Hybrid Quadrupole-orbitrap Mass Spectrometer Research Articles

O1.7. PROTEOMIC ANALYSIS OF BLOOD BASED SAMPLES FROM THE OPTiMiSE (OPTIMIZATION OF TREATMENT AND MANAGEMENT OF SCHIZOPHRENIA IN EUROPE) STUDY POINT TOWARDS COMPLEMENT PATHWAY PROTEIN CHANGES

BackgroundThe OPTiMiSE (Optimization of Treatment and Management of Schizophrenia in Europe) trial may help in the identification of predictors of treatment response. Medication naïve patients with first episode schizophrenia or schizophreniform disorder were enrolled in the study and treated open-label for a four-week period with amisulpride. PANSS ratings were undertaken at baseline and following the four-week treatment. 30 non-remitters (as defined by the Andreasen criteria) with the worst change in PANSS scores and the 30 remitters with the best change in PANSS scores were selected to represent good and poor outcome groups.MethodsWe compared proteomic markers in serum collected prior to treatment in 30 patients who subsequently showed a good response to amisulpride (“responders”, and 30 patients who did not show a good response (“non-responders”). Serum samples were depleted using High Performance Liquid Chromatography (HPLC) attached to a MARS column to remove the 14 most abundant plasma proteins (albumin, IgG, antitrypsin, IgA, transferrin, haptoglobin, fibrinogen, alpha2-macroglobulin, alpha1-acid glycoprotein, IgM, apolipoprotein AI, apolipoprotein AII, complement C3, and transthyretin). The groups were matched for ethnicity, gender and age.50 µg from each sample were reduced, alkylated, tryptically digested, then zip-tipped to concentrate and purify. Samples were then run for a 90-min gradient on a Thermo Scientific Q- Exactive Hybrid Quadrupole-Orbitrap Mass Spectrometer. Raw MS data were processed by MaxQuant software and searched against the human Uniprot database, for label free quantitation of peptides and proteins. False discovery rates (FDR) were set at 1% for both peptide and protein levels in target/decoy to minimize false positives. The match between runs feature was utilized.ResultsFour hundred and sixty-four protein identifications were obtained. Samples were excluded where >30% of proteins were missing, and we used imputation, where the missingness depends upon a threshold of detection. This left 228 proteins for analysis. Of these, 21 were significantly different between responders and non-responders (p<0.05), one was FDR positive, one at trend FDR level. Pathway analysis (KEGG, David NIH) of the significant proteins determined “complement and coagulation cascades” to be the top pathway affected with six proteins from the list assigned to the pathway. These were CFI, C4A, C6, F9, VWF and SERPING1, all found to be up-regulated and with p-values ranging from 0.002 to 0.044. C6 is a constituent of the membrane attack complex (MAC) that plays a key role in the innate and adaptive immune response, while CFI belongs to the alternative pathway, C4A belongs to the classical pathway and F9 and VWF play roles in the intrinsic coagulation pathway.DiscussionThese data complement results by Sekar et al., implicating excessive complement activity in the development of schizophrenia. Our data identifies the complement proteins in treatment response and this is also consistent with our previous findings of up-regulation of the complement pathway among those at risk of future psychotic experiences.

Proteomics-based insights into mitogen-activated protein kinase inhibitor resistance of cerebral melanoma metastases

BackgroundMAP kinase inhibitor (MAPKi) therapy for BRAF mutated melanoma is characterized by high response rates but development of drug resistance within a median progression-free survival (PFS) of 9–12 months. Understanding mechanisms of resistance and identifying effective therapeutic alternatives is one of the most important scientific challenges in melanoma. Using proteomics, we want to specifically gain insight into the pathophysiological process of cerebral metastases.MethodsCerebral metastases from melanoma patients were initially analyzed by a LC–MS shotgun approach performed on a QExactive HF hybrid quadrupole-orbitrap mass spectrometer. For further validation steps after bioinformatics analysis, a targeted LC-QQQ-MS approach, as well as Western blot, immunohistochemistry and immunocytochemistry was performed.ResultsIn this pilot study, we were able to identify 5977 proteins by LC–MS analysis (data are available via ProteomeXchange with identifier PXD007592). Based on PFS, samples were classified into good responders (PFS ≥ 6 months) and poor responders (PFS le 3 months). By evaluating these proteomic profiles according to gene ontology (GO) terms, KEGG pathways and gene set enrichment analysis (GSEA), we could characterize differences between the two distinct groups. We detected an EMT feature (up-regulation of N-cadherin) as classifier between the two groups, V-type proton ATPases, cell adhesion proteins and several transporter and exchanger proteins to be significantly up-regulated in poor responding patients, whereas good responders showed an immune activation, among other features. We identified class-discriminating proteins based on nearest shrunken centroids, validated and quantified this signature by a targeted approach and could correlate parts of this signature with resistance using the CPL/MUW proteome database and survival of patients by TCGA analysis. We further validated an EMT-like signature as a major discriminator between good and poor responders on primary melanoma cells derived from cerebral metastases. Higher immune activity is demonstrated in patients with good response to MAPKi by immunohistochemical staining of biopsy samples of cerebral melanoma metastases.ConclusionsEmploying proteomic analysis, we confirmed known extra-cerebral resistance mechanisms in the cerebral metastases and further discovered possible brain specific mechanisms of drug efflux, which might serve as treatment targets or as predictive markers for these kinds of metastasis.

