Coupling immuno-magnetic capture with LC-MS/MS(MRM) as a sensitive, reliable, and specific assay for SARS-CoV-2 identification from clinical samples.

Recently, numerous diagnostic approaches from different disciplines have been developed for SARS-CoV-2 diagnosis to monitor and control the COVID-19 pandemic. These include MS-based assays, which provide analytical information on viral proteins. However, their sensitivity is limited, estimated to be 5 × 104 PFU/ml in clinical samples. Here, we present a reliable, specific, and rapid method for the identification of SARS-CoV-2 from nasopharyngeal (NP) specimens, which combines virus capture followed by LC-MS/MS(MRM) analysis of unique peptide markers. The capture of SARS-CoV-2 from the challenging matrix, prior to its tryptic digestion, was accomplished by magnetic beads coated with polyclonal IgG-α-SARS-CoV-2 antibodies, enabling sample concentration while significantly reducing background noise interrupting with LC-MS analysis. A sensitive and specific LC-MS/MS(MRM) analysis method was developed for the identification of selected tryptic peptide markers. The combined assay, which resulted in S/N ratio enhancement, achieved an improved sensitivity of more than 10-fold compared with previously described MS methods. The assay was validated in 29 naive NP specimens, 19 samples were spiked with SARS-CoV-2 and 10 were used as negative controls. Finally, the assay was successfully applied to clinical NP samples (n = 26) pre-determined as either positive or negative by RT-qPCR. This work describes for the first time a combined approach for immuno-magnetic viral isolation coupled with MS analysis. This method is highly reliable, specific, and sensitive; thus, it may potentially serve as a complementary assay to RT-qPCR, the gold standard test. This methodology can be applied to other viruses as well.

Differentiation of leucine and isoleucine residues in peptides using charge transfer dissociation mass spectrometry (CTD-MS).

RATIONALE: The function of a protein or the binding affinity of an antibody can be substantially altered by the replacement of leucine (Leu) with isoleucine (Ile), and vice versa, so the ability to identify the correct isomer using mass spectrometry can help resolve important biological questions. Tandem mass spectrometry approaches for Leu/Ile (Xle) discrimination have been developed, but they all have certain limitations. METHODS: Four model peptides and two wild-type peptide sequences containing either Leu or Ile residues were subjected to charge transfer dissociation (CTD) mass spectrometry on a modified three-dimensional ion trap. The peptides were analyzed in both the 1+ and 2+ charge states, and the results were compared to conventional collision-induced dissociation spectra of the same peptides obtained using the same instrument. RESULTS: CTD resulted in 100% sequence coverage for each of the studied peptides and provided a variety of side-chain cleavages, including d, w and v ions. Using CTD, reliable d and w ions of Xle residues were observed more than 80% of the time. When present, d ions are typically greater than 10% of the abundance of the corresponding a ions from which they derive, and w ions are typically more abundant than the z ions from which they derive. CONCLUSIONS: CTD has the benefit of being applicable to both 1+ and 2+ precursor ions, and the overall performance is comparable to that of other high-energy activation techniques like hot electron capture dissociation and UV photodissociation. CTD does not require chemical modifications of the precursor peptides, nor does it require additional levels of isolation and fragmentation.

Development and Validation of a LC-MS/MS Technique for the Analysis of Short Chain Fatty Acids in Tissues and Biological Fluids without Derivatisation Using Isotope Labelled Internal Standards.

The gut microbiota is critical to the maintenance of physiological homeostasis and as such is implicated in a range of diseases such as colon cancer, ulcerative colitis, diabetes, cardiovascular diseases, and neurodegenerative diseases. Short chain fatty acids (SCFAs) are key metabolites produced by the gut microbiota from the fermentation of dietary fibre. Here we present a novel, sensitive, and direct LC-MS/MS technique using isotopically labelled internal standards without derivatisation for the analysis of SCFAs in different biological matrices. The technique has significant advantages over the current widely used techniques based on sample derivatization and GC-MS analysis, including fast and simple sample preparation and short LC runtime (10 min). The technique is specific and sensitive for the quantification of acetate, butyrate, isobutyrate, isovalerate, lactate, propionate and valerate. The limits of detection were all 0.001 mM except for acetate which was 0.003 mM. The calibration curves for all the analytes were linear with correlation coefficients r2 > 0.998. The intra- and inter-day precisions in three levels of known concentrations were <12% and <20%, respectively. The quantification accuracy ranged from 92% to 120%. The technique reported here offers a valuable analytical tool for use in studies of SCFA production in the gut and their distribution to host tissues.

Free Open Source Software for Protein and Peptide Mass Spectrometry- based Science.

