Hybrid Mass Spectrometer Research Articles

Direct Approach for Qualitative and Quantitative Characterization of Glycoproteins Using Tandem Mass Tags and an LTQ Orbitrap XL Electron Transfer Dissociation Hybrid Mass Spectrometer

The application of multiplexed isobaric tandem mass tag (TMT) labeling and an LTQ Orbitrap XL ETD (electron transfer dissociation) hybrid mass spectrometer as a direct approach for qualitative and quantitative characterization of glycoproteins is reported. Bovine fetuin was used as a model glycoprotein in this study. For online liquid chromatography-mass spectrometry (LC-MS) analysis, high-resolution, mass accurate full scan MS spectra were acquired in the Orbitrap mass analyzer followed by data-dependent tandem mass spectrometry (MS/MS) with alternating collision-induced dissociation (CID), ETD, and higher-energy collisional dissociation (HCD) scans. An additional in-source dissociation scan was used as a highly sensitive and selective detection method for eluting glycosylated peptides. By alternatively using three different dissociation methods, 23 glycoforms from all 5 corresponding glycopeptides were identified from a trypsin digest of bovine fetuin. With ETD, labile glycans were retained without any signs of carbohydrate cleavage with concurrent fragmentation of the peptide backbone. Glycosylation sites were clearly localized from the ETD fragmentation data. Glycan structure elucidation was accomplished using CID. The CID experiments generated fragment ions predominantly from cleavage of glycosidic bonds without breaking the peptide bond. Novel to this method, the TMT labeling protocol was modified and adapted for higher labeling efficiency, and a TriVersa NanoMate was used to reinfuse samples to improve ETD and HCD spectra of glycopeptides. Quantification with TMT was verified based on the HCD spectra from multiple nonglycopeptides and glycopeptides. This method can be used as a qualitative and quantitative technique for direct characterization of glycoproteins and has applicability for detection of counterfeit glycoprotein drug products.

Differential effects of lipoprotein apheresis by lipidfiltration or dextran sulfate adsorption on lipidomic profile

Objective and methodsAcute modification of plasma lipidomic profile was assessed by top-down shotgun profiling on a LTQ Orbitrap hybrid mass spectrometer in 14 patients treated with two different apheresis techniques: plasma lipidfiltration (LF) and whole blood dextran sulfate adsorption (DSA). ResultsPatients treated with DSA revealed a significantly more pronounced reduction of LDL-cholesterol (LDL-C), a diminished decrease of HDL-cholesterol (HDL-C) and triglycerides (TG), and a similar reduction in lipoprotein (a) (Lp(a)) level. Against the overall tendency of reduction of lipid metabolites of all lipid classes in post-apheresis plasma, independent of apheresis technology applied, a highly significant increase of phosphatidylethanolamines (PE) in response to DSA was observed. ConclusionThese data indicate that DSA technology may be associated with an activation or damage of blood cells at contact surface which subsequently leads to a massive liberation of cellular and membrane PE's. Pathophysiological consequences, especially with respect to coagulation system and oxidative stress, have to be further elucidated.

Use of modified Doehlert‐type experimental design in optimization of a hybrid electrospray ionization ion trap time‐of‐flight mass spectrometry technique for glutathione determination

The capabilities of modified non-spherical Doehlert-type experimental designs to optimize the performance of a hybrid mass spectrometer were investigated in this paper for the first time. The optimization process was completed in three successive steps with groups of variables avoiding any univariable approach. Glutathione (GSH, reduced) was selected as a very interesting analyte since it is considered to be one of the most abundant tripeptides in human organism and its action against xenobiotics and oxidative radicals is well known. In particular, a tandem technique based on sequential ion management by an ion trap followed by a time-of-flight mass analyzer (ITTOFMS) was introduced commercially in recent years and investigated for glutathione determination. Glutathione was injected in a stream of a typical mobile phase used in liquid chromatography (LC) and analyzed after electrospray ionization (ESI) in tandem MS. The three main steps of the LC/MS system, namely the LC mobile phase, the ESI interface and the MS analyzer, were independently optimized in terms of maximum sensitivity. In this context quadratic models were found and their prediction power was evaluated. A calibration study was performed at default and optimum conditions in order to quantitatively estimate the sensitivity enhancement of the employed technique for this analyte. Satisfactory precision (RSD 5.7%) and detectability (LOD 0.07 µmol L(-1) ) were achieved. The results highlighted the possibilities offered by the employment of multifactorial optimization towards the improvement of performance of tandem mass spectrometry techniques.

