-EPI approach is suitable for high-throughput screening of reactive metabolites in the drug discovery process.     

Detection and structural characterization of glutathione-trapped reactive metabolites using liquid chromatography-high-resolution mass spectrometry and mass defect filtering

The present study was designed to apply the mass defect filter (MDF) approach to the screening and identification of reactive metabolites using high-resolution mass spectrometry. Glutathione (GSH)-trapped reactive metabolites of acetaminophen, diclofenac, carbamazepine, clozapine, p-cresol, 4-ethylphenol, and 3-methylindole in human liver microsomes (HLM) were analyzed by HPLC coupled with Orbitrap or Fourier transform ion cyclotron resonance mass spectrometry. Through the selective removal of all ions that fall outside of the GSH adduct MDF template windows, the processed full scan MS chromatograms displayed GSH adducts as major components with no or a few interference peaks. The accurate mass LC-MS data sets were also utilized for the elimination of false positive peaks, detection of stable oxidative metabolites with other MDF templates, and determination of metabolite molecular formulas. Compared to the neutral loss scan by a triple quadrupole instrument, the MDF approach was more sensitive and selective in screening for GSH-trapped reactive metabolites in HLM and rat bile and far more effective in detecting GSH adducts that do not afford the neutral loss of 129 Da as a significant fragmentation pathway. The GSH adduct screening capability of the MDF approach, together with the utility of accurate mass MS/MS information in structural elucidation, makes high-resolution LC-MS a useful tool for analyzing reactive metabolites.     

Conjugation of keto fatty acids to glutathione in plant tissues. Characterization and quantification by HPLC-tandem mass spectrometry

Both biotic and abiotic stress activate the oxylipin pathway in plants. As reactive electrophile species (RES), some oxylipins are expected to bind cellular nucleophiles in a Micha?l-type addition reaction. Using the HPLC-tandem mass spectrometry techniques, we have established the analytical basis for the investigation of oxylipin conjugation to glutathione (GSH) in plant extracts. The GSH adducts to the four keto fatty acid isomers issued from both linoleic and linolenic acids were first produced and their mass spectrometric features analyzed in the positive electrospray ionization mode. In all cases, the main fragmentation (MS2 mode) of the pseudomolecular ion leads to the neutral loss of a glutamyl moiety (-129 Da), affording an ion that gives structural information upon an additional fragmentation (MS3 mode). The glutamyl loss was confirmed by the analysis of other GSH adducts to oxylipin RES and appeared as being characteristic of GSH adducts. It is thus proposed to search GSH adducts in plant extracts by HPLC-MS/MS, using initially the neutral loss mode and then the MS2 mode to further characterize the identified compounds. This methodology was successfully applied to the analysis of GSH adducts upon infiltration into leaves of the four previous keto fatty acids at 5 mM, a concentration inducing cell death. The production of GSH adducts to oxylipin RES was observed for the first time in plant tissues. Furthermore, the levels of adduct production explain in part the observed GSH depletion. These results support the role of RES in altering protein activities and cellular redox balance of plant cells, via addition reactions to cellular nucleophiles.     

Enhanced screening of glutathione-trapped reactive metabolites by in-source collision-induced dissociation and extraction of product ion using UHPLC-high resolution mass spectrometry

A selective and sensitive approach, called extraction of product ion (XoPI) method, was developed for the detection of l-glutathione (GSH)-trapped reactive metabolites employing an Orbitrap high resolution mass spectrometer. Fragmentation of GSH conjugates in the negative ion mode leads to a product ion, deprotonated γ-glutamyl-dehydroalanyl-glycine (m/z 272.0888). As a means of utilizing this property, negative ion high resolution MS data were collected from in vitro incubations by monitoring ions from m/z 269.5 to 274.5 under in-source collision-induced dissociation. Extraction of product ions at m/z 272.0888 ± 5 ppm from this data resulted in a chromatogram exhibiting deprotonated γ-glutamyl-dehydroalanyl-glycine as the major peaks with no or very few interferences. Therefore, peaks in this extracted product ion chromatogram potentially came from GSH-trapped reactive metabolites. The GSH conjugate parent ions were then confirmed in the corresponding full scan MS data, and their structures were identified from their MS(2) fragmentation patterns. The effectiveness of the approach was assessed with four model compounds, amodiaquine, clozapine, diclofenac, and fipexide, all well-known to form GSH-trapped reactive metabolites, following incubation in human liver microsomes supplemented with β-nicotinamide adenine dinucleotide 2'-phosphate reduced tetrasodium salt (NADPH) and GSH. The results from XoPI method were compared to two other commonly employed liquid chromatography-mass spectrometry (LC-MS) methods: precursor ion scan method and mass defect filter method. Overall, the XoPI method was more selective and sensitive in detecting the GSH conjugates. Many GSH conjugates previously not reported were detected and characterized in this study.     

