Abstract: The analysis of intact protein assemblies in native-like states by mass spectrometry offers a wealth of information on their biochemical and biophysical properties. Here we show that the Orbitrap mass analyzer can be used to measure protein assemblies of molecular weights approaching one megadalton with sensitivity down to the detection of single ions. Minor instrumental modifications enabled the measurement of various protein assemblies with outstanding mass-spectral resolution.

Abstract: 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 t...

Abstract: Advances in proteomics are continually driven by the introduction of new mass spectrometric instrumentation with improved performances. The recently introduced quadrupole Orbitrap (Q Exactive) tandem mass spectrometer allows fast acquisition of high-resolution higher-energy collisional dissociation (HCD) tandem mass spectra due to the parallel mode of operation, where the generation, filling, and storage of fragment ions can be performed while simultaneously measuring another ion packet in the Orbitrap mass analyzer. In this study, data-dependent acquisition methods for “fast” or “sensitive” scanning were optimized and assessed by comparing stable isotope labeled yeast proteome coverage. We discovered that speed was the most important parameter for sample loads above 125 ng, where a 95 ms HCD scanning method allowed for identification and quantification of more than 2000 yeast proteins from 1 h of analysis time. At sample loads below 125 ng, a 156 ms HCD acquisition method improved the sensitivity, mass a...

Abstract: During the last two decades, mass spectrometry (MS) has been increasingly used in the environmental sciences with the objective of investigating the presence of organic pollutants. MS has been widely coupled with chromatographic techniques, both gas chromatography (GC) and liquid chromatography (LC), because of their complementary nature when facing a broad range of organic pollutants of different polarity and volatility. A clear trend has been observed, from the very popular GC–MS with a single quadrupole mass analyser, to tandem mass spectrometry (MS–MS) and, more recently, high-resolution mass spectrometry (HRMS). For years GC has been coupled to HR magnetic sector instruments, mostly for dioxin analysis, although in the last ten years there has been growing interest in HRMS with time-of-flight (TOF) and Orbitrap mass analyzers, especially in LC–MS analysis. The increasing interest in the use of HRMS in the environmental sciences is because of its suitability for both targeted and untargeted analysis, owing to its sensitivity in full-scan acquisition mode and high mass accuracy. With the same instrument one can perform a variety of tasks: pre- and post-target analysis, retrospective analysis, discovery of metabolite and transformation products, and non-target analysis. All these functions are relevant to the environmental sciences, in which the analyst encounters thousands of different organic contaminants. Thus, wide-scope screening of environmental samples is one of the main applications of HRMS. This paper is a critical review of current use of HRMS in the environmental sciences. Needless to say, it is not the intention of the authors to summarise all contributions of HRMS in this field, as in classic descriptive reviews, but to give an overview of the main characteristics of HRMS, its strong potential in environmental mass spectrometry and the trends observed over the last few years. Most of the literature has been acquired since 2005, coinciding with the growth and popularity of HRMS in this field, with a few exceptions that deserve to be mentioned because of their relevance.

Abstract: Top–down shotgun lipidomics relies on direct infusion of total lipid extracts into a high-resolution tandem mass spectrometer and implies that individual lipids are recognized by their accurately determined m/z. Lipid ionization efficiency and detection specificity strongly depend on the acquisition polarity, and therefore it is beneficial to analyze lipid mixtures in both positive and negative modes. Hybrid LTQ Orbitrap mass spectrometers are widely applied in top–down lipidomics; however, rapid polarity switching was previously unfeasible because of the severe and immediate degradation of mass accuracy. Here, we report on a method to rapidly acquire high-resolution spectra in both polarity modes with sub-ppm mass accuracy and demonstrate that it not only simplifies and accelerates shotgun lipidomics analyses but also improves the lipidome coverage because more lipid classes and more individual species within each class are recognized. In this way, shotgun analysis of total lipid extracts of human blood plasma enabled to quantify 222 species from 15 major lipid classes within 7 min acquisition cycle. Copyright © 2012 John Wiley & Sons, Ltd.