Sensitive Detection of Neonicotinoid Insecticides and Other Selected Pesticides in Pollen and Nectar Using Nanoflow Liquid Chromatography Orbitrap Tandem Mass Spectrometry.

In this work, a new method based on nanoflow LC with high-resolution MS was developed for the determination of eight pesticides in pollen and nectar samples, including neonicotinoid insecticides and other selected pesticides commonly found in bees and beeswax. Detection was undertaken with a hybrid quadrupole-Orbitrap mass spectrometer (Q Exactive™) equipped with a commercial nanospray ion source. The extraction of pesticides from pollen samples was performed by a modified micro-QuEChERS method scaled down to Eppendorf tubes, whereas nectar samples were simply diluted with a water-methanol (95 + 5, v/v) solution. Good linearity (>0.999 in all cases) was obtained between 0.05 and 500 µg/kg and between 0.04 and 400 µg/kg for pollen and nectar, respectively. Recovery rates in pollen ranged from 85 to 97%, with RSDs <12%. Matrix effect was evaluated and showed negligible effects for all studied pesticides. The lowest concentration levels tested and validated were 0.5 and 0.4 µg/kg for pollen and nectar matrixes, respectively. In addition, selected incurred samples were studied, obtaining several positive findings in pollen and nectar samples, demonstrating the sensitivity and applicability of the proposed method.

QUALITATIVE ANALYSIS OF THE COMPOSITION NON-VOLATILE BRAZILIAN WHOLE GRAPE JUICES USING QEXACTIVE™ (HYBRID QUADRUPOLE-ORBITRAP MASS SPECTROMETER) AND PRINCIPAL COMPONENT ANALYSIS (PCA)

The main constituents of grape juice are non-volatile compounds and are responsible, in most cases, the organoleptic characteristics of the product, particularly flavor and color, attributes related to quality and acceptance product by consumers. The aim of this paper was to study, in a qualitative way, the non-volatile compounds of Brazilian whole grape juices using QExactive ™ (Hybrid Quadrupole-Orbitrap Mass Spectrometer) and principal component analysis (PCA). With QExactive™, high resolution and precision equipment, it was possible to identify organic acids, phenolic acids and certain amino acids in the samples. The PCA proved to be an important tool, because through it, it was possible to obtain information about the group juices studied correlating them with the compounds identified in QExactive™.

Chromatographic column evaluation for the untargeted profiling of glucosinolates in cauliflower by means of ultra-high performance liquid chromatography coupled to high resolution mass spectrometry

The untargeted profiling is a promising approach for the characterization of secondary metabolites in biological matrices. Thanks to the recent rapid development of high-resolution mass spectrometry (HRMS) instrumentations, the number of applications by untargeted approaches for biological samples profiling has widely increased in the recent years. Despite the high potentialities of HRMS, however, a major issue in natural products analysis often arises in the upstream process of compounds separation. A separation technique is necessary to avoid phenomena such as signal suppression, and it is especially needed in the presence of isomeric metabolites, which are otherwise indistinguishable. Glucosinolates (GLSs), a group of secondary metabolites widely distributed among plants, resulted to be associated to the prevention of some serious diseases, such as cancer. This led to the development of several methods for the analysis of GLSs in vegetables tissues. The issue of GLSs chromatographic separation has been widely studied in the past because of the difficulty in the analysis of this highly polar and variable class of compounds. Several alternatives to reversed phase (RP) chromatography, sometimes not compatible with the coupling of liquid chromatography with mass spectrometry, have been tested for the analysis of intact GLSs. However, the availability of new stationary phases, in the last years, could allow the re-evaluation of RP chromatography for the analysis of intact GLSs.In this work, a thorough evaluation of four RP chromatographic columns for the analysis of GLSs in cauliflower (Brassica oleracea L. var. botrytis) extracts by an ultra-high performance liquid chromatographic system coupled via electrospray source to a hybrid quadrupole-Orbitrap mass spectrometer is presented. The columns tested were the following: one column Luna Omega polar C18, one column Kinetex Biphenyl, one column Kinetex core-shell XB-C18, two columns Kinetex core-shell XB-C18. After a previous optimization of the extraction method, cauliflower extracts were analyzed testing four different mobile phases onto the four columns for a total of sixteen different chromatographic conditions. The chromatographic systems were evaluated based on the number of detected and tentatively identified GLSs. Luna Polar stationary phase resulted to be the most suitable for the analysis of GLSs compared to Kinetex XB and Kinetex Biphenyl columns stationary phase. However, two in series Kinetex XB columns increased the number of tentatively identified GLSs compared to one Kinetex XB, showing the importance of column length in the analysis of complex mixtures. The data obtained with the best chromatographic system were deeply analyzed by MS/MS investigation for the final identification. Fiflty-one GLSs were tentatively identified, 24 of which have never been identified in cauliflower. Finally the linearity of the analytes response over the analyzed range of concentration was checked, suggesting that the developed method is suitable for both qualitative and quantitative analysis of GLSs in phytochemical mixtures.