Exploration of CP12 conformational changes and of quaternary structural properties using electrospray ionization traveling wave ion mobility mass spectrometry

CP12 is a small chloroplast protein involved in the Benson-Calvin cycle. Since it was demonstrated that the CP12 protein shared different conformational properties between reduced and oxidized states we took advantage of the segregational properties of the Traveling Wave Ion Mobility (TWIM) guide to study subtle conformational changes related to redox changes. Electrospray ionization mass (ESI-MS) spectra of the CP12 protein were recorded in the positive ion mode using an ESI source fitted on a quadrupole time-of-flight (QToF) hybrid mass spectrometer equipped with a TWIM cell (Synapt HDMS G1, Waters Corp., Manchester) under non-denaturing conditions. Non-covalent experiments were performed using the same instrument without the use of the TWIM device. Whatever the CP12 form studied, our results showed that CP12 protein was represented by two conformers in equilibrium that displayed very slight differences. These observations led us to propose that CP12 protein structure is rather undergoing transient subtle structural changes than having two different conformational populations in solution. In addition, using non-denaturing experiments, NAD and CP12 stoichiometry were determined with respect to the GAPDH tetramer and the redox state of CP12. In this study we showed that the use of the segregational property of the ion mobility (TWIM, Synapt G1 HDMS, Waters, Manchester, UK) allowed differentiation of subtle conformational changes between redox states of the CP12 protein. Standard non-denaturing experiments revealed different binding stoichiometry according to the redox state of the CP12 protein.

Discrimination of cyclic and linear oligosaccharides by tandem mass spectrometry using collision‐induced dissociation (CID), pulsed‐Q‐dissociation (PQD) and the higher‐energy C‐trap dissociation modes

Carbohydrates have essential functions in living organisms and cells, but, due to the presence of numerous linkage combinations, substituent sites and possible conformations, they are the class of biomolecules which exhibits the huge structural diversity found in nature. Thereby, due to such diversity and poor ionization, their structural deciphering by mass spectrometry is still a very challenging task. Here, we studied a series of linear and cyclic neutral oligosaccharides using electrospray with collision-induced dissociation (CID), pulsed-Q-dissociation (PQD) and the higher-energy C-trap dissociation (HCD) feature of a linear ion trap Orbitrap hybrid mass spectrometer (LTQ-Orbitrap). The collision energy necessary to obtain 50% fragmentation (CE(50) values) in CID, PQD and HCD was used to correlate both size and structures. The default settings for activation time and/or activation Q are the most appropriate, except for HCD, where 100 ms instead of 30 ms gave more intense fragment ions. PQD exhibits a 2-8-fold lower sensitivity than CID. HCD provides signals closer or slightly superior by 1.5-fold than PQD, and offers a more balanced ion distribution through the spectrum. Furthermore, HCD offers the possibility to make fine adjustments of the energy via the eV scale to further increase the yield of low-mass fragments. The complementarity of CID, PQD and HCD was clearly demonstrated by obtaining structural information on hexa-, hepta- and octasaccharides. Together, these results clearly indicate the usefulness of the CID/HCD pair for further structural deciphering, and analysis of more complex structures such as multi-antennary carbohydrates or glycoconjuguates alone or in mixture.