Abstract: Clinical and forensic toxicology and doping control deal with hundreds or thousands of drugs that may cause poisoning or are abused, are illicit, or are prohibited in sports. Rapid and reliable screening for all these compounds of different chemical and pharmaceutical nature, preferably in a single analytical method, is a substantial effort for analytical toxicologists. Combined chromatography-mass spectrometry techniques with standardised reference libraries have been most commonly used for the purpose. In the last ten years, the focus has shifted from gas chromatography-mass spectrometry to liquid chromatography-mass spectrometry, because of progress in instrument technology and partly because of the polarity and low volatility of many new relevant substances. High-resolution mass spectrometry (HRMS), which enables accurate mass measurement at high resolving power, has recently evolved to the stage that is rapidly causing a shift from unit-resolution, quadrupole-dominated instrumentation. The main HRMS techniques today are time-of-flight mass spectrometry and Orbitrap Fourier-transform mass spectrometry. Both techniques enable a range of different drug-screening strategies that essentially rely on measuring a compound's or a fragment's mass with sufficiently high accuracy that its elemental composition can be determined directly. Accurate mass and isotopic pattern acts as a filter for confirming the identity of a compound or even identification of an unknown. High mass resolution is essential for improving confidence in accurate mass results in the analysis of complex biological samples. This review discusses recent applications of HRMS in analytical toxicology.

Abstract: Background. Gas chromatography (GC) and liquid chromatography (LC) coupled with mass spectrometry (MS) are widely used to confirm drug screening results and for urine screening in presumed intoxicated patients. These techniques are better suited to targeted analysis than to general unknown screening and, due to the complexity of testing, results are seldom available rapidly enough to contribute to the immediate care of the patient. High resolution (HR)/MS with time-of-flight (TOF) or orbitrap instruments offer potential advantages in clinical toxicology. Comparison of GC-MS, LC-MS/MS and LC-HR/MS. For unknown analyses, GC-MS and LC-MS/MS require comparison of full-scan spectra against preestablished libraries. Operation in full-scan mode greatly reduces sensitivity and some drugs present in low but significant concentrations may be missed. Selected ion monitoring (SIM) in GC/MS and selected reaction monitoring (SRM) in LC-MS/MS, where only targeted ions are monitored, increase sensitivity but require prio...

Abstract: In the present study, a new type of mass spectrometer combining a quadrupole mass filter, a higher collision dissociation (HCD) cell and an Orbitrap detector, was evaluated for the analysis of dried blood spots (DBS) in doping controls. DBS analysis is characterized by the necessity to detect prohibited compounds in sub-nanogram-per-milliliter levels with high identification capacity. After extraction of DBS with an organic solvent and liquid chromatographic separation (using a regular C18-RP-analytical UHPLC-column) of target analytes, mass spectrometry is performed with a high-resolution full scan in positive and negative mode by means of electrospray ionisation. Single-product ion mass spectra are acquired using the data-dependent analysis mode (employing an inclusion list) for previously selected precursors of known prohibited compounds with fixed retention time ranges. Besides, a sensitive screening in a targeted approach, non-targeted analysis for retrospective data evaluation is thus possible. The chosen experimental design enables the determination of various drugs from different classes with one generic sample preparation which is shown for 26 selected model compounds (Δ9-tetrahydrocannabinol (THC), tetrahydrocannabinol-9-carboxylic acid (THC-COOH), methylhexaneamine, methylphenidate, cocaine, nikethamide, 3,4-methylenedioxyamphetamine, N-methyl-3,4-methylenedioxyamphetamine, strychnine, mesocarb, salbutamol, formoterol, clenbuterol, metandienone, stanozolol, bisoprolol, propranolol, metoprolol, anastrazole, clomiphene, exemestane, dexamethasone, budesonide, selective androgen receptor modulator (SARM) S4 (andarine), SARM S1, hydrochlorothiazide). Generally, only qualitative result interpretation was focussed upon, but for target analytes with deuterium-labelled internal standards (salbutamol, clenbuterol, cocaine, dexamethasone, THC-COOH and THC) quantitative analysis was also possible. Especially the most challenging analytes, THC and its carboxy metabolite, were detected in DBS at relevant concentrations (<0.5 ng/mL) using targeted HCD experiments. The method was validated for the parameters: specificity, linearity (0–20 ng/mL), precision (<25%), recovery (mean 60%), limit of detection/quantification, ion suppression, stability and accuracy (80–120%). Six isotope-labelled analogues used as internal standards facilitate a quantitative result interpretation which is of utmost importance especially for in-competition drug sports testing.

Abstract: The capabilities of a high-resolution (HR), accurate mass spectrometer (Exactive-MS) operating in full scan MS mode was investigated for the quantitative LC/MS analysis of drugs in patients' plasma samples. A mass resolution of 50 000 (FWHM) at m/z 200 and a mass extracted window of 5 ppm around the theoretical m/z of each analyte were used to construct chromatograms for quantitation. The quantitative performance of the Exactive-MS was compared with that of a triple quadrupole mass spectrometer (TQ-MS), TSQ Quantum Discovery or Quantum Ultra, operating in the conventional selected reaction monitoring (SRM) mode. The study consisted of 17 therapeutic drugs including 8 antifungal agents (anidulafungin, caspofungin, fluconazole, itraconazole, hydroxyitraconazole posaconazole, voriconazole and voriconazole-N-oxide), 4 immunosuppressants (ciclosporine, everolimus, sirolimus and tacrolimus) and 5 protein kinase inhibitors (dasatinib, imatinib, nilotinib, sorafenib and sunitinib). The quantitative results obtained with HR-MS acquisition show comparable detection specificity, assay precision, accuracy, linearity and sensitivity to SRM acquisition. Importantly, HR-MS offers several benefits over TQ-MS technology: absence of SRM optimization, time saving when changing the analysis from one MS to another, more complete information of what is in the samples and easier troubleshooting. Our work demonstrates that U/HPLC coupled to Exactive HR-MS delivers comparable results to TQ-MS in routine quantitative drug analyses. Considering the advantages of HR-MS, these results suggest that, in the near future, there should be a shift in how routine quantitative analyses of small molecules, particularly for therapeutic drugs, are performed. Copyright © 2012 John Wiley & Sons, Ltd.

Abstract: This paper presents an application of ultrahigh-performance liquid chromatography and electrospray ionization quadrupole Orbitrap high-resolution mass spectrometry (UHPLC/ESI Q-Orbitrap) for determination of 166 pesticide residues in fruits and vegetables. Pesticides were extracted from the samples using the QuEChERS (quick, easy, cheap, effective, rugged, and safe) procedure. UHPLC/ESI Q-Orbitrap MS (i.e., full MS scan) acquired full MS data for quantification, and UHPLC/ESI Q-Orbitrap dd-MS2 (i.e., data-dependent scan) obtained product-ion spectra for confirmation. UHPLC/ESI Q-Orbitrap MS quantification was achieved using matrix-matched standard calibration curves with isotopically labeled standards or chemical analogues as internal standards. The method performance characteristics that included overall recovery, intermediate precision, and measurement uncertainty were evaluated according to a nested experimental design. For the matrices studied, about 90.3–91.5% of the pesticides had recoveries between...

Abstract: Modern mass spectrometry-based proteomics can produce millions of peptide fragmentation spectra, which are automatically identified in databases using sequence-specific b- or y-ions. Proteomics projects have mainly been performed with low resolution collision-induced dissociation (CID) in ion traps and beam-type fragmentation on triple quadrupole and QTOF instruments. Recently, the latter has also become available with Orbitrap instrumentation as higher energy collisional dissociation (HCD), routinely providing full mass range fragmentation with high mass accuracy. To systematically study the nature of HCD spectra, we made use of a large scale data set of tryptic peptides identified with an FDR of 0.0001, from which we extract a subset of more than 16 000 that have little or no contribution from cofragmented precursors. We employed a newly developed computer-assisted “Expert System”, which distills our experience and literature knowledge about fragmentation pathways. It aims to automatically annotate the ...

Abstract: Metabolic profiles of biofluids obtained by atmospheric pressure ionization mass spectrometry-based technologies contain hundreds to thousands of features, most of them remaining unknown or at least not characterized in analytical systems. We report here on the annotation of the human adult urinary metabolome and metabolite identification from electrospray ionization mass spectrometry (ESI-MS)-based metabolomics data sets. Features of biological interest were first of all annotated using the ESI-MS database of the laboratory. They were also grouped, thanks to software tools, and annotated using public databases. Metabolite identification was achieved using two complementary approaches: (i) formal identification by matching chromatographic retention times, mass spectra, and also product ion spectra (if required) of metabolites to be characterized in biological data sets to those of reference compounds and (ii) putative identification from biological data thanks to MS/MS experiments for metabolites not available in our chemical library. By these means, 384 metabolites corresponding to 1484 annotated features (659 in negative ion mode and 825 in positive ion mode) were characterized in human urine samples. Of these metabolites, 192 and 66 were formally and putatively identified, respectively, and 54 are reported in human urine for the first time. These lists of features could be used by other laboratories to annotate their ESI-MS metabolomics data sets.

Abstract: The analytical capabilities of liquid chromatography with single-stage high-resolution mass spectrometry have been investigated with emphasis on qualitative aspects related to selective detection during screening and to identification. The study involved 21 different vegetable and fruit commodities, a screening database of 556 pesticides for evaluation of false positives, and a test set of 130 pesticides spiked to the commodities at 0.01, 0.05, and 0.20 mg/kg for evaluation of false negatives. The final method involved a QuEChERS-based sample preparation (without dSPE clean up) and full scan acquisition using alternating scan events without/with fragmentation, at a resolving power of 50,000. Analyte detection was based on extraction of the exact mass (±5 ppm) of the major adduct ion at the database retention time ±30 s and the presence of a second diagnostic ion. Various options for the additional ion were investigated and compared (other adduct ions, M + 1 or M + 2 isotopes, fragments). The two-ion approach for selective detection of the pesticides in the full scan data was compared with two alternative approaches based on response thresholds. Using the two-ion approach, the number of false positives out of 11,676 pesticide/commodity combinations targeted was 36 (0.3 %). The percentage of false negatives, assessed for 2,730 pesticide/commodity combinations, was 13 %, 3 %, and 1 % at the 0.01-, 0.05-, and 0.20-mg/kg level, respectively (slightly higher with fully automated detection). Following the SANCO/12495/2011 protocol for validation of screening methods, the screening detection limit was determined for 130 pesticides and found to be 0.01, 0.05, and ≥0.20 mg/kg for 86, 30, and 14 pesticides, respectively. For the detected pesticides in the spiked samples, the ability for unambiguous identification according to EU criteria was evaluated. A proposal for adaption of the criteria was made.

Abstract: Mass spectrometric imaging (MSI) has emerged as a powerful technique to obtain spatial arrangement of individual molecular ions in animal tissues. Ambient desorption electrospray ionization (DESI) technique is uniquely suited for such imaging experiments, as it can be performed on animal tissues in their native environment without prior treatments. Although MSI has become a rapid growing technique for localization of proteins, lipids, drugs, and endogenous compounds in different tissues, quantification of imaged targets has not been explored extensively. Here we present a novel MSI approach for localization and quantification of drugs in animal thin tissue sections. DESI-MSI using an Orbitrap mass analyzer in full scan mode was performed on 6 μm coronal brain sections from rats that were administered 2.5 mg/kg clozapine. Clozapine was localized and quantified in individual brain sections 45 min postdose. External calibration curves were prepared by micropipetting standards with internal standard (IS) on top of the tissues, and average response factors were calculated for the scans in which both clozapine and IS were detected. All response factors were normalized to area units. Quantifications from DESI-MSI revealed 0.2-1.2 ng of clozapine in individual brain sections, results that were further confirmed by extraction and liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis.

Abstract: Suwannee River fulvic acid (SRFA) was dialyzed through a 100-500 molecular weight cutoff dialysis membrane, and the dialysate and retentate were analyzed by UV-visible absorption and high-resolution Orbitrap mass spectrometry (MS). A significant fraction (36% based on dissolved organic carbon) of SRFA passed through the dialysis membrane. The fraction of SRFA in the dialysate had a different UV-visible absorption spectrum and was enriched in low molecular weight molecules with a more aliphatic composition relative to the initial SRFA solution. Comparison of the SRFA spectra collected by Orbitrap MS and Fourier transform ion cyclotron resonance MS (FT-ICR MS) demonstrated that the mass accuracy of the Orbitrap MS is sufficient for determination of unique molecular formulas of compounds with masses <600 Da in a complex mixture, such as SRFA. The most intense masses detected by Orbitrap MS were found in the 100-200 Da mass range. Many of these low molecular masses corresponded to molecular formulas of previously identified compounds in organic matter, lignin, and plants, and the use of the standard addition method provided an upper concentration estimate of selected target compounds in SRFA. Collectively, these results provide evidence that SRFA contains low molecular weight components that are present individually or in loosely bound assemblies.

Abstract: Mass spectrometry based proteomics generally seeks to identify and fully characterize protein species with high accuracy and throughput. Recent improvements in protein separation have greatly expanded the capacity of Top Down Proteomics (TDP) to identify a large number of intact proteins. To date, TDP has been most tightly associated with Fourier Transform Ion Cyclotron Resonance (FT-ICR) mass spectrometry. Here, we couple the improved separations to a Fourier-Transform instrument based not on ICR but using the Orbitrap Elite mass analyzer. Application of this platform to H1299 human lung cancer cells resulted in the unambiguous identification of 690 unique proteins and over 2000 proteoforms identified from proteins with intact masses 500 identifications) in an Orbitrap mass spectrometer and exemplifies an accessible platform for whole protein mass spectrometry.

Abstract: RATIONALE Similar to other mass spectrometric technologies based on ion trapping in a spatially restricted area, the performance of Orbitrap Fourier transform mass spectrometry (FTMS) is affected by the interaction between the trapped ion clouds. One of the effects associated with Coulombic interaction inside the ion trap is the ion cloud coupling, known in ion cyclotron resonance (ICR) FTMS as coalescence, or a phase-locking phenomenon. Nevertheless, the direct observation of ion coalescence has not been reported for Orbitrap FTMS yet. METHODS We have performed experiments on ion coalescence with a pair of isobaric peptides in the state-of-the-art hybrid linear ion trap high-field compact Orbitrap Elite FT mass spectrometer using both standard and advanced signal processing modes. RESULTS For the instrument configuration employed in this work we found that ion coalescence occurs when two singly charged peptides with the mass difference of 22?mDa and molecular weight of about 1060?Da have the total abundance of at least 7.5*104 charges. CONCLUSIONS We experimentally demonstrate the existence of the ion coalescence phenomenon in Orbitrap FTMS for peptides for a wide range of total trapped ion population. Using the applicable modeling of the phase-locking threshold we estimate the effect of ion coalescence on the performance of Orbitrap FTMS. Copyright (C) 2012 John Wiley & Sons, Ltd.

Abstract: Although liquid chromatography/mass spectrometry using selected reaction monitoring (LC/SRM-MS) holds great promise for targeted protein analysis, quantification of therapeutic monoclonal antibody (mAb) in tissues represents a daunting challenge due to the extremely low tissue levels, complexity of tissue matrixes, and the absence of an efficient strategy to develop an optimal LC/SRM-MS method. Here we describe a high-throughput, streamlined strategy for the development of sensitive, selective, and reliable quantitative methods of mAb in tissue matrixes. A sensitive nano-LC/nanospray-MS method was employed to achieve a low lower limit of quantification (LOQ). For selection of signature peptides (SP), the SP candidates were identified by a high-resolution Orbitrap and then optimal SRM conditions for each candidate were obtained using a high-throughput, on-the-fly orthogonal array optimization (OAO) strategy, which is capable of optimizing a large set of SP candidates within a single nano-LC/SRM-MS run. Usi...

Abstract: Fungal contamination of food causes health and economic concerns. Several species of lactic acid bacteria (LAB) have antifungal activity which may inhibit food spoilage fungi. LAB have GRAS (generally recognised as safe) status, allowing them to be safely integrated into food systems as natural food preservatives. A method is described herein that enables rapid screening of LAB cultures for 25 known antifungal compounds associated with LAB. This is the first chromatographic method developed which enables the rapid identification of a wide range of antifungal compounds by a single method with a short analysis time (23 min). Chromatographic separation was achieved on a Phenomenex Gemini C18 100A column (150 mm × 2.0 mm; 5 μm) by use of a mobile-phase gradient prepared from (A) water containing acetic acid (0.1%) and (B) acetonitrile containing acetic acid (0.1%), at a flow rate of 0.3 µL min−1. The gradient involved a progressive ramp from 10–95% acetonitrile over 13 min. The LC was coupled to a hybrid LTQ Orbitrap XL fourier-transform mass spectrometer (FTMS) operated in negative ionisation mode. High mass accuracy data (<3 ppm) obtained by use of high resolution (30,000 K) enabled unequivocal identification of the target compounds. This method allows comprehensive profiling and comparison of different LAB strains and is also capable of the identification of additional compounds produced by these bacteria.