In the field of biology, and specifically in protein and peptide science, the power of mass spectrometry is that it is applicable to a vast spectrum of applications. Mass spectrometry can be applied to identify proteins and peptides in complex mixtures, to identify and locate post-translational modifications, to characterize the structure of proteins and peptides to the most detailed level or to detect protein-ligand non-covalent interactions. Thanks to the Free and Open Source Software (FOSS) movement, scientists have limitless opportunities to deepen their skills in software development to code software that solves mass spectrometric data analysis problems. After the conversion of raw data files into open standard format files, the entire spectrum of data analysis tasks can now be performed integrally on FOSS platforms, like GNU/Linux, and only with FOSS solutions. This review presents a brief history of mass spectrometry open file formats and goes on with the description of FOSS projects that are commonly used in protein and peptide mass spectrometry fields of endeavor: identification projects that involve mostly automated pipelines, like proteomics and peptidomics, and bio-structural characterization projects that most often involve manual scrutiny of the mass data. Projects of the last kind usually involve software that allows the user to delve into the mass data in an interactive graphics-oriented manner. Software projects are thus categorized on the basis of these criteria: software libraries for software developers vs desktop-based graphical user interface, software for the end-user and automated pipeline-based data processing vs interactive graphics-based mass data scrutiny.

Matrix-Assisted Ionization and Tandem Mass Spectrometry Capabilities in Protein Biomarker Characterization-An Initial Study Using the Small Cell Lung Cancer Biomarker Progastrin Releasing Peptide as a Model Compound.

This initial study evaluates vacuum matrix-assisted ionization (vMAI) mass spectrometry (MS) for identification and determination of tryptic peptides from the biomarker protein progastrin releasing peptide (ProGRP). Similar peptides and charge states were observed as in liquid chromatography (LC) electrospray ionization (ESI) MS. The prolonged ion duration in vMAI with similar charge states as in ESI was advantageous for determining the MS/MS fragmentation conditions compared to MAI. It is assumed that the vacuum ionization conditions lower the detection limits of the experiment. This may be the reason vMAI combined with high resolution MS enabled detection of tryptic peptides from more digested proteins than MAI selected reaction monitoring MS. Additionally, MAI ion mobility spectrometry MS (MAI-IMS-MS) was evaluated for differentiation of intact protein isoforms, successfully enabling differentiation of the isoforms by drift time selection. Examples are both shown for model proteins bovine serum albumin, cytochrome C, and lysozyme and the clinically relevant small cell lung cancer protein biomarker ProGRP, which exists in three isoforms. Coupling with the vacuum ionization conditions using a dedicated vacuum-probe source MAI enables information to be extracted readily as with conventional approaches, just faster.

Mass Spectrometry Techniques: Principles and Practices for Quantitative Proteomics.

In the current omics-age of research, major developments have been made in technologies that attempt to survey the entire repertoire of genes, transcripts, proteins, and metabolites present within a cell. While genomics has led to a dramatic increase in our understanding of such things as disease morphology and how organisms respond to medications, it is critical to obtain information at the proteome level since proteins carry out most of the functions within the cell. The primary tool for obtaining proteome-wide information on proteins within the cell is mass spectrometry (MS). While it has historically been associated with the protein identification, developments over the past couple of decades have made MS a robust technology for protein quantitation as well. Identifying quantitative changes in proteomes is complicated by its dynamic nature and the inability of any technique to guarantee complete coverage of every protein within a proteome sample. Fortunately, the combined development of sample preparation and MS methods have made it capable of quantitatively comparing many thousands of proteins obtained from cells and organisms.

Collision energies on QTof and Orbitrap instruments: How to make proteomics measurements comparable?

Quadrupole time-of-flight (QTof) collision-induced dissociation (CID) and Orbitrap higher-energy collisional dissociation (HCD) are the most commonly used fragmentation techniques in mass spectrometry-based proteomics workflows. The information content of the MS/MS spectra is first and foremost determined by the applied collision energy. How can we set up the two instrument types to achieve maximum transferability? To answer this question, we compared MS/MS spectra obtained on a Bruker QTof CID and a Thermo Q-Exactive Focus Orbitrap HCD instrument as a function of collision energy using the similarity index. Results show that with a few eV lower collision energy setting on HCD (Orbitrap-specific CID) than on QTof CID, nearly identical MS/MS spectra can be obtained for leucine enkephalin pentapeptide standard, for selected +2 and +3 enolase tryptic peptides and for a large number of peptides in a HeLa protein digest. The Bruker QTof was able to produce colder ions, which may be significant to study inherently labile compounds. Further, we examined energy dependence of peptide identification confidence, as characterized by Mascot scores, on the HeLa peptides. In line with earlier QTof results, this dependence shows one or two maxima (unimodal or bimodal behavior) on Orbitrap. The fraction of bimodal peptides is lower on Orbitrap. Optimal energies as a function of m/z show a similar linear trend on both instruments, which suggests that with appropriate collision energy adjustment, matching conditions for proteomics can be achieved. Data have been deposited in the MassIVE repository (MSV000086434).