Screening and sequencing of glycated proteins by neutral loss scan LC/MS/MS method

Nonenzymatic protein glycation is caused by a Schiff's base reaction between the aldehyde groups of reducing sugars and the primary amines of proteins. A reversed-phase liquid chromatography method followed by a neutral loss scan mass spectrometric method was developed for the screening of glycation in proteins. The neutral loss scan was based on a unique sugar moiety neutral loss (-162 Da) that we observed in the fragmentation spectra of glycated peptides on Q-Tof type mass spectrometers. The collision energy was optimized for this neutral loss using a glycated synthetic peptide, and 20 eV was found to be the optimum collision energy. The neutral loss scan experiment was composed of two segments. In the first segment, the glycated peptides were identified based on the signature neutral loss of 162 Da when the collision energy was elevated to 20 eV. In the second segment, the glycated peptides were selected as the parent ions and fragmented at higher collision energy to break the peptide bonds. The fragmentation spectra of the selected glycated peptides revealed both the amino acid sequences and the sites of glycation. This neutral loss scan method was used to study the glycation in human serum albumin (HSA). The glycation sites in HSA were identified based on the retention time shift of glycated peptides, the mass accuracy from the MS scan, the signature neutral loss, and MS/MS information. Using this method, we were able to identify that 31 lysine residues were partially glycated from the glycated HSA sample, which has a total of 59 lysine residues.     

Detection of in vitro kinase generated protein phosphorylation sites using gamma[18O4]-ATP and mass spectrometry

A novel stable-isotope labeling approach for identification of phosphopeptides that utilizes adenosine triphosphate, in which four oxygen-16 atoms attached to the terminal phosphate group are substituted with oxygen-18 [gamma((18)O4)-ATP], has been developed. The ability to use gamma((18)O4)-ATP to monitor phosphorylation modification within various proteins was conducted by performing in vitro kinase reactions in the presence of a 1:1 mixture of gamma((18)O4)-ATP and normal isotopic abundance ATP (ATP). After tryptic digestion, the peptides were analyzed using mass spectrometry (MS). Phosphorylated peptides are easily recognized within the MS spectrum owing to the presence of doublets separated by 6.01 Da; representing versions of the peptide modified by ATP and gamma((18)O4)-ATP. Standard peptides phosphorylated using gamma((18)O4)-ATP via in vitro kinase reactions showed no exchange loss of (18)O with (16)O. The identity of these doublets as phosphorylated peptides could be readily confirmed using tandem MS. The method described here provides the first direct stable-isotope labeling method to definitely detect phosphorylation sites within proteins.     

Generation and identification of reactive metabolites by electrochemistry and immobilized enzymes coupled on-line to liquid chromatography/mass spectrometry

The detection of reactive metabolites using conventional in vivo and in vitro techniques is hampered because the intermediately formed reactive species are prone to covalent binding to cellular macromolecules. Therefore, the application of improved methods is required. The on-line coupling of an electrochemical reactor and horseradish peroxidase immobilized on magnetic microparticles with liquid chromatography/mass spectrometry (EC/LC/MS or HRP/LC/MS) allows the direct detection of reactive metabolites of the model compounds amodiaquine, amsacrine, and mitoxantrone, which are all known for readily binding to cellular macromolecules after metabolization by cytochrome P450. EC/LC/MS and HRP/LC/MS experiments were compared to rat liver microsome incubations and proved to be valuable complementary methods since reactive quinone, quinone imine, and quinone diimine species could be detected directly and not only after trapping with glutathione. Furthermore, N-dealkylation and N-oxidation of amodiaquine were successfully simulated by electrochemical oxidation reactions, as well as the formation of an aldehyde. Therefore, EC/LC/MS and HRP/LC/MS are promising tools for the identification of both reactive and stable metabolites in drug development.     