Identification of Novel Tumor-Associated Antigens for Chronic Lymphocytic Leukemia (CLL) Based On HLA Ligandome Analysis – New Targets for Peptide Based Immunotherapy

AbstractAbstract 4119Recurrent disease, selection for chemo resistant clones and inhibition of immune effector functions are limitations of chemotherapeutic treatment of cancer including CLL. This is even more since graft-versus-leukemia effects and remissions after donor lymphocyte infusions have been correlated to long-term CLL free survival. Clonotype analysis of such cases suggested clonally expanded CD8+ T cells that recognize tumor associated antigens (TAAs) presented on HLA (human leukocyte antigen) as mediators of the observed effects, thus making CLL an attractive target for peptide vaccine based immunotherapy. For this goal we established the approach of direct isolation and identification of naturally processed and presented HLA ligands from tissues of interest by affinity chromatography and mass spectrometry. Comparative, semi-quantitative analysis of the HLA ligandomes of malignant and benign samples provided the rationale for selection of potential targets. 42 CLL patients (ages 48–85; median 70 years) of different HLA types and disease stages (Binet A, 24 patients; Binet B, 11 patients; Binet C, 7 patients) were enrolled in this study. Furthermore we collected blood samples from 50 healthy volunteers. HLA class I ligands were isolated from CLL cells as well as benign B cells from healthy donors and unsorted healthy PBMC using a standard affinity chromatography protocol implementing the pan-HLA class I specific antibody W6/32. Liquid chromatography coupled mass spectrometry (LC-MS/MS) based peptide analysis was performed on an LTQ Orbitrap hybrid mass spectrometer followed by annotation of fragment spectra using the MASCOT search algorithm against the human proteome comprised in the SwissProt database. Spectral counting based analysis provided semi-quantitative information regarding the abundance of HLA ligands and their source proteins in the respective ligandomes. In addition, HLA quantification experiments on the cell surface of CLL cells and autologous healthy B cells were performed using a flow cytometric indirect immunofluorescence assay. For this interim analysis we completely analyzed the HLA ligandomes of 7 CLL patients and 10 healthy controls. In total, we identified more than 15.000 different HLA ligands representing more than 7300 different proteins. This comprised more than 6,500 different ligands from CLL cells representing a total of 4,149 source proteins. Comparative analysis of representation in the HLA ligandomes of CLL cells, healthy B cells and healthy PBMC identified 1741 different source proteins as being exclusively expressed in CLL. Semi-quantitative evaluation revealed 138 of these proteins as being highly expressed on CLL (e.g. SET proto-oncogene, Pim-1 Oncogene, Mucin 1). Flow cytomerty based quantification of surface HLA expression revealed similar amounts of HLA class I (p=0.23, unpaired t-test) and II (p=0.33, unpaired t-test) molecules on CLL cells and autologous benign B lymphocytes, with a trend to higher HLA expression on CLL cells.Taken together, the presented strategy enabled the identification of a vast array of both known and novel TAAs and their corresponding naturally processed and presented HLA ligands in CLL. It pinpointed highly overrepresented TAAs for further analysis and immunogenicity testing. Expansion of the dataset after analysis of all enrolled patients will provide an unprecedented in-depth characterization of the HLA ligandome of CLL for future immunotherapeutic approaches. Disclosures:No relevant conflicts of interest to declare.

Selectivity of LC-MS/MS analysis: Implication for proteomics experiments

The recent development of hybrid mass spectrometers with high resolution and accurate mass capabilities has opened new avenues in quantitative proteomics. A systematic study was performed to assess the quantification performances of a novel quadrupole-Orbitrap instrument operated in MS/MS mode (parallel reaction monitoring). It included the analyses of 35 isotopically labeled peptides spiked in urine samples to establish their dilution curves. The results were evaluated by replicating the analyses on a triple quadrupole instrument operated in selected reaction monitoring (SRM; often referred as multiple reaction monitoring, MRM) mode to assess and compare the gain in selectivity resulting from high resolution fragment ion analysis. The high resolving power dramatically increased the selectivity of measurements by separating ions of interest from interferences, which occurred in several cases, and thus improved the quantification performance. In addition, an experiment to assess the "co-habitation" of fragment ions in specific regions of the LC-MS/MS spectral space of a complex proteome digest was carried out. The study included the evaluation of the fragmentation patterns acquired under various experimental conditions (i.e., quadrupole isolation windows and Orbitrap resolving powers) for more than 200 peptides, which provided an experimental baseline to guide the development of methods for parallel reaction monitoring acquisition. This article is part of a Special Issue entitled: From protein structures to clinical applications.

The determination of phenolic profiles of Serbian unifloral honeys using ultra-high-performance liquid chromatography/high resolution accurate mass spectrometry

Polyphenolic profiles of 44 unifloral Serbian honeys were analyzed using ultra-high-performance liquid chromatography (UHPLC) coupled with hybrid mass spectrometer which combines the Linear Trap Quadrupole (LTQ) and OrbiTrap mass analyzer. Rapid UHPLC method was developed in combination with a high sensitivity accurate mass scan and a simultaneous data dependent scan. The honey samples were of different botanical origin: acacia (Robinia pseudoacacia), sunflower (Helianthus annuus), linden (Tilia cordata), basil (Ocimum basilicum), buckwheat (Fagopyrum esculentum), oilseed rape (Brassica napus), and goldenrod (Solidago virgaurea). The presence of 43 compounds, mainly flavonoids, was proven in all honey samples by their characteristic mass spectra and fragmentation pattern. Relatively high amounts of chrysin, pinocembrin and galangin were identified in all honey extracts. p-Coumaric acid was not detected in basil, buckwheat and goldenrod honey extracts. A larger amount of gallic acid (max value 1.45mg/kg) was found in the sunflower honey, while a larger amount of apigenin (0.97mg/kg) was determined in the buckwheat honey in comparison with other honeys. The samples were classified according to the botanical origin using pattern recognition technique, Principal Component Analysis (PCA). The LTQ OrbiTrap technique was proven to be reliable for the unambiguous detection of phenolic acids, their derivatives, and flavonoid aglycones based on their molecular masses and fragmentation pattern.

Automated Gas-Phase Purification for Accurate, Multiplexed Quantification on a Stand-Alone Ion-Trap Mass Spectrometer

Isobaric tagging enables the acquisition of highly multiplexed proteome quantification, but it is hindered by the pervasive problem of precursor interference. The elimination of coisolated contaminants prior to reporter tag generation can be achieved through the use of gas-phase purification via proton transfer ion/ion reactions (QuantMode); however, the original QuantMode technique was implemented on the high-resolution linear ion-trap-Orbitrap hybrid mass spectrometer enabled with electron transfer dissociation (ETD). Here we extend this technology to stand-alone linear ion-trap systems (trapQuantMode, trapQM). Facilitated by the use of inlet beam-type activation (i.e., trapHCD) for production and observation of the low mass-to-charge reporter region, this scan sequence comprises three separate events to maximize peptide identifications, minimize duty cycle requirements, and increase quantitative accuracy, precision, and dynamic range. Significant improvements in quantitative accuracy were attained over standard methods when using trapQM to analyze an interference model system comprising tryptic peptides of yeast that we contaminated with human peptides. Finally, we demonstrate practical benefits of this method by analysis of the proteomic changes that occur during mouse skeletal muscle myoblast differentiation. While the reduced duty cycle of trapQM led to the identification of fewer proteins than conventional operation (4050 vs 2964), trapQM identified more significant differences (>1.5 fold, 1362 vs 1132, respectively; p < 0.05) between the proteomes of undifferentiated myoblasts and differentiated myotubes and nearly 10-fold more differences with changes greater than 5-fold (96 vs 12). We further show that our trapQM dataset is superior for identifying changes in protein abundance that are consistent with the metabolic and structural changes known to accompany myotube formation.

The Potassium Channel Interacting Protein 3 (DREAM/KChIP3) Heterodimerizes with and Regulates Calmodulin Function

Downstream regulatory element antagonistic modulator (DREAM/KChIP3), a neuronal EF-hand protein, modulates pain, potassium channel activity, and binds presenilin 1. Using affinity capture of neuronal proteins by immobilized DREAM/KChIP3 in the presence and absence of calcium (Ca(2+)) followed by mass spectroscopic identification of interacting proteins, we demonstrate that in the presence of Ca(2+), DREAM/KChIP3 interacts with the EF-hand protein, calmodulin (CaM). The interaction of DREAM/KChIP3 with CaM does not occur in the absence of Ca(2+). In the absence of Ca(2+), DREAM/KChIP3 binds the EF-hand protein, calcineurin subunit-B. Ca(2+)-bound DREAM/KChIP3 binds CaM with a dissociation constant of ∼3 μM as assessed by changes in DREAM/KChIP3 intrinsic protein fluorescence in the presence of CaM. Two-dimensional (1)H,(15)N heteronuclear single quantum coherence spectra reveal changes in chemical shifts and line broadening upon the addition of CaM to (15)N DREAM/KChIP3. The amino-terminal portion of DREAM/KChIP3 is required for its binding to CaM because a construct of DREAM/KChIP3 lacking the first 94 amino-terminal residues fails to bind CaM as assessed by fluorescence spectroscopy. The addition of Ca(2+)-bound DREAM/KChIP3 increases the activation of calcineurin (CN) by calcium CaM. A DREAM/KChIP3 mutant incapable of binding Ca(2+) also stimulates calmodulin-dependent CN activity. The shortened form of DREAM/KChIP3 lacking the NH(2)-terminal amino acids fails to activate CN in the presence of calcium CaM. Our data demonstrate the interaction of DREAM/KChIP3 with the important EF-hand protein, CaM, and show that the interaction alters CN activity.

An ion-current-based, comprehensive and reproducible proteomic strategy for comparative characterization of the cellular responses to novel anti-cancer agents in a prostate cell model

Proteome-level investigation of the molecular targets in anticancer action of promising pharmaceutical candidates is highly desirable but remains challenging due to the insufficient proteome coverage, limited capacity for biological replicates, and largely unregulated false positive biomarker discovery of current methods. This study described a practical platform strategy to address these challenges, using comparison of drug response proteomic signatures by two promising anti-cancer agents (KX01/KX02) as the model system for method development/optimization. Drug-treated samples were efficiently extracted followed by precipitation/on-pellet-digestion procedure that provides high, reproducible peptide recovery. High-resolution separations were performed on a 75-cm-long, heated nano-LC column with a 7-h gradient, with a highly reproducible nano-LC/nanospray configuration. An LTQ Orbitrap hybrid mass spectrometer with a charge overfilling approach to enhance sensitivity was used for detection. Analytical procedures were optimized and well-controlled to achieve high run-to-run reproducibility that permits numerous replicates in one set, and an ion-current-based approach was utilized for quantification. The false positives of biomarker discovery arising from technical variability was controlled based on FBDR measurement by comparing biomarker numbers in each drug-treated group vs. "sham samples", which were analyzed in an order randomly interleaved with the analysis drug-treated samples. More than 1500 unique protein groups were quantified under stringent criteria, and of which about 30% displayed differential expression with FBDR of 0.3-2.1% across groups. Comparison of drug-response proteomic signatures and the subsequent immunoassay revealed that the action mechanisms of KX01/KX02 are similar but significantly different from vinblastine, which correlates well with clinical and pre-clinical observations. Furthermore, the results strongly supported the hypothesis that KX01/KX02 are dual-action agents (through inhibition of tubulin and Src). Moreover, informative insights into the drug-actions on cell cycle, growth/proliferation, and apoptosis were obtained. This platform technology provides extensive evaluation of drug candidates and facilitates in-depth mechanism studies.

Enhanced Peptide Identification by Electron Transfer Dissociation Using an Improved Mascot Percolator

Peptide identification using tandem mass spectrometry is a core technology in proteomics. Latest generations of mass spectrometry instruments enable the use of electron transfer dissociation (ETD) to complement collision induced dissociation (CID) for peptide fragmentation. However, a critical limitation to the use of ETD has been optimal database search software. Percolator is a post-search algorithm, which uses semi-supervised machine learning to improve the rate of peptide spectrum identifications (PSMs) together with providing reliable significance measures. We have previously interfaced the Mascot search engine with Percolator and demonstrated sensitivity and specificity benefits with CID data. Here, we report recent developments in the Mascot Percolator V2.0 software including an improved feature calculator and support for a wider range of ion series. The updated software is applied to the analysis of several CID and ETD fragmented peptide data sets. This version of Mascot Percolator increases the number of CID PSMs by up to 80% and ETD PSMs by up to 60% at a 0.01 q-value (1% false discovery rate) threshold over a standard Mascot search, notably recovering PSMs from high charge state precursor ions. The greatly increased number of PSMs and peptide coverage afforded by Mascot Percolator has enabled a fuller assessment of CID/ETD complementarity to be performed. Using a data set of CID and ETcaD spectral pairs, we find that at a 1% false discovery rate, the overlap in peptide identifications by CID and ETD is 83%, which is significantly higher than that obtained using either stand-alone Mascot (69%) or OMSSA (39%). We conclude that Mascot Percolator is a highly sensitive and accurate post-search algorithm for peptide identification and allows direct comparison of peptide identifications using multiple alternative fragmentation techniques.

Assessment of benzophenone-4 reactivity with free chlorine by liquid chromatography quadrupole time-of-flight mass spectrometry

The stability of the UV filter benzophenone-4 (BP-4) in free chlorine-containing water was investigated, for the first time, by liquid chromatography quadrupole time-of-flight mass spectrometry (LC–QqTOF-MS). High mass accuracy and resolution capabilities of this hybrid mass spectrometer were used for the reliable assignation of empirical formulae and chemical structures of BP-4 derivatives. Time-course profiles of the parent compound and its by-products were simultaneously recorded by direct injection of sample aliquots, after quenching the excess of chlorine, in the LC–QqTOF-MS system. At neutral pHs, in excess of chlorine, BP-4 showed a limited stability fitting a pseudo-first-order degradation kinetics. A noticeable reduction in the half-lives of BP-4 was observed when increasing the sample pH between 6 and 8 units and also in presence of bromide traces. The reaction pathway of this UV filter involved a first electrophilic substitution of hydrogen per chlorine (or bromide) in the phenolic ring, followed by oxidation of the carbonyl moiety to an ester group, which induced a further electrophilic substitution in the same aromatic ring. Above reactions were also noticed when mixing a BP-4 containing personal care product with chlorinated tap water and in chlorinated swimming pool and sewage water, previously spiked with a BP-4 standard.

Identification and characterization of urinary prenylamine metabolites by means of liquid chromatography‐tandem mass spectrometry

Prenylamine is a vasodilator of phenylalkylamine structure and was used for the treatment of angina pectoris, until reports of undesirable effects including ventricular tachycardia led to a decreasing use of the drug in the 1980s. Metabolic N-dealkylation of orally ingested prenylamine can liberate amphetamine in humans and cause positive findings for amphetamine in doping and forensic analysis. In 2010, the World Anti-Doping Agency (WADA) classified prenylamine as a non-specified stimulant according to the 2010 Prohibited List, thus banning its use in sports in-competition. Supporting the development of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) based detection method, a post-administration urine sample following a single oral prenylamine ingestion (Segontin(®) 60 mg) was analyzed for urinary metabolites. The LC-separated analytes were ionized in positive electrospray ionization (ESI) mode and detected as protonated ions using an AB Sciex TripleTOF 5600 quadrupole-time-of-flight hybrid mass spectrometer. Over 40 phase I metabolites were detected, including previously unknown mono- bis-, tris- and tetra-hydroxylated prenylamine, several hydroxylated and methoxylated prenylamine metabolites and (hydroxylated) diphenylpropylamine. Investigation of the collision-induced dissociation behaviours of the metabolites by high resolution/high accuracy mass spectrometry allowed for the assignment of the nature and the site of observed metabolic transformations. The most abundant phase I metabolite was confirmed as p-hydroxy-prenlyamine by chemical synthesis and stable isotope labelling of reference material. An existing routine screening assay based on direct injection and LC-MS/MS analysis of urine was modified and validated according to common guidelines, in order to allow for the detection of p-hydroxy-prenylamine in sports drug testing. The assay demonstrated the ability to detect the target metabolite at 0.1 ng/ml at intra- and inter-day imprecisions below 10%.

Cholesteryl ester acyl oxidation and remodeling in murine macrophages: formation of oxidized phosphatidylcholine

Cholesterol is an essential component of eukaryotic cell membranes, regulating fluidity and permeability of the bilayer. Outside the membrane, cholesterol is esterified to fatty acids forming cholesterol esters (CEs). Metabolism of CEs is characterized by recurrent hydrolysis and esterification as part of the CE cycle; however, since recombinant 15-lipoxygenase (15-LO) was shown to oxidize cholesteryl linoleate of LDL, there has been interest in CE oxidation, particularly in the context atherogenesis. Studies of oxidized CE (oxCE) metabolism have focused on hydrolysis and subsequent reverse cholesterol transport with little emphasis on the fate the newly released oxidized fatty acyl component. Here, using mass spectrometry to analyze lipid oxidation products, CE metabolism in murine peritoneal macrophages was investigated. Ex vivo macrophage incubations revealed that cellular 15-LO directly oxidized multiple CE substrates from intracellular stores and from extracellular sources. Freshly harvested murine macrophages also contained 15-LO-specific oxCEs, suggesting the enzyme may act as a CE-oxidase in vivo. The metabolic fate of oxCEs, particularly the hydrolysis and remodeling of oxidized fatty acyl chains, was also examined in the macrophage. Metabolism of deuterated CE resulted in the genesis of deuterated, oxidized phosphatidylcholine (oxPC). Further experiments revealed these oxPC species were formed chiefly from the hydrolysis of oxidized CE and subsequent reacylation of the oxidized acyl components into PC.

Proteomic Analysis of Bacillus thuringiensis at Different Growth Phases by Using an Automated Online Two-Dimensional Liquid Chromatography-Tandem Mass Spectrometry Strategy

The proteome of a new Bacillus thuringiensis subsp. kurstaki strain, 4.0718, from the middle vegetative (T(1)), early sporulation (T(2)), and late sporulation (T(3)) phases was analyzed using an integrated liquid chromatography (LC)-based protein identification system. The system comprised two-dimensional (2D) LC coupled with nanoscale electrospray ionization (ESI) tandem mass spectrometry (MS/MS) on a high-resolution hybrid mass spectrometer with an automated data analysis system. After deletion of redundant proteins from the different batches and B. thuringiensis subspecies, 918, 703, and 778 proteins were identified in the respective three phases. Their molecular masses ranged from 4.6 Da to 477.4 Da, and their isoelectric points ranged from 4.01 to 11.84. Function clustering revealed that most of the proteins in the three phases were functional metabolic proteins, followed by proteins participating in cell processes. Small molecular and macromolecular metabolic proteins were further classified according to the Kyoto Encyclopedia of Genes and Genome and BioCyc metabolic pathway database. Three protoxins (Cry2Aa, Cry1Aa, and Cry1Ac) as well as a series of potential intracellular active factors were detected. Many significant proteins related to spore and crystal formation, including sporulation proteins, help proteins, chaperones, and so on, were identified. The expression patterns of two identified proteins, CotJc and glutamine synthetase, were validated by Western blot analysis, which further confirmed the MS results. This study is the first to use shotgun technology to research the proteome of B. thuringiensis. Valuable experimental data are provided regarding the methodology of analyzing the B. thuringiensis proteome (which can be used to produce insecticidal crystal proteins) and have been added to the related protein database.

Identification of isomeric 5-hydroxytryptophan- and oxindolylalanine-containing peptides by mass spectrometry

Cells continuously produce reactive oxidative species that can modify all cellular components. In proteins, for example, cysteine, methionine, tryptophan (Trp), and tyrosine residues are particularly prone to oxidation. Here, we report two new approaches to distinguish two isomeric oxidation products of Trp residues, i.e. 5-hydroxytryptophan (5-HTP) and oxindolylalanine (Oia) residues, in peptides. First, 2-nitrobenzenesulfenyl chloride, known to derivatize Trp residues in position 2 of the indole ring, was used to label 5-HTP residues. The mass shift of 152.98 m/z units allowed identifying 5-HTP- besides Trp-containing peptides by mass spectrometry, whereas Oia residues were not labeled. Second, fragmentation of the Oia- and 5-HTP-derived immonium ions at m/z 175.08 produced ions characteristic for each residue that allowed their identification even in the presence of y(1) ions at m/z 175.12 derived from peptides with C-terminal arginine residues. The pseudo MS(3) spectra acquired on a quadrupole time-of-flight hybrid mass spectrometer displayed two signals at m/z 130.05 and m/z 132.05 characteristic for Oia-containing peptides and a group of six signals (m/z 103.04, 120.04, 130.04, 133.03, 146.04, and 148.04) for 5-HTP-cointaining peptides. In both cases, the relative signal intensities appeared to be independent of the sequence providing a specific fingerprint of each oxidative modification.