Identification-Free Control over the Precursor Isotopic Mass Misassignment in Orbitrap-Based Proteomics.

Selection of a precursor ion from a peptide isotopic cluster to obtain a fragmentation mass spectrum is a crucial step in data-dependent proteome analysis. However, the monoisotopic mass assignment performed in this step is often an issue confronted by the data acquisition software of hybrid Orbitrap FTMS that is most widely used in proteomics. To address the problem, many data processing tools, such as raw data converters and search engines, have optional accounting for the precursor mass shift due to the isotopic error. These solutions require additional data preprocessing steps and lead to an increase in the search space, thus making the analysis longer and/or less reliable. In this work, we processed 100 Orbitrap-based LC-MS/MS runs from 10 publicly available data sets to examine the rate of precursor isotope misassignment. The effect from taking the isotope error into account during the search on the number of identified peptides varied in a wide range from 0 to 33%. Thus, it may be tempting to spend extra time before or during a search to account for the mass assignment issue. Alternatively, this effect can be predicted a priori using an identification-free metric, which can be a part of data quality control software. Based on the results obtained in this work, we propose such a metric be further added into the visual and intuitive quality control software, viQC, developed previously and available at https://github.com/lisavetasol/viQC. It takes about a minute to calculate and plot nine quality metrics, including the proposed one for typical proteome analysis.

Metabolomics Analysis of Viral Therapeutics.

Virotherapy, enabled by recent advances in the transdisciplinary field of biotechnology, has emerged as a powerful tool for use in anticancer treatment, gene therapy, immunotherapy, etc. Examining the effects of viruses and virus-derived immune-modulating therapeutics is of great fundamental and clinical interest. Here we describe a sample preparation protocol for metabolite extraction from virus-infected tissue, in addition to liquid chromatography-mass spectrometry conditions essential for subsequent analysis. This metabolomics approach delivers highly sensitive and specific metabolite information on various biospecimens. Such an approach may be adopted to monitor biological changes in over 30 relevant metabolic pathways in response to viral infection and also viral therapeutics.

Rapid Analysis of Reduced Antibody Drug Conjugate by Online LC-MS/MS with Fourier Transform Ion Cyclotron Resonance Mass Spectrometry.

Antibody drug conjugates (ADCs), which harness the high targeting specificity of monoclonal antibodies (mAb) with the potency of small molecule therapeutics, are one of the fastest growing pharmaceutical classes. Nevertheless, ADC conjugation techniques and processes may introduce intrinsic heterogeneity including primary sequence variants, varied drug-to-antibody ratio (DAR) species, and drug positional isomers, which must be monitored to ensure the safety and efficacy of ADCs. Liquid chromatography coupled to mass spectrometry (LC-MS) is a powerful tool for characterization of ADCs. However, the conventional bottom-up MS analysis workflows require an enzymatic digestion step which can be time consuming and may introduce artifactual modifications. Herein, we develop an online LC-MS/MS method for rapid analysis of reduced ADCs without digestion, enabling determination of DAR, characterization of the primary sequence, and localization of the drug conjugation site of the ADC using high-resolution Fourier transform ion cyclotron resonance (FTICR) MS. Specifically, a model cysteine-linked ADC was reduced to generate six unique subunits: light chain (Lc) without drug (Lc0), Lc with 1 drug (Lc1), heavy chain (Hc) without drug (Hc0), and Hc with 1-3 drugs (Hc1-3, respectively). A concurrent reduction strategy is applied to assess ADC subunits in both the partially reduced (intrachain disulfide bonds remain intact) and fully reduced (all disulfide bonds are cleaved) forms. The entire procedure including the sample preparation and LC-MS/MS takes less than 55 min, enabling rapid multiattribute analysis of ADCs.

Saliva versus Plasma Therapeutic Drug Monitoring of Gentamicin in Jordanian Preterm Infants. Development of a Physiologically-Based Pharmacokinetic (PBPK) Model and Validation of Class II Drugs of Salivary Excretion Classification System.

Gentamicin has proven to be a very successful treatment for bacterial infection, but it also can cause adverse effects, especially ototoxicity, which is irreversible. Therapeutic drug monitoring (TDM) in saliva is a more convenient non-invasive alternative compared to plasma. A physiologically-based pharmacokinetic (PBPK) model of gentamicin was built and validated using previously-published plasma and saliva data. The validated model was then used to predict experimentally-observed plasma and saliva gentamicin TDM data in Jordanian pediatric preterm infant patients measured using sensitive LCMS/MS method. A correlation was established between plasma and saliva exposures. The developed PBPK model predicted previously reported gentamicin levels in plasma, saliva and those observed in the current study. A good correlation was found between plasma and saliva exposures. The PBPK model predