Data-dependent Tandem Mass Spectrometry Research Articles

A Carrier-Based Quantitative Proteomics Method Applied to Biomarker Discovery in Pericardial Fluid

Data-dependent liquid chromatography tandem mass spectrometry is challenged by the large concentration range of proteins in plasma and related fluids. We adapted the SCoPE method from single-cell proteomics to pericardial fluid, where a myocardial tissue carrier was used to aid protein quantification. The carrier proteome and patient samples were labeled with distinct isobaric labels, which allowed separate quantification. Undepleted pericardial fluid from patients with type 2 diabetes mellitus and/or heart failure undergoing heart surgery was analyzed with either a traditional liquid chromatography tandem mass spectrometry method or with the carrier proteome. In total, 1398 proteins were quantified with a carrier, compared to 265 without, and a higher proportion of these proteins were of myocardial origin. The number of differentially expressed proteins also increased nearly four-fold. For patients with both heart failure and type 2 diabetes mellitus, pathway analysis of upregulated proteins demonstrated the enrichment of immune activation, blood coagulation, and stress pathways. Overall, our work demonstrates the applicability of a carrier for enhanced protein quantification in challenging biological matrices such as pericardial fluid, with potential applications for biomarker discovery. Mass spectrometry data are available via ProteomeXchange with identifier PXD053450.

Mass spectrometry detection of organophosphorus pesticide adducts on butyrylcholinesterase and albumin

This study established a method to prepare and detect OPs adducts on butyrylcholinesterase (BChE) and human serum albumin (HSA). OPs (methyl paraoxon, ethyl paraoxon, methyl parathion, parathion) were incubated with BChE or HSA in vitro, and the adducts of OPs-BChE or OPs-HSA were prepared and qualitatively analyzed by ultra-performance liquid chromatography data-dependent high-resolution tandem mass spectrometry (UPLC-ddHRMS/MS). The amounts of BChE and HSA in the incubating systems were varied and the resulting amounts of the adducts were determined using linear regression. OPs-BChE in the blood were isolated by immunomagnetic separation (IMS), and then digested into the OPs-nonapeptide adduct by pepsin. The proteins in the remaining blood plasma were precipitated and digested by pronase to OPs-tyrosines(OPs-Tyr), which were quantified by UPLC-ddHRMS/MS. 4 OPs-nonapeptides and 4 OPs-Tyr adducts were obtained through the process above. The relative mass deviation of incubated adducts between the actual and theoretical exact masses was less than 10 ppm, and further confirmed by fragmentation mass spectra analysis. Calibration curves were linear for all adducts with a coefficient of determination value (R2) ≥0.995. The limits of detection (LOD) and limits of quantification (LOQ) for adducts detected by MS ranged from 0.05 to 1.0 ng/mL, and from 0.1 to 2.0 ng/mL, respectively. The recovery percentages for adducts ranged from 76.1 % to 107.1 %, matrix effects ranged from 83.4 % to 102.1 %. The inter-day and intra-day precision were 6.1–10.1 % and 6.9–12.9 % for adducts. This study provides a new reference method for the detection of organophosphorus pesticide poisoning. In addition, two blood samples with organophosphorus poisoning were tested by the designed method, and the corresponding adducts were detected in both samples.

Targeted Workflow Investigating Variations in the Tear Proteome by Liquid Chromatography Tandem Mass Spectrometry.

Proteins in tears have an important role in eye health and have been shown as a promising source of disease biomarkers. The goal of this study was to develop a robust, sensitive, and targeted method for profiling tear proteins to examine the variability within a group of healthy volunteers over three days. Inter-individual and inter-day variabilities were examined to contribute to understanding the normal variations in the tear proteome, as well as to establish which proteins may be better candidates as eventual biomarkers of specific diseases. Tear samples collected on Schirmer strips were subjected to bottom-up proteomics, and resulting peptides were analyzed using an optimized targeted method measuring 226 proteins by liquid chromatography-scheduled multiple reaction monitoring. This method was developed using an in-house database of identified proteins from tears compiled from high-resolution data-dependent liquid chromatography tandem mass spectrometry data. The measurement of unique peptide signals can help better understand the dynamics of each of these proteins in tears. Some interesting trends were seen in specific pathways or protein classes, including higher variabilities for those involved in glycolysis, glutathione metabolism, and cytoskeleton proteins and lower variation for those involving the degradation of the extracellular matrix. The overall aim of this study was to contribute to the field of tear proteomics with the development of a novel and targeted method that is highly amenable to the clinical laboratory using high flow LC and commonly used triple quadrupole mass spectrometry while ensuring that protein quantitation was reported based on unique peptides for each protein and robust peak areas with data normalization. These results report on variabilities on over 200 proteins that are robustly detected in tear samples from healthy volunteers with a simple sample preparation procedure.

Comparative proteomic analysis of ventricular and cisternal cerebrospinal fluid in haemorrhagic stroke patients

BackgroundAnalysis of cerebrospinal fluid (CSF) using mass spectrometry is a relatively novel analytical tool, and comparisons of ventricular and cisternal proteomes are yet to be performed. This may have implications for clinical medicine, particularly in demonstrating continuity of the ventricular system with preserved flow in the presence of ventricular blood. Other uses include the identification of novel biomarkers, including for diagnosis of subarachnoid haemorrhage and of aetiology. The primary objective was therefore to characterise and compare the proteomes of ventricular and CSF after haemorrhagic stroke. MethodsPaired CSF samples were prospectively collected from the optico-carotid cistern and the frontal horn of the lateral ventricle at the time of craniotomy and clipping in 8 patients with haemorrhagic stroke. Six patients had an aneurysmal subarachnoid haemorrhage (aSAH) from a ruptured saccular aneurysm, one patient had an aSAH after rupture of a mycotic aneurysm and one patient had a spontaneous intracerebral haemorrhage (IPH) with an adjacent unruptured saccular aneurysm. Samples were processed and proteins identified and quantified using data-dependent liquid chromatography tandem mass spectrometry (DDA LC-MSMS). ResultsThere was no systematic difference between the cisternal and ventricular proteomes. However, blinded principal component analysis (PCA) of the cisternal and ventricular samples separated patients according to pathophysiology. Additionally CSF D-Dimer levels were not detected in the IPH patient but were reliably measured in aSAH patients. ConclusionsVentricular CSF is representative of cisternal CSF after aSAH. CSF proteomic PCA analysis can distinguish between haemorrhage types. CSF D-dimer levels may represent a novel diagnostic marker for aSAH. Label free DDA LC-MSMS CSF analysis may inform possible biomarkers.

Simultaneous measurement of six biomarkers of dichlorvos in blood by ultra performance liquid chromatography-quadrupole/electrostatic field orbitrap mass spectrometry

A protocol was developed, via a data-dependent high-resolution tandem mass spectrometry (ddHRMS/MS), to detect six biomarkers of dichlorvos in its metabolites and blood adducts of butyrylcholinesterase and albumin without using standard synthetic peptides. Firstly, the adducts of dimethoxy phosphonate (DMP-BChE) and the aged adducts of methoxy phosphonate (MxP-BChE) were isoloated by immunomagnetic separation (IMS), and then digested to DMP-nonapeptide and MxP-nonapeptide by pepsin. The dichlorvos and its metabolites (Trimethyl phosphate, Dimethyl phosphate) were analyzed in the supernatant of IMS treatment after protein precipitation. The precipitate was digested by pronase to phosphorylated tyrosine (DMP-Tyr), which were quantified by ultra performance liquid chromatography-quadrupole/electrostatic field orbitrap mass spectrometry (UPLC-MS). The linearity of detector response of all biomarkers was studied in their respective ranges, and the correlation coefficients (R2) were all greater than 0.9981. The limits of detection (LOD) and limits of quantification (LOQ) were 0.2–10 ng/mL and 0.5–20 ng/mL, respectively. The recoveries of the six biomarkers were 78.6–110.0 %, matrix effect were 64.1–106.8 %. Inter- and intra-assay precision had coefficients of variation of ≤13.2 % and ≤9.7 %, respectively. In addition, MxP- BChE, DMP-Tyr, TMP, DMP and DDVP were detected in the blood of three cases who died from DDVP poisoning, but DMP- BChE was not detected in all of them, which may be caused by the instability of DMP- BChE and its easy aging to form MxP-BCHE.

Multi-Omic Analysis to Characterize Metabolic Adaptation of the E. coli Lipidome in Response to Environmental Stress.

As an adaptive survival response to exogenous stress, bacteria undergo dynamic remodelling of their lipid metabolism pathways to alter the composition of their cellular membranes. Here, using Escherichia coli as a well characterised model system, we report the development and application of a ‘multi-omics’ strategy for comprehensive quantitative analysis of the temporal changes in the lipidome and proteome profiles that occur under exponential growth phase versus stationary growth phase conditions i.e., nutrient depletion stress. Lipidome analysis performed using ‘shotgun’ direct infusion-based ultra-high resolution accurate mass spectrometry revealed a quantitative decrease in total lipid content under stationary growth phase conditions, along with a significant increase in the mol% composition of total cardiolipin, and an increase in ‘odd-numbered’ acyl-chain length containing glycerophospholipids. The inclusion of field asymmetry ion mobility spectrometry was shown to enable the enrichment and improved depth of coverage of low-abundance cardiolipins, while ultraviolet photodissociation-tandem mass spectrometry facilitated more complete lipid structural characterisation compared with conventional collision-induced dissociation, including unambiguous assignment of the odd-numbered acyl-chains as containing cyclopropyl modifications. Proteome analysis using data-dependent acquisition nano-liquid chromatography mass spectrometry and tandem mass spectrometry analysis identified 83% of the predicted E. coli lipid metabolism enzymes, which enabled the temporal dependence associated with the expression of key enzymes responsible for the observed adaptive lipid metabolism to be determined, including those involved in phospholipid metabolism (e.g., ClsB and Cfa), fatty acid synthesis (e.g., FabH) and degradation (e.g., FadA/B,D,E,I,J and M), and proteins involved in the oxidative stress response resulting from the generation of reactive oxygen species during β-oxidation or lipid degradation.

Liquid chromatography-Orbitrap Tribrid high-resolution mass spectrometry using data dependent-tandem mass spectrometry with triple stage fragmentation as a screening tool to perform identification and risk assessment of unknown substances in food contact epoxy resin.

A new, fast, and automatic approach has been applied for the tentative identification of unknown substances released by food contact epoxy resin after performing a migration test with food simulant. This approach combines intelligent data acquisition with AcquireX linked to liquid chromatography-Orbitrap Tribrid high-resolution mass spectrometry using data dependent-tandem mass spectrometry with triple stage fragmentation coupled to Compound Discoverer™ software for automated data processing and compound identification. The identification of the observed features was performed using a set of identification criteria, including exact mass, isotope pattern, tandem mass spectrometry spectra match, and retention time. With these criteria, 263 substances were tentatively identified. Most of the identified compounds were additives, such as plasticisers, stabilizers, and antioxidants, used in different plastic applications. However, metabolites, biological constituents with pharmacological activity, and other substances with industrial applications were also detected. In order to perform a risk assessment of the food contact epoxy resin, threshold of toxicological concern approach was applied for the identified compounds. There was not risk associated with the migration of the identified substances.

Sensitive Screening of New Psychoactive Substances in Serum Using Liquid Chromatography-Quadrupole Time-of-Flight Mass Spectrometry.

Analysis of new psychoactive substances (NPS) still poses a challenge for many institutions due to the number of available substances and the constantly changing drug market. Both new and well-known substances keep appearing and disappearing on the market, making it hard to adapt analytical methods in a timely manner. In this study we developed a qualitative screening approach for serum samples by means of liquid chromatography--quadrupole time-of-flight mass spectrometry. Samples were measured in data-dependent auto tandem mass spectrometry mode and identified by fragment spectra comparison, retention time and accurate mass. Approximately 500 NPS, including 195 synthetic cannabinoids, 180 stimulants, 86 hallucinogens, 26 benzodiazepines and 7 others were investigated. Serum samples were fortified to 1 ng/mL and 10 ng/mL concentrations to estimate approximate limits of identification (LOIs). Samples were extracted using solid-phase extraction with non-endcapped C18 material and elution in two consecutive steps. Benzodiazepines were eluted in the first step, while substances of other NPS subclasses were distributed among both extracts. To determine LOIs, both extracts were combined. Ninety-six percent (470/492) of investigated NPS were detected in 10 ng/mL samples and 88% (432/492) were detected in 1 ng/mL samples. Stimulants stood out with higher LOIs, possibly due to instability of certain methcathinone derivatives. However, considering relevant blood concentrations, the method provided sufficient sensitivity for stimulants as well as other NPS subclasses. Data-dependent acquisition was proven to provide high sensitivity and reliability when combined with an information-dependent preferred list, without losing its untargeted operation principle. Summarizing, the developed method fulfilled its purpose as a sensitive untargeted screening for serum samples and allows uncomplicated expansion of the spectral library to include thousands of targets.

Characterization of galacto-oligosaccharides using high-performance anion exchange chromatography-tandem mass spectrometry.

The analysis of complex oligosaccharide mixtures remains a challenge in the field of analytical chemistry. In this work, two commercial galacto-oligosaccharides samples were characterized using high-performance anion exchange chromatography coupled to mass spectrometry. The isomeric oligosaccharides were resolved with high resolution. The structures of the individual isomers with a degree of polymerization up to 6 were analyzed using targeted selected ion monitoring with data-dependent tandem mass spectrometry, with additional in-source collision-induced dissociation.

Combination of different chromatographic and sampling modes for high-resolution mass spectrometric screening of organic microcontaminants in water.

This study explores the combination of two sampling strategies (polar organic compounds integrative sampler (POCIS) vs. spot sampling) and four chromatographic retention modes (reversed-phase liquid chromatography (RPLC), hydrophilic interaction liquid chromatography (HILIC), mixed-mode liquid chromatography (MMLC) and supercritical fluid chromatography (SFC)) for high-resolution mass spectrometry (HRMS) screening of organic pollutants in water samples. To this end, a suspect screening approach, using iterative data-dependent tandem mass spectrometry (MS/MS) driven by a library of 3227 chemicals (including pharmaceuticals, pesticides, drugs of abuse, human metabolites, industrial chemicals and other pollutants), was employed. Results show that POCIS can afford a larger number of positive identifications as compared to spot sampling. On the other hand, the best suited retention mechanisms, in terms of identified analytes, are SFC, and followed by RPLC, MMLC and HILIC. However, the best combination (POCIS + SFC) would only allow the identification of 67% of the detected analytes. Thus, the combination of the two sampling strategies, spot and passive sampling, with two orthogonal retention mechanisms, RPLC and SFC, is proposed in order to maximize the number of analytes detected (89%). This strategy was applied to different surface water (river and estuary) samples from Galicia (NW Spain). A total of 155 compounds were detected at a confidence level 2a, from which the major class was pharmaceuticals (61%).

Sensitive Immunopeptidomics by Leveraging Available Large-Scale Multi-HLA Spectral Libraries, Data-Independent Acquisition, and MS/MS Prediction

Mass spectrometry (MS) is the state-of-the-art methodology for capturing the breadth and depth of the immunopeptidome across human leukocyte antigen (HLA) allotypes and cell types. The majority of studies in the immunopeptidomics field are discovery driven. Hence, data-dependent tandem MS (MS/MS) acquisition (DDA) is widely used, as it generates high-quality references of peptide fingerprints. However, DDA suffers from the stochastic selection of abundant ions that impairs sensitivity and reproducibility. In contrast, in data-independent acquisition (DIA), the systematic fragmentation and acquisition of all fragment ions within given isolation m/z windows yield a comprehensive map for a given sample. However, many DIA approaches commonly require generating comprehensive DDA-based spectrum libraries, which can become impractical for studying noncanonical and personalized neoantigens. Because the amount of HLA peptides eluted from biological samples such as small tissue biopsies is typically not sufficient for acquiring both meaningful DDA data necessary for generating comprehensive spectral libraries and DIA MS measurements, the implementation of DIA in the immunopeptidomics translational research domain has remained limited. We implemented a DIA immunopeptidomics workflow and assessed its sensitivity and accuracy by matching DIA data against libraries with growing complexity—from sample-specific libraries to libraries combining 2 to 40 different immunopeptidomics samples. Analyzing DIA immunopeptidomics data against a complex multi-HLA spectral library resulted in a two-fold increase in peptide identification compared with sample-specific library and in a three-fold increase compared with DDA measurements, yet with no detrimental effect on the specificity. Furthermore, we demonstrated the implementation of DIA for sensitive personalized neoantigen discovery through the analysis of DIA data with predicted MS/MS spectra of clinically relevant HLA ligands. We conclude that a comprehensive multi-HLA library for DIA approach in combination with MS/MS prediction is highly advantageous for clinical immunopeptidomics, especially when low amounts of biological samples are available.

Novel neuroprotective peptides in the venom of the solitary scoliid wasp Scolia decorata ventralis

BackgroundSolitary wasp venoms may be a rich source of neuroactive substances, since their venoms are used for paralyzing preys. We have been exploring bioactive constituents of solitary wasp venoms and, in this study, the component profile of the venom from a solitary scoliid wasp, Scolia decorata ventralis, was investigated through a comprehensive analysis using LC-MS. Two peptides were synthesized, and their neuroprotective properties were evaluated.MethodsA reverse-phase HPLC connected to ESI-MS was used for LC-MS analyses. Online mass fingerprinting was performed from TIC, and data-dependent tandem mass spectrometry gave the MS/MS spectra. The sequences of two major peptide components were determined by MALDI-TOF/TOF MS analysis, confirmed by solid phase synthesis. Using the synthetic peptides, biological activities were assessed. Cell integrity tests and neuroprotection analyzes using H2O2 as an oxidative stress inducer were performed for both peptides.ResultsOnline mass fingerprinting revealed that the venom contains 123 components, and the MS/MS analysis resulted in 33 full sequences of peptide components. The two main peptides, α-scoliidine (DYVTVKGFSPLR) and β-scoliidine (DYVTVKGFSPLRKA), present homology with the bradykinin C-terminal. Despite this, both peptides did not behave as substrates or inhibitors of ACE, indicating that they do not interact with this metallopeptidase. In further studies, β-scoliidine, but not α -scoliidine, showed protective effects against oxidative stress-induced neurotoxicity in PC12 cells through integrity and metabolism cell assays. Interestingly, β-scoliidine has the extension of the KA dipeptide at the C-terminal in comparison with α-scoliidine.ConclusionComprehensive LC-MS and MS/MS analyses from the Scolia decorata ventralis venom displayed the component profile of this venom. β-scoliidine showed an effective cytoprotective effect, probably due to the observed increase in the number of cells. This is the first report of solitary wasp venom peptides showing neuroprotective activity.

Targeted Quantitation Mode Comparison of Haloacetic Acids, Bromate, and Dalapon in Drinking Water Using Ion Chromatography Coupled to High-Resolution (Orbitrap) Mass Spectrometry

A highly selective, sensitive, and simple analytical method for identification and quantification of nine haloacetic acids, bromate, and dalapon has been developed. This method uses ion chromatography (IC) coupled with electrospray ionization-high-resolution mass spectrometry (IC-ESI-HRMS) to directly analyze water samples on a high capacity anion-exchange column, eliminating the need for sample pretreatment/derivatization. Our study compared the following three types of targeted quantitation experiments using a quadrupole–orbitrap hybrid mass spectrometer, full-scan MS with data-dependent tandem mass spectrometry (full MS/dd-MS2 with inclusion list), targeted selected ion monitoring (SIM) with data-dependent tandem mass spectrometry (t-SIM/dd-MS2), and parallel reaction monitoring (PRM). Sensitivity, linearity, accuracy, and precision were validated following the guidelines of U.S. EPA Method 557. Single laboratory lowest concentration minimum reporting levels (LCMRLs) for the analytes using three different acquisition modes ranged from 0.0011 to 0.18 μg/L. All three quantitation modes showed good linearity for the eleven analytes with coefficients of determination of 0.9981- 0.9993. This IC-ESI-HRMS method was successfully applied to the analysis of commercial bottled water, tap water from San Francisco Bay Area, and the same tap water that has been through a filtered drinking water faucet. Both t-SIM/dd-MS2 and PRM modes were sensitive to confirm the trace-level presence of all nine HAAs, bromate, and dalapon in the tap water sample. Full-scan HRMS data acquisition provided the benefits of simultaneous data collection for both targeted and non-targeted components, and thus, suitability for simultaneous quantification of an unlimited number of compounds. Data-dependent MS/MS (dd-MS2) product-ion spectra were used for confirmation. All three modes showed good quantitative performance and obtained similar values. Single laboratory precision and accuracy data are presented for three water matrices: reagent water, laboratory synthetic sample matrix (LSSM), and tap water. Single laboratory precision was 0.078- 8.04%, and accuracy was in the range 70–130% for the three MS modes.

Simultaneous separation and determination of 32 fentanyl-related substances, including seven sets of isomeric fentanyl analogues, by ultra-high-performance liquid chromatography coupled with high-resolution mass spectrometry.

A method for separation and determination of 32 fentanyl-related substances, including seven sets of isomeric fentanyl analogues, was developed using ultra-high-performance liquid chromatography coupled with quadrupole-orbitrap high-resolution mass spectrometry. The collision energy, chromatographic column, and mobile phase were optimized. All compounds were efficiently flushed out of a universal C18 column with a soft gradient consisting of solvent A (2mM ammonium formate and 0.1% formic acid in water) and solvent B (2mM ammonium formate and 0.1% formic acid in methanol) in only 20 min, achieving excellent resolution. Detection and analysis were carried out simultaneously in the positive ion mode using the full scan and data-dependent tandem mass spectrometry modes with a normalized collision energy of 40. The method was validated in terms of limit of detection, limit of quantification, linearity, accuracy, and precision. For all fentanyl-related substances, the limit of detection (0.5ng/mL) and limit of quantification (1ng/mL) were adequate for screening and quantification in daily drug control. Calibration curves for all compounds were established in the range of 1-500ng/mL. The intra- and interday precision (RSD%) were within 0.4-2.3 and 0.7-2.7%, respectively. The accuracy ranged from 99 to 106%. The method was applied to analyze seized drug samples.

Shortlisting SARS-CoV-2 Peptides for Targeted Studies from Experimental Data-Dependent Acquisition Tandem Mass Spectrometry Data.

Detection of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) is a crucial tool for fighting the COVID‐19 pandemic. This dataset brief presents the exploration of a shotgun proteomics dataset acquired on SARS‐CoV‐2 infected Vero cells. Proteins from inactivated virus samples were extracted, digested with trypsin, and the resulting peptides were identified by data‐dependent acquisition tandem mass spectrometry. The 101 peptides reporting for six viral proteins were specifically analyzed in terms of their analytical characteristics, species specificity and conservation, and their proneness to structural modifications. Based on these results, a shortlist of 14 peptides from the N, S, and M main structural proteins that could be used for targeted mass‐spectrometry method development and diagnostic of the new SARS‐CoV‐2 is proposed and the best candidates are commented.

Trace analysis of multi-class phytohormones in Oryza sativa using different scan modes in high-resolution Orbitrap mass spectrometry: method validation, concentration levels, and screening in multiple accessions.

Phytohormones are signaling and regulating metabolites involved in numerous plant processes, including growth, development, and responses to stress. Currently, the focus is on the analysis of multiple phytohormones in order to characterize crosstalk and hormone signaling networks. In this paper, representative phytohormones of the major classes are simultaneously determined in rice tissues by a generic solid-liquid extraction, followed by liquid chromatography and electrospray ionization high-resolution tandem mass spectrometry using a Q-Exactive™ instrument. After a thorough optimization of the sample preparation, the analytical method was fully validated toward the ultra-trace quantification of six a priori selected plant hormones using three scan modes of the quadrupole-Orbitrap instrument: full-scan high-resolution mass spectrometry, targeted single ion monitoring (t-SIM), and t-SIM followed by data-dependent tandem mass spectrometry. Overall, a similar quantitative performance was noticed for the different scan modes. The analytical method was successfully applied to measure basal phytohormone levels in six different rice accessions, comprising Oryza sativa ssp. japonica, indica, and Oryza glaberrima. Hormone concentrations were higher in shoots than in roots or at least similar. Except for a lower level of salicylic acid in shoots of O. glaberrima versus O. sativa, no other differences in hormone levels could be noticed that were dependent of the (sub)species assignment of the analyzed accessions. Making use of the benefits of full-scan high-resolution mass spectrometry, a first post-run suspect screening was performed, suggesting - based on accurate mass measurements and isotopic patterns - the possible presence of about 50 additional plant hormones in the rice tissues. Graphical abstract ᅟ.

Mass Spectrometric Identification of Cardiac Troponin T in Urine of Patients Suffering from Acute Myocardial Infarction.

Because of its high cardiospecificity, cardiac troponin T (cTnT) is one of the first-choice biomarkers to diagnose acute myocardial infarction (AMI). cTnT is extensively fragmented in serum of patients suffering from AMI. However, it is currently unknown whether all cTnT is completely degraded in the body or whether some cTnT fragments can leave the body via urine. The aim of the present study is to develop a method for the detection of cTnT in urine and to examine whether cTnT is detectable in patient urine. Proteins in urine samples of 20 patients were precipitated using a cTnT-specific immunoprecipitation technique and a nonspecific acetonitrile protein precipitation. After in-solution digestion of the precipitated proteins, the resulting peptides were separated and analyzed using HPLC and mass spectrometry with a targeted selected ion monitoring assay with data-dependent tandem mass spectrometry (t-SIM/dd-MS2). The t-SIM/dd-MS2 assay was validated using a synthetic peptide standard containing 10 specific cTnT peptides of interest and with purified human intact cTnT spiked in urine from healthy individuals. Using this assay, 6 different cTnT-specific peptides were identified in urine samples from 3 different patients, all suffering from AMI. We show here for the first time that cTnT can be present in the urine of AMI patients using a targeted LC-MS/MS assay. Whether the presence of cTnT in urine reflects a physiological or pathophysiological process still needs to be elucidated.

DnaJ Homolog Subfamily B Member 9 Is a Putative Autoantigen in Fibrillary GN.

Fibrillary GN is a rare form of GN of uncertain pathogenesis that is characterized by the glomerular accumulation of randomly arranged, nonbranching fibrils (12-24 nm) composed of Ig and complement proteins. In this study, we used mass spectrometry to comprehensively define the glomerular proteome in fibrillary GN compared with that in controls and nonfibrillary GN renal diseases. We isolated glomeruli from formalin-fixed and paraffin-embedded biopsy specimens using laser capture microdissection and analyzed them with liquid chromatography and data-dependent tandem mass spectrometry. These studies identified DnaJ homolog subfamily B member 9 (DNAJB9) as a highly sampled protein detected only in fibrillary GN cases. The glomerular proteome of fibrillary GN cases also contained IgG1 as the dominant Ig and proteins of the classic complement pathway. In fibrillary GN specimens only, immunofluorescence and immunohistochemistry with an anti-DNAJB9 antibody showed strong and specific staining of the glomerular tufts in a distribution that mimicked that of the immune deposits. Our results identify DNAJB9 as a putative autoantigen in fibrillary GN and suggest IgG1 and classic complement effector pathways as likely mediators of the destructive glomerular injury in this disease.

Peptidomic analysis of the venom of the solitary bee Xylocopa appendiculata circumvolans

BackgroundAmong the hymenopteran insect venoms, those from social wasps and bees – such as honeybee, hornets and paper wasps – have been well documented. Their venoms are composed of a number of peptides and proteins and used for defending their nests and themselves from predators. In contrast, the venoms of solitary wasps and bees have not been the object of further research. In case of solitary bees, only major peptide components in a few venoms have been addressed. Therefore, the aim of the present study was to explore the peptide component profile of the venom from the solitary bee Xylocopa appendiculata circumvolans by peptidomic analysis with using LC-MS.MethodsA reverse-phase HPLC connected to ESI-OrbiTrap MS was used for LC-MS. On-line mass fingerprinting was made from TIC, and data-dependent tandem mass spectrometry gave MSMS spectra. A major peptide component was isolated by reverse-phase HPLC by conventional way, and its sequence was determined by Edman degradation, which was finally corroborated by solid phase synthesis. Using the synthetic specimen, biological activities (antimicrobial activity, mast cell devaluation, hemolysis, leishmanicidal activity) and pore formation in artificial lipid bilayer were evaluated.ResultsOn-line mass fingerprinting revealed that the crude venom contained 124 components. MS/MS analysis gave 75 full sequences of the peptide components. Most of these are related to the major and novel peptide, xylopin. Its sequence, GFVALLKKLPLILKHLH-NH2, has characteristic features of linear cationic α-helical peptides; rich in hydrophobic and basic amino acids with no disulfide bond, and accordingly, it can be predicted to adopt an amphipathic α-helix secondary structure. In biological evaluation, xylopin exhibited broad-spectrum antimicrobial activity, and moderate mast cell degranulation and leishmanicidal activities, but showed virtually no hemolytic activity. Additionally, the peptide was able to incorporate pores in artificial lipid bilayers of azolectin, confirming the mechanism of the cytolytic activity by pore formation in biological membranes.ConclusionsLC-ESI-MS and MS/MS analysis of the crude venom extract from a solitary bee Xylocopa appendiculata circumvolans revealed that the component profile of this venom mostly consisted of small peptides. The major peptide components, xylopin and xylopinin, were purified and characterized in a conventional manner. Their chemical and biological characteristics, belonging to linear cationic α-helical peptides, are similar to the known solitary bee venom peptides, melectin and osmin. Pore formation in artificial lipid bilayers was demonstrated for the first time with a solitary bee peptide.

Comprehensive solid-phase extraction of multitudinous bioactive peptides from equine plasma and urine for doping detection

The ability to analyze biological samples for multitudinous exogenous peptides with a single analytical method is desired for doping control in horse racing. The key to achieving this goal is the capability of extracting all target peptides from the sample matrix. In the present study, theory of mixed-mode solid-phase extraction (SPE) of peptides from plasma is described, and a generic mixed-mode SPE procedure has been developed for recovering multitudinous exogenous peptides with remarkable sequence diversity, from equine plasma and urine in a single procedure. Both the theory and the developed SPE procedure have led to the development of a novel analytical method for comprehensive detection of multitudinous bioactive peptides in equine plasma and urine using liquid chromatography coupled to high resolution mass spectrometry (LC-HRMS). Thirty nine bioactive peptides were extracted with strong anion-exchange mixed-mode SPE sorbent, separated on a reversed-phase C18 column and detected by HRMS and data-dependent tandem mass spectrometry. The limit of detection (LOD) was 10–50 pg mL−1 in plasma for most of the peptides and 100 pg mL−1 for the remaining. For urine, LOD was 20–400 pg mL−1 for most of the peptides and 1–4 ng mL−1 for the others. In vitro degradation of the peptides in equine plasma and urine was examined at ambient temperature; the peptides except those with a D-amino acid at position 2 were unstable not only in plasma but also in urine. The developed method was successful in analysis of plasma and urine samples from horses administered dermorphin. Additionally, dermorphin metabolites were identified in the absence of reference standards. The developed SPE procedure and LC-HRMS method can theoretically detect virtually all peptides present at a sufficient concentration in a sample. New peptides can be readily included in the method to be detected without method re-development. The developed method also generates such data that can be retrospectively analyzed for peptides unknown at the time of sample analysis. It is the first generic analytical method for comprehensive detection of multitudinous exogenous peptides in biological samples, to the authors' knowledge.