Systematic Comparison of Label-Free, Metabolic Labeling, and Isobaric Chemical Labeling for Quantitative Proteomics on LTQ Orbitrap Velos

A variety of quantitative proteomics methods have been developed, including label-free, metabolic labeling, and isobaric chemical labeling using iTRAQ or TMT. Here, these methods were compared in terms of the depth of proteome coverage, quantification accuracy, precision, and reproducibility using a high-performance hybrid mass spectrometer, LTQ Orbitrap Velos. Our results show that (1) the spectral counting method provides the deepest proteome coverage for identification, but its quantification performance is worse than labeling-based approaches, especially the quantification reproducibility; (2) metabolic labeling and isobaric chemical labeling are capable of accurate, precise, and reproducible quantification and provide deep proteome coverage for quantification; isobaric chemical labeling surpasses metabolic labeling in terms of quantification precision and reproducibility; and (3) iTRAQ and TMT perform similarly in all aspects compared in the current study using a CID-HCD dual scan configuration. On the basis of the unique advantages of each method, we provide guidance for selection of the appropriate method for a quantitative proteomics study.

Identification of in Vivo Disulfide Conformation of TRPA1 Ion Channel

TRPA1 (transient receptor potential ankyrin 1) is an ion channel expressed in the termini of sensory neurons and is activated in response to a broad array of noxious exogenous and endogenous thiol-reactive compounds, making it a crucial player in chemical nociception. A number of conserved cysteine residues on the N-terminal domain of the channel have been identified as critical for sensing these electrophilic pungent chemicals, and our recent EM structure with modeled domains predicts that these cysteines form a ligand-binding pocket, allowing for the possibility of disulfide bonding between the cysteine residues. Here, we present a comprehensive mass spectrometry investigation of the in vivo disulfide bonding conformation and in vitro reactivity of 30 of the 31 cysteine residues in the TRPA1 ion channel. Four disulfide bonds were detected in the in vivo TRPA1 structure: Cys-666-Cys-622, Cys-666-Cys-463, Cys-622-Cys-609, and Cys-666-Cys-193. All of the cysteines detected were reactive to N-methylmaleimide (NMM) in vitro, with varying degrees of labeling efficiency. Comparison of the ratio of the labeling efficiency at 300 μM versus 2 mM NMM identified a number of cysteine residues that were outliers from the mean labeling ratio, suggesting that protein conformation changes rendered these cysteines either more or less protected from labeling at the higher NMM concentrations. These results indicate that the activation mechanism of TRPA1 may involve N-terminal conformation changes and disulfide bonding between critical cysteine residues.

Axonal Gradient of Arachidonic Acid-containing Phosphatidylcholine and Its Dependence on Actin Dynamics

Phosphatidylcholine (PC) is the most abundant component of lipid bilayers and exists in various molecular forms, through combinations of two acylated fatty acids. Arachidonic acid (AA)-containing PC (AA-PC) can be a source of AA, which is a crucial mediator of synaptic transmission and intracellular signaling. However, the distribution of AA-PC within neurons has not been indicated. In the present study, we used imaging mass spectrometry to characterize the distribution of PC species in cultured neurons of superior cervical ganglia. Intriguingly, PC species exhibited a unique distribution that was dependent on the acyl chains at the sn-2 position. In particular, we found that AA-PC is enriched within the axon and is distributed across a proximal-to-distal gradient. Inhibitors of actin dynamics (cytochalasin D and phallacidin) disrupted this gradient. This is the first report of the gradual distribution of AA-PC along the axon and its association with actin dynamics.