Phenolic profiles and antioxidant activity of Turkish Tombul hazelnut samples (natural, roasted, and roasted hazelnut skin)

The phenolic profiles and antioxidant status of hazelnut samples [natural (raw) hazelnut, roasted hazelnut, and roasted hazelnut skin] were compared. Free and bound (ester-linked and glycoside-linked) phenolic acids were examined using liquid chromatography–tandem mass spectrometry (LC–MS/MS). Comprehensive identification of phenolics was carried out using Q-exactive hybrid quadrupole-orbitrap mass spectrometer (Q-OT-MS). Samples were also assessed for their total phenolics and antioxidant activities using three different assays. Ten free and bound phenolic acids were quantified in hazelnut samples. Roasted hazelnut skin contained the highest content of total phenolic acids, followed by natural and roasted hazelnuts. The majority of phenolic acids were present in the bound form. Using a Q-OT-MS, 22 compounds were tentatively identified, 16 of which were identified for the first time in hazelnut samples. The newly identified compounds consisted of flavonoids, phenolic acids and related compounds, hydrolysable tannins and related compounds, and other phenolics. Three antioxidant assays demonstrated similar trends that roasted hazelnut skin rendered the highest activity. The present work suggests that roasted hazelnut skin is a rich source of phenolics and can be considered as a value-added co-product for use as functional food ingredient and antioxidant.

Spectrum Effect Relationship and Component Knock-Out in Angelica Dahurica Radix by High Performance Liquid Chromatography-Q Exactive Hybrid Quadrupole-Orbitrap Mass Spectrometer.

Different extracts of Angelica dahuricae were available for whitening or treating vitiligo clinically. They showed inhibitory or activating effects on tyrosinase, a rate-limiting enzyme of melanogenesis. This study aimed to identify active compounds on tyrosinase in water extract of Angelica dahurica Radix. We applied spectrum-effect relationship and component knock-out methods to make it clear. HPLC was used to obtain the specific chromatograms. The effects on tyrosinase activity were examined by measuring the oxidation rate of levodopa in vitro. Partial least squares method was used to examine the spectrum-effect relationships. The knocked-out samples were prepared by HPLC method, and the identification of knocked-out compounds was conducted by the high performance liquid chromatography-four stage rod-electrostatic field orbit trap high resolution mass spectrometry. Results showed that S6, S14, S18, S21, S35, S36, S37, S40, and S41 were positively correlated to inhibitory activity of Angelica dahuricae on tyrosinase whereas S9, S11, S8, S12, S22, and S30 were negatively correlated. When the concentration of each sample was 1 g·mL−1, equal to the amount of raw medicinal herbs, oxypeucedanin hydrate, imperatorin, cnidilin, and isoimperatorin had inhibitory effects on tyrosinase activity whereas byakangelicin and bergapten had activating effects.

Evaluation of column length and particle size effect on the untargeted profiling of a phytochemical mixture by using UHPLC coupled to high-resolution mass spectrometry.

Liquid chromatography coupled to high-resolution mass spectrometry is the technique of choice for the untargeted profiling of food matrices. Despite the high potential of high-resolution mass spectrometry, when dealing with complex mixtures, an efficient separation technique is also needed. The novel core-shell chromatographic columns packed with sub-2μm sized particles are claimed to show very good resolution. However, the analytes retention can be significantly altered when working under ultra-high performance chromatographic conditions. In this work, an evaluation of four chromatographic systems, with either a single or two in-series Kinetex™ C18 columns, either packed with 2.6 or 1.7μm particles, is presented for the targeted analysis of a standard mixture and the untargeted analysis of a strawberry extract. An ultra-high performance chromatographic system coupled via an electrospray source to a hybrid quadrupole-Orbitrap mass spectrometer was used. From the extensive comparison, a surprising result was obtained, namely, that the system identifying the largest number of features was the one with two in-series connected columns with the larger particle size. The inconsistency among the theoretical assumptions and the applicative findings points out the importance of an extensive chromatographic evaluation for the comprehensive untargeted profiling of complex real samples.

Proteomic analysis of protein purified derivative of Mycobacterium bovis

BackgroundTuberculin skin test based on in vivo intradermal inoculation of purified protein derivative from Mycobacterium bovis (bPPD) is the diagnostic test for the control and surveillance of bovine tuberculosis (bTB).MethodsProteomic analysis was performed on different bPPD preparations from M. bovis, strain AN5. Proteins were precipitated from bPPD solutions by TCA precipitation. The proteome of bPPD preparations was investigated by bottom-up proteomics, which consisted in protein digestion and nano-LC–MS/MS analysis. Mass spectrometry analysis was performed on a Q-exactive hybrid quadrupole-Orbitrap mass spectrometer coupled online to an Easy nano-LC1000 system.ResultsThree hundred and fifty-six proteins were identified and quantified by at least 2 peptides (99% confidence per peptide). One hundred and ninety-eight proteins, which had not been previously described, were detected; furthermore, the proteomic profile shared 80 proteins with previous proteomes from bPPDs from the United Kingdom and Brazil and 139 protein components from bPPD from Korea. Locus name of M. bovis (Mb) with orthologs from M. tuberculosis H37Rv, comparative gene and protein length, molecular mass, functional categories, gene name and function of each protein were reported. Ninety-two T cell mycobacterial antigens responsible for delayed-type hypersensitivity were detected, fifty-two of which were not previously reported in any bPPD proteome. Data are available via ProteomeXchange with identifier PXD005920.ConclusionsThis study represents the highest proteome coverage of bPPD preparations to date. Since proteins perform cellular functions essential to health and/or disease, obtaining knowledge of their presence and variance is of great importance in understanding disease states and for advancing translational studies. Therefore, to better understand Mycobacterium tuberculosis complex biology during infection, survival, and persistence, the reproducible evaluation of the proteins that catalyze and control these processes is critically important. More active and more specific tuberculins would be desirable. Indeed, many antigens contained within bPPD are currently responsible for the cross-reactivity resulting in false-positive results as they are shared between non-tuberculous and tuberculous mycobacteria.

Comprehensive polyphenol profiling of a strawberry extract (Fragaria × ananassa) by ultra-high-performance liquid chromatography coupled with high-resolution mass spectrometry.

The aim of metabolic untargeted profiling is to detect and identify unknown compounds in a biological matrix to achieve the most comprehensive metabolic coverage. In phytochemical mixtures, however, the complexity of the sample could present significant difficulties in compound identification. In this case, the optimization of both the chromatographic and the mass-spectrometric conditions is supposed to be crucial for the detection and identification of the largest number of compounds. In this work, a systematic investigation of different chromatographic and mass-spectrometric conditions is presented to achieve a comprehensive untargeted profiling of a strawberry extract (Fragaria × ananassa). To fulfill this aim, an ultra-high-pressure liquid chromatography system coupled via an electrospray source to a hybrid quadrupole-Orbitrap mass spectrometer was used. Spectra were acquired in data-dependent mode, and several parameters were investigated to acquire the largest possible number of both mass spectrometry (MS) features and MS2 mass spectra for unique metabolites. The main classes of polyphenols studied were flavonoids, phenolic acids, dihydrochalcones, ellagitannins, and proanthocyanidins. Method optimization allowed to us identify and tentatively identify 18 and 113 compounds, respectively, among which 74have never been reported before in strawberries and, to the best of our knowledge, 22 of them have never been reported before. The results show the importance of an extended investigation of the chromatographic and mass-spectrometric method before a complete untargeted profiling of complex phytochemical mixtures.

Determination of Protein Markers for Beef Tenderness in U.S. Select Beef

ObjectivesBeef tenderness is a primary quality feature that defines the consumer’s perception of the product quality. Therefore definition of chemical characteristics that contribute to variation in tenderness within a specific USDA grade can help refine our understanding of meat tenderness. The objective of this experiment was to identify novel protein markers for tenderness in Longissimus dorsi sarcoplasmic fractions utilizing proteomics and a valuable library of beef samples. Materials and MethodsSteaks (USDA Select grade) used in this trial were classified as low shear force (LSF, n = 12; 11.3 ± 1.6 kg) and high shear force (HSF, n = 12; 34.3 ± 4.7 kg), according to their slice shear force (SSF, kg) measurements at 14 d postmortem. Western blotting of whole-muscle samples was used to determine troponin-T degradation (indicator of overall proteolysis). Sarcoplasmic protein samples from d 2 postmortem were prepared for two-dimensional difference in gel electrophoresis (2D-DIGE). Samples from LSF and HSF were sorted according to SSF values and were matched to a pooled internal reference (representative of all samples). Protein samples were loaded (75 µg) on 24 cm strips, pH 6 to 9, and focused in immobilized pH gradient in the first dimension. For the second dimension the strips were run on 12.5% acrylamide gels (n = 12). After 2D-DIGE, protein spots found to be different in relative abundance between HSF and LSF samples, and with other proteins of interest, were identified using Q Exactive Hybrid Quadrupole-Orbitrap Mass Spectrometer (LC-MS/MS). Immunoblots were performed to confirm protein identity. ResultsThe LSF samples exhibited greater troponin-T degradation (as evidenced by abundance of a 30 kDa fragment) than the HSF samples (LSF ratio to reference was 1.8, HSF ratio to reference was < 0.01; P < 0.01). A total of 31 spots showed a significant difference (P < 0.1) in relative abundance across groups (HSF vs. LSF). Among these, 18 spots were identified as 8 different proteins (Table 1). Proteins that were more abundant in the LSF samples are metabolic enzymes involved in the glycolytic pathway (glyceraldehyde-3-phosphate dehydrogenase, fructose bisphosphate aldolase A). Among the six proteins that showed greater abundance in the HSF samples, four belong to the oxidative energy metabolism pathways (malate dehydrogenase, creatine kinase M-type, myoglobin, carbonic anhydrase 3), one is involved in redox pathways (peroxiredoxin-1) and another one in protein turnover (polyubiquitin). The greater abundance of spots corresponding to oxidative metabolism enzymes suggests that aerobic metabolism may decrease potential for postmortem proteolysis. Table 1Differentially abundant proteins in high and low slice shear force groups Protein% CoverageRatio (LSF/HSF)P-valueGlyceraldehyde-3-phosphate dehydrogenase (GAPDH)26.731.980.060Fructose-bisphosphate aldolase A62.361.79< 0.050Polyubiquitin (Fragment)21.22–1.180.100Malate dehydrogenase, mitochondrial63.31–1.18< 0.050Creatine kinase M-type70.87–1.190.080Myoglobin99.35–1.290.095Carbonic anhydrase 379.12–1.56< 0.010Perociredoxin-170.85–1.240.055ConclusionThe results demonstrate that variation in tenderness within U.S. Select Top loin steaks is due in large part to variation in proteolysis. The variation in tenderness and proteolysis in these defined groups may be partially explained by a greater abundance of metabolic proteins involved in oxidative metabolism. This project was funded in part by grants from the Beef Checkoff (Denver, CO) and the Iowa Beef Industry Council (Ames, IA). The scholarship for the first author was granted by CNPq-Brazil.

Determination of Protein Markers for Beef Tenderness in U.S. Select Beef

ObjectivesBeef tenderness is a primary quality feature that defines the consumer’s perception of the product quality. Therefore definition of chemical characteristics that contribute to variation in tenderness within a specific USDA grade can help refine our understanding of meat tenderness. The objective of this experiment was to identify novel protein markers for tenderness in Longissimus dorsi sarcoplasmic fractions utilizing proteomics and a valuable library of beef samples. Materials and MethodsSteaks (USDA Select grade) used in this trial were classified as low shear force (LSF, n = 12; 11.3 ± 1.6 kg) and high shear force (HSF, n = 12; 34.3 ± 4.7 kg), according to their slice shear force (SSF, kg) measurements at 14 d postmortem. Western blotting of whole-muscle samples was used to determine troponin-T degradation (indicator of overall proteolysis). Sarcoplasmic protein samples from d 2 postmortem were prepared for two-dimensional difference in gel electrophoresis (2D-DIGE). Samples from LSF and HSF were sorted according to SSF values and were matched to a pooled internal reference (representative of all samples). Protein samples were loaded (75 µg) on 24 cm strips, pH 6 to 9, and focused in immobilized pH gradient in the first dimension. For the second dimension the strips were run on 12.5% acrylamide gels (n = 12). After 2D-DIGE, protein spots found to be different in relative abundance between HSF and LSF samples, and with other proteins of interest, were identified using Q Exactive Hybrid Quadrupole-Orbitrap Mass Spectrometer (LC-MS/MS). Immunoblots were performed to confirm protein identity. ResultsThe LSF samples exhibited greater troponin-T degradation (as evidenced by abundance of a 30 kDa fragment) than the HSF samples (LSF ratio to reference was 1.8, HSF ratio to reference was < 0.01; P < 0.01). A total of 31 spots showed a significant difference (P < 0.1) in relative abundance across groups (HSF vs. LSF). Among these, 18 spots were identified as 8 different proteins (Table 1). Proteins that were more abundant in the LSF samples are metabolic enzymes involved in the glycolytic pathway (glyceraldehyde-3-phosphate dehydrogenase, fructose bisphosphate aldolase A). Among the six proteins that showed greater abundance in the HSF samples, four belong to the oxidative energy metabolism pathways (malate dehydrogenase, creatine kinase M-type, myoglobin, carbonic anhydrase 3), one is involved in redox pathways (peroxiredoxin-1) and another one in protein turnover (polyubiquitin). The greater abundance of spots corresponding to oxidative metabolism enzymes suggests that aerobic metabolism may decrease potential for postmortem proteolysis. Table 1Differentially abundant proteins in high and low slice shear force groups Protein% CoverageRatio (LSF/HSF)P-valueGlyceraldehyde-3-phosphate dehydrogenase (GAPDH)26.731.980.060Fructose-bisphosphate aldolase A62.361.79< 0.050Polyubiquitin (Fragment)21.22–1.180.100Malate dehydrogenase, mitochondrial63.31–1.18< 0.050Creatine kinase M-type70.87–1.190.080Myoglobin99.35–1.290.095Carbonic anhydrase 379.12–1.56< 0.010Perociredoxin-170.85–1.240.055ConclusionThe results demonstrate that variation in tenderness within U.S. Select Top loin steaks is due in large part to variation in proteolysis. The variation in tenderness and proteolysis in these defined groups may be partially explained by a greater abundance of metabolic proteins involved in oxidative metabolism. This project was funded in part by grants from the Beef Checkoff (Denver, CO) and the Iowa Beef Industry Council (Ames, IA). The scholarship for the first author was granted by CNPq-Brazil.

Synthesis of stable isotopically labeled peptides with filter-assisted enzymatic labeling for the diagnosis of hepatitis B virus infection utilizing mass spectrometry-based proteomics strategy

A facile method for the preparation of stable isotopically labeled peptides was developed by means of filter-assisted tryptic 16O/18O water labeling, which could be directly applied to the determination of hepatitis B virus infection from human serum with tandem mass spectrometry. Tryptic peptides of hepatitis B surface antigen or hepatitis B e antigen from different subtypes of hepatitis B virus were synthesized with traditional solid-phase peptide synthesis as potential biomarkers. Trypsin catalyzed oxygen-18 exchange at their amidated c-terminus of arginine or lysine residue. The protease catalyzed oxygen-18 to oxygen-16 back exchange reaction was eliminated due to the complete removal of trypsin by the centrifugal filter containing a thin membrane associated with molecular weight cut-off of 10 KDa. The synthetic isotopic peptides were spiked into trichloroacetic acid/acetone precipitated human serum as internal standards and were selectively detected with multiplexed parallel reaction monitoring on a hybrid quadrupole-orbitrap mass spectrometer. The limit of detection for all synthetic peptides were in the range of 0.09 fmol–1.13 fmol. The results indicated that the peptide YLWEWASVR derived from hepatitis B surface antigen was quantified approximately 200 fmol per μl serum and may serve as a diagnostic biomarker for the detection of hepatitis B virus infected disease.

Neutral losses of sodium benzoate and benzoic acid in the fragmentation of the [M+Na]+ ions of methoxyfenozide and tebufenozide via intramolecular rearrangement in electrospray ionization tandem mass spectrometry.

Electrospray ionization (ESI) tandem mass spectrometry can be applied to determine structural information about organic compounds. The [M+Na]+ ion is one of the major precursor ions in ESI mass spectrometry, but its fragmentation mechanism study is still insufficient. This study reveals the interesting fragmentation reactions of the [M+Na]+ ions of methoxyfenozide and tebufenozide. The fragmentations of the [M+Na]+ , [M+Li]+ , and [M+H]+ ions of methoxyfenozide and tebufenozide were studied using a hybrid quadrupole-orbitrap mass spectrometer and an ion trap mass spectrometer. A hydrogen/deuterium (H/D)-exchange experiment in the amide group of methoxyfenozide allowed for the confirmation of the fragmentation mechanism. Density functional theory (DFT) calculations were performed for a further understanding of the fragmentation mechanism of the [M+Na]+ ion of methoxyfenozide. Neutral losses of sodium benzoate and benzoic acid in the fragmentation of the [M+Na]+ ions of methoxyfenozide and tebufenozide were observed as the major fragmentation pathways. In contrast, similar fragmentations were not observed or minor pathways in the fragmentation of the [M+Li]+ and [M+H]+ ions of methoxyfenozide and tebufenozide. In addition, a minor product ion resulting from loss of NaOH was identified, which was the first reported example in the fragmentation of sodiated compounds in mass spectrometry. Losses of sodium benzoate and benzoic acid in the fragmentation of the [M+Na]+ ions of methoxyfenozide and tebufenozide are proposed to be formed through an intramolecular rearrangement reaction, which is supported by DFT calculations. An H/D-exchange experiment confirms that the carboxyl hydrogen of benzoic acid and the hydrogen of NaOH exclusively derive from the amide hydrogen of the precursor ion. This study enriches our knowledge on the Na+ -induced fragmentation reactions. Copyright © 2016 John Wiley & Sons, Ltd.

Comparative Proteomic Profiling of Sera from Patients with Refractory Multiple Myeloma Reveals Pathways and Biomarkers Predicting Response to Bortezomib-Based Therapy

Introduction: Identification of drug-resistance biomarkers in multiple myeloma (MM) is a first step to individualization of treatment. Human serum is easily collected and does not require expensive preparation to analyze, therefore it seems the most suitable material to search for biomarkers.Objective: The goal of this study was to establish serum biomarkers predicting response/resistance to PAD (bortezomib, adriamycin, dexamethasone) chemotherapy in patients with refractory MM.Material and Methods:Comparative proteome analysis was performed on pretreatment serum samples obtained from 56 proteasome inhibitor-naïve, transplant-eligible patients qualified for PAD chemotherapy due to CTD (cyclophosphamide, thalidomide, dexamethasone) refractory disease. Samples were collected after patients signed informed consent according to Declaration of Helsinki. Subjects were classified into four groups based on the response to PAD: 13 patients achieved complete response (CR group), 10 revealed very good partial response (VGPR group), 17 - partial response (PR group) and 16 - stable disease or progressive disease (SD/PD group). Serum proteins after trypsin digestion were analyzed using Q-Exactive hybrid quadrupole-Orbitrap mass spectrometer coupled to the chromatograph Dionex 3000 Ultimate nanoLC (Thermo Scientific). Obtained data were analyzed using MaxQuant/Perseus software.Results and discussion: 632 proteins were identified with a false discovery rate of 1% (FDR). A protein was considered to be differentially accumulated if the difference between experimental groups was statistically significant (p< 0.05) and the minimum fold change was ± 1.5. Proteins identified with a minimum of 2 peptides were considered significant. The proteomic signature revealed 52 proteins, which differentiated all analyzed experimental groups (ANOVA significant). The most differential proteins revealed comparison between CR/VGPR vs PD/SD groups. Eleven up-regulated and 35 down-regulated proteins were identified in sera of patients derived from PD/SD (non responders) group compared to CR/VGPR (responders) among them: sulfhydryl oxidase (fold change 2.56), kallistatin (fold change 0.32), gelsolin (fold change 0.50), thyroxine-binding globulin (fold change 0.28), PDZ and LIM domain protein 1 (fold change 2.38), corticosteroid-binding globulin (fold change 0.32), and phosphatidylinositol-glycan-specific phospholipase D (fold change 0.42). Also principal component analysis (PCA) differentiated the CR/VGPR and PD/SD patients. Proteins like thyroxine-binding globulin or corticosteroid-binding globulin seem to be nonspecifically deregulated, however sulfhydryl oxidase may have a causative relation to bad prognosis. Sulfhydryl oxidase plays an important role in growth regulation and has metastatic potential. Overexpression of this enzyme was presented in pancreatic, prostate and breast cancer. Moreover, it was showed that up-regulation of this enzyme predicts a poor prognosis in patients with breast cancer (Katchman et al. 2013).Conclusions: We identified proteins, some of which may serve as potential prognostic factors predicting response to bortezomib-based regimens in patients with refractory MM. Label-free proteomic analysis is a useful method to screen for proteins differentially expressed in the sera of multiple myeloma patients revealed different response to chemotherapy. DisclosuresDytfeld:Celgene: Research Funding; Janssen Poland: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees. Jakubowiak:Karyopharm: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Sanofi: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; SkylineDx: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen Inc.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees.

Direct Visualization of Neurotransmitters in Rat Brain Slices by Desorption Electrospray Ionization Mass Spectrometry Imaging (DESI - MS).

Mass spectrometry imaging (MSI) of neurotransmitters has so far been mainly performed by matrix-assisted laser desorption/ionization (MALDI) where derivatization reagents, deuterated matrix and/or high resolution, or tandem MS have been applied to circumvent problems with interfering ion peaks from matrix and from isobaric species. We herein describe the application of desorption electrospray ionization mass spectrometry imaging (DESI)-MSI in rat brain coronal and sagittal slices for direct spatial monitoring of neurotransmitters and choline with no need of derivatization reagents and/or deuterated materials. The amino acids γ-aminobutyric (GABA), glutamate, aspartate, serine, as well as acetylcholine, dopamine, and choline were successfully imaged using a commercial DESI source coupled to a hybrid quadrupole-Orbitrap mass spectrometer. The spatial distribution of the analyzed compounds in different brain regions was determined. We conclude that the ambient matrix-free DESI-MSI is suitable for neurotransmitter imaging and could be applied in studies that involve evaluation of imbalances in neurotransmitters levels. Graphical Abstract ᅟ.

Ceramide lipids in alive and thermally stressed mussels: an investigation by hydrophilic interaction liquid chromatography-electrospray ionization Fourier transform mass spectrometry.

Hydrophilic interaction liquid chromatography coupled to electrospray ionization-Fourier transform mass spectrometry was employed to study ceramide lipids occurring in mussels of sp. Mytilus galloprovincialis. Lipid extracts from alive mussels and mussels deliberately subjected to specific thermal treatments were analyzed. In particular, single and tandem MS measurements were performed on a hybrid quadrupole-Orbitrap mass spectrometer and then complemented by MS(n) analyses (n = 2, 3) achieved by a linear ion trap mass spectrometer. This approach enabled the characterization of 66 ceramide lipids, encompassing ceramide phosphoethanolamines (CPE), ceramide aminoethylphosphonates (CAEP) and N-monomethylated CAEP. The sphingoid and acyl chains of each ceramide lipid could be distinctly recognized in terms of numbers of carbon atoms and C=C bonds, and indications on the possible location of the latter on the sphingoid chain could be often inferred from fragmentation patterns. The occurrence of several species hydroxylated on the α carbon of the acyl chain was also discovered. On the other hand, the sphingoid chain of ceramide lipids was never found to be involved in oxidation processes, unless forced exposure of the mussel lipid extracts to atmospheric oxygen was performed. CPE(d19:3/16:0) and its hydroxylated form, CPE(d19:3/2-OH-16:0), were found to be the prevailing species among CPE, whereas CAEP(d18:2/16:0), CAEP(d19:3/16:0) and CAEP(d19:3/2-OH-16:0) were the most abundant CAEP. Finally, ceramide lipids showed a remarkably higher stability, compared with glycerophospholipids, in mussels subjected to different thermal treatments. This finding opens interesting perspectives on the role of ceramide-based lipids in the adaptation of aquatic organisms to thermal stresses. Copyright © 2016 John Wiley & Sons, Ltd.

Targeted and untargeted profiling of alkaloids in herbal extracts using online solid-phase extraction and high-resolution mass spectrometry (Q-Orbitrap).

The biological activity of alkaloids (ALKs) and the different content of these natural products in herbs and plants have made them an attractive field for chemical studies. A screening method automatically combining online solid-phase purification and concentration of samples with analysis using ultra-high performance liquid chromatography coupled with a hybrid quadrupole orbitrap mass spectrometer was developed and is reported in this paper. The proposed quantification method was validated for 35 ALKs with reference to pure analytical standards. A further 48 ALKs were identified on the basis of their accurate mass and characterised for chromatographic retention time and fragmentation profile, following their confirmation in extracts of herbs already well documented in the literature. More than 250 other untargeted ALKs were also tentatively identified using literature information, such as exact mass and isotopic pattern. The mass spectrometer operated in positive ion mode and mass spectra were acquired, with full MS-data-dependent MS/MS analysis (full MS-dd MS/MS) at a resolution of 140 000. The method was linear up to an ALK concentration of 1000/3000 µg l(-1) , with R(2) always >0.99 and limits of detection ranging between 0.04 and 10 µg l(-1) . Accuracy, expressed as the recovery relative error, had a median value of 7.4%, and precision (relative standard deviation %) was generally lower than 10% throughout the quantitation range. The proposed method was then used to investigate the targeted and untargeted ALK profile of a selection of 18 alpine herbal plants, establishing that pyrrolizidine, pyrrolidine and piperidine ALKs were the most well represented. Copyright © 2016 John Wiley & Sons, Ltd.

Metabolomic profiling predicts outcome of rituximab therapy in rheumatoid arthritis

ObjectiveTo determine whether characterisation of patients' metabolic profiles, utilising nuclear magnetic resonance (NMR) and mass spectrometry (MS), could predict response to rituximab therapy. 23 patients with active, seropositive rheumatoid arthritis...

Abstract 3981: In situ mass spectrometry using desorption electrospray ionization mass spectrometry (DESI-MS) can detect specific lipid profiles across breast cancer molecular subtypes

Abstract INTRODUCTION Lipid profiling as a source for biomarkers identification is an evolving field with some enthusiastic results in cancer. Alterations on lipid metabolism have been increasingly recognized as a hallmark of cancer cells and may help to understand breast cancer carcinogenesis. Analytical chemistry and Pathology have been recently combined in the development of chemical microscopy using in situ mass spectrometry (MS) and may help to decipher breast cancer molecular initiation and progression. OBJECTIVE We aimed to use in situ Desorption Electrospray Ionization Mass Spectrometry (DESI-MS) to investigate lipid profiles across invasive breast cancer (IBC) molecular subtypes and related normal parenchyma (NBP) and ductal carcinoma in situ (DCIS). METHODS: We selected frozen samples from 9 IBC with various ER/PR/HER2 status (ER 0 to 100%, PR 0 to 90%, HER2 0 and 3+, Ki67 10 to 70%). Unstained slides obtained from frozen tissue at 10μm were submitted to DESI-MS analysis with no pretreatment and spatial resolution of 100μm. Molecular profiles (mass to charge [m/z] ratio versus signal intensity) were generated in triplicate directly from tissues to detect changes from areas of interest: IBC (and adjacent normal breast parenchyma and DCIS whenever present) regions were identified by a breast pathologist on HE-stained sequential slides. Experiment was conducted in full-scan mode and m/z range of 100-1200. DESI mass spectra were obtained using a Thermo ScientificTM Q ExactiveTM Hybrid Quadrupole Orbitrap Mass Spectrometer in the positive ion mode. The Biomap software was used to visualize the images and to export the spectrums to metaboanalyst, where the data were statiscally analyzed. RESULTS: The images obtained by lipid profiling presented a direct correlation with HE stained slides, with some ions m/z clearly overlaying areas of normal parenchyma, DCIS and invasive breast cancer. The unsupervised hierarchical clustering analysis showed all technical triplicates remained grouped. All NBP but one clustered together. Triple negative IBC and its related NBP formed a distinct cluster from all other ER+ samples. The ER+/HER2 + tumor segregated from other ER+/HER2- tumors. DCIS and IBC from ER+/PR+/HER2- tumor clustered together. The Ions with m/z 186.9 and m/z 303.2 were consistently related to NBP and IBC respectively. CONCLUSIONS: Chemical microscopy using DESI-MS is a powerful tool to identify specific lipid profiles across molecular subtypes of IBC and can contribute to understand breast cancer carcinogenesis. Citation Format: Victor P. Andrade, Adriana L. Santoro, Sabrina T. Moretti, Nicolas V. Schwab, Marcos N. Eberlin. In situ mass spectrometry using desorption electrospray ionization mass spectrometry (DESI-MS) can detect specific lipid profiles across breast cancer molecular subtypes. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3981.