Enzyme-DNA biocolloids for DNA adduct and reactive metabolite detection by chromatography-mass spectrometry

Silica microbead bioreactors (0.5 microm diameter) coated with DNA and enzymes were fabricated to measure reactive metabolite and DNA-adduct formation rates relevant to genotoxicity screening. Cytochrome (cyt) P450 2E1, cyt P450(cam), and myoglobin (Mb) were incorporated into thin films with DNA using the electrostatic layer-by-layer (LbL) method. The utility of these biocolloids was demonstrated by oxidation of guaiacol, styrene, and (4-methylnitrosoamino)-1-(3-pyridyl)-1-butanone (NNK). Enzyme turnover rates for formation of reactive metabolites were monitored using gas chromatography/mass spectrometry (GC/MS) and liquid chromatography-mass spectrometry (LC-MS). Capillary LC-MS/MS was employed to determine DNA nucleobase adducts after catalyzing the reactive metabolite formation with DNA-enzyme biocolloids and then using neutral thermal hydrolysis on the biocolloids. Dramatic improvements in surface area to volume ratio over similar films on macroscopic surfaces opens new avenues for genotoxicity screening and enabled the first use of pure cyt P450 enzymes in enzyme-DNA films to produce DNA adducts. The method makes possible identification and formation rate measurements of major and minor DNA adducts as well as the metabolites themselves in <5 min of reaction time using relevant human liver enzymes.     

Electrochemical genotoxicity screening for arylamines bioactivated by N-acetyltransferase

Genotoxicity screening sensors that measure DNA damage from metabolism of arylamines were developed and evaluated. The sensors feature ultrathin films containing DNA and N-acetyltransferase (NAT) on pyrolytic graphite (PG) electrodes. NAT in the film catalyzed the conversion of the arylamine 2-aminofluorene (2-AF) to 2-acetylaminofluorene (2-AAF) by acetyl coenzyme A (AcCoA) dependent N-acetylation, as verified by liquid chromatography. DNA damage in the films from exposure to reactive 2-AF metabolites was measured subsequent to the enzyme reaction using catalytic voltammetric oxidation with Ru(bpy)32+. Square wave voltammetric (SWV) peaks increased with enzyme reaction time, and relative DNA damage rates at pH 5.8 were measured within 2 min. Control incubations of DNA/NAT films without AcCoA gave no significant sensor response. CapLC-MS/MS analysis of 2-AAF/DNA reaction products was consistent with 2-AF-guanine adducts formed in the films. DNA damage occurred more rapidly under weakly acidic conditions (pH 5.5-5.8) than at neutral pH, suggesting that genotoxicity from arylamine metabolism by NAT could be more significant in slightly acidic environments.     

De novo sequencing of neuropeptides using reductive isotopic methylation and investigation of ESI QTOF MS/MS fragmentation pattern of neuropeptides with N-terminal dimethylation

A stable-isotope dimethyl labeling strategy was previously shown to be a useful tool for quantitative proteomics. More recently, N-terminal dimethyl labeling was also reported for peptide sequencing in combination with database searching. Here, we extend these previous studies by incorporating N-terminal isotopic dimethylation for de novo sequencing of neuropeptides directly from tissue extract without any genomic information. We demonstrated several new sequencing applications of this method in addition to the identification of the N-terminal residue using the enhanced a(1) ion. The isotopic labeling also provides easier and more confident de novo sequencing of peptides by comparing similar MS/MS fragmentation patterns of the isotopically labeled peptide pairs. The current study on neuropeptides shows several distinct fragmentation patterns after N-terminal dimethylation which have not been reported previously. The y((n-1)) ion is enhanced in multiply charged peptides and is weak or missing in singly charged peptides. The MS/MS spectra of singly charged peptides are simplified due to the enhanced N-terminal fragments and suppressed internal fragments. The neutral loss of dimethylamine is also observed. The mechanisms for the above fragmentations are proposed. Finally, the structures of the immonium ion and related ions of N(alpha), N(epsilon)-tetramethylated lysine and N(epsilon)-dimethylated lysine are explored.     

Electrochemical detection of glutathione using redox indicators

The nucleophilic addition of the aminothiols homocysteine (HCY), cysteine (CYS), and glutathione (GSH) to the electrogenerated quinone of fluorone black (1) via the ECE mechanism is reported. It is demonstrated that 1selectively reacts with GSH to form the bis-GSH adduct, 1-(GSH)(2), while only the monothiol adducts were generated in the presence of HCY and CYS (1-HCY and 1-CYS, respectively). The more anodic E(pa) of 1-(GSH)2 relative to 1 and 1-GSH (DeltaE(pa) approximately +0.14) is the voltammetric signature that allows the discrimination of GSH from HCY and CYS. It is also shown that the presence of structurally similar aminothiols-HCY and CYS-posed no interference to the signature voltammetric response of 1-(GSH)2.     

Identification strategy using combined mass spectrometric techniques for elucidation of phase I and phase II in vitro metabolites of lipophilic marine biotoxins

Combining mass spectrometric tools, a total of 47 in vitro metabolites of okadaic acid (OA), dinophysistoxins 1 and 2 (DTX1 and DTX2), yessotoxin (YTX), azaspiracid1 (AZA1), and pectenotoxin 2 (PTX2) could be detected and confirmed after an incubation with rat liver S9-mix. In a first step, liquid chromatography (LC) combined with tandem mass spectrometry (MS/MS) was used as a screening tool for the identification of in vitro metabolites of lipophilic marine biotoxins. Metabolic phase I and phase II reactions were screened for metabolites by calculating and subsequently monitoring theoretical MS transitions. In a second step, metabolites were confirmed by determination of accurate masses using high resolution MS provided by Orbitrap technology. Subsequently, product ion spectra, precursor ion spectra, and MS3 spectra were recorded for structure elucidation of metabolites. While all investigated toxins were found to form various oxygenated metabolites during the oxidative phase I metabolism, those metabolites varied in the number of added oxygen atoms and in the number of individual isomers. No hints were obtained concerning the formation of glutathione adducts, and a conjugation with glucuronic acid was detected for AZA1 only.     

Acylic sugar derivatives for GC/MS analysis of 13C-enrichment during carbohydrate metabolism

Metabolic profiling with stable-isotope tracers in combination with gas chromatography/mass spectrometry (GC/MS) is a well-established technique for measuring substrate redistribution within metabolic pathways. This analysis relies on the ability to localize and quantify the fractional incorporation of 13C isotope into each carbon atom of precursor-derived metabolites. In this paper, several carbohydrate derivatization procedures (peracetylation, deuterioalditol acetates, and aldononitrile acetates) are evaluated for the positional isotopic information obtained by gas chromatography/electron impact mass spectrometry (GC/EI-MS). These derivatives have been compared for the quantitative evaluation of 13C distribution into isotopomers of 13C-labeled aldoses and ketoses, and the fragmentation pathways for 15 hexoses, pentoses, and amino sugars of biological origin have been assessed. In addition, a new type of carbohydrate derivative (dialkyldithioacetal acetates) has been developed for GC/MS that retains the charge on the anomeric carbon of the original monosaccharide. Electron impact ionization of these derivatives generates well-resolved base peaks arising from C1-C2 bond cleavage with charge retention at the C1 thiol groups. The dialkyldithioacetal acetates are uniquely well suited for measuring isotopic enrichment into the characteristic anomeric carbon of aldose sugars and will facilitate the global analysis of metabolic flux in carbohydrate pathways.     

Development of mass spectrometric method for analysis of cyclic nitramine explosives DTIW and HNIW

Mass spectrometric (MS) methods are used for the analysis of two novel nitramine explosives-hexanitrohexaazaisowurzitane (HNIW) and 4,10-dinitro-2,6,8,12-tetraoxa-4,10-diazaisowurzitane (DTIW). The methods include electrospray (ESI) and atmospheric pressure chemical ionization techniques for liquid chromatography/MS (LC/MS), chemical ionization for direct introduction (DCI), and gas chromatography/MS (CI-GC/MS). It is found that HNIW (438 Da) is detectable using both positive and negative modes of DCI and in the negative mode ESI-MS. Several anions were found to complex with HNIW, e.g., CF3CO2-, Cl-, Br-, I-, NO3-, and NO2-. On the other hand, DTIW could only be detected using positive DCI and CI-GC/MS, where an MH+ ion (m/z 263) was formed. The fragmentation pathways of the two nitramines were further studied by MS2 experiments. Apparently, the main fragmentation pathway of the MH+ ion of DTIW involves the loss of nitrous acid. Several anion adducts of HNIW that were studied dissociate to afford neutral HNIW and the added anions. However, Cl-, Br-, I-, and NO2- afford a series of fragments that resulted from the dissociation of the isowurzitane structure. For these anions, limit of detection was also found. To understand some of the HNIW fragmentation pathways, DFT calculations were used.     

Is GSH Chelated Pt Molecule Inactive in Anti‐Cancer Treatment? A Case Study of Pt6GS4

Platinum (Pt) drugs are widely used in anti‐cancer treatment although many reports advocated that tumor cells could inactivate Pt drugs via glutathione‐Pt (GSH‐Pt) adducts formation. To date, GSH chelated Pt molecules have not been assessed in cancer treatment because GSH‐Pt adducts are not capable of killing cancer cells, which is widely accepted and well followed. In this report, endogenous biothiol is utilized to precisely synthesize a GSH chelated Pt molecule (Pt₆GS₄). This Pt₆GS₄ molecule can be well taken up by aggressive triple negative breast cancer (TNBC) cells. Subsequently, its metabolites could enter nuclei to interact with DNA, finally the DNA‐Pt complex triggers TNBC cell apoptosis via the p53 pathway. Impressively, high efficacy for anti‐cancer treatment is achieved by Pt₆GS₄ both in vitro and in vivo when compared with traditional first‐line carboplatin in the same dosage. Compared with carboplatin, Pt₆GS₄ keeps tumor bearing mice alive for a longer time and is non‐toxic for the liver and kidneys. This work opens a route to explore polynuclear Pt compound with accurate architecture for enhancing therapeutic effects and reducing systemic toxicity.     

Stable-isotope dimethyl labeling for quantitative proteomics

In this paper, we report a novel, stable-isotope labeling strategy for quantitative proteomics that uses a simple reagent, formaldehyde, to globally label the N-terminus and epsilon-amino group of Lys through reductive amination. This labeling strategy produces peaks differing by 28 mass units for each derivatized site relative to its nonderivatized counterpart and 4 mass units for each derivatized isotopic pair. This labeling reaction is fast (less than 5 min) and complete without any detectable byproducts based on the analysis of MALDI and LC/ESI-MS/MS spectra of both derivatized and nonderivatized peptide standards and tryptic peptides of hemoglobin molecules. The intensity of the a(1) and y(n-1) ions produced, which were not detectable from most of the nonderivatized fragments, was substantially enhanced upon labeling. We further tested the method based on the analysis of an isotopic pair of peptide standards and a pair of defined protein mixtures with known H/D ratios. Using LC/MS for quantification and LC/MS/MS for peptide sequencing, the results show a negligible isotopic effect, a good mass resolution between the isotopic pair, and a good correlation between the experimental and theoretical data (errors 0-4%). The relative standard deviation of H/D values calculated from peptides deduced from the same protein are less than 13%. The applicability of the method for quantitative protein profiling was also explored by analyzing changes in nuclear protein abundance in an immortalized E7 cell with and without arsenic treatment.     

Protocol for liquid chromatography/mass spectrometry of glutathione conjugates using postcolumn solvent modification

A novel protocol for thermospray liquid chromatography/mass spectrometry (LC/MS) analysis of mixtures of glutathione conjugates is reported. Solvent conditions for optimal high-performance liquid chromatography are not always the same as for optimal thermospray ionization mass spectrometry. Labile glutathione conjugates that give poor spectra in aqueous ammonium acetate yield more intense molecular ion signals with increased percentages of acetonitrile. Direct injection thermospray ionization using 30-60% acetonitrile in aqueous ammonium acetate produced protonated molecular ions for glutathione conjugates of menadione, styrene oxide, pentachlorophenyl methyl sulfone, chlorodinitrobenzene, and chlorambucil. Since, the high percentages of organic modifier needed for good molecular ion intensity preclude chromatographic separation of these polar compounds, successful graphic separation of these polar compounds, successful LC/MS was facilitated by postcolumn addition of organic modifiers to the mobile phase. This new methodology allowed excellent chromatographic separations and thermospray ionization mass spectra to be obtained for a mixture of haloalkane glutathione conjugates. Moreover, cleavage of the gamma-glutamyl-cysteine amide bond of glutathione results in class-characteristic fragment ions. Changes in the fragmentation pathways in spectra acquired with and without organic modifiers shed light on the importance of the desolvation process in obtaining good molecular ion sensitivity in thermospray.     

Strategy for the identification of sites of phosphorylation in proteins: neutral loss triggered electron capture dissociation

We have previously demonstrated the suitability of data-dependent electron capture dissociation (ECD) for incorporation into proteomic strategies. The ability to directly determine sites of phosphorylation is a major advantage of electron capture dissociation; however, the low stoichiometry associated with phosphorylation means that phosphopeptides are often overlooked in data-dependent ECD analyses. In contrast, collision-induced dissociation (CID) tends to result in loss of the labile phosphate group, often at the expense of sequence fragments. Here, we demonstrate a novel strategy for the characterization of phosphoproteins which exploits the neutral loss feature of CID such that focused ECD of phosphopeptides is achieved. Peptides eluting from a liquid chromatograph are first subjected to CID, and if a neutral loss of 98 Da (corresponding to H3PO4) from the precursor is observed, ECD of that same precursor is performed; i.e., the method comprises neutral loss triggered ECD (NL-ECD-MS/MS). The method was applied to tryptic digests of beta-casein and alpha-casein. For alpha-casein, four sites of phosphorylation were identified with NL-ECD-MS/MS compared with a single site identified by ECD-MS/MS. The method also resulted in ECD of a doubly phosphorylated peptide. A further benefit of the method is that overall protein sequence coverage is improved. Sequence information from nonphosphorylated peptides is obtained as a result of the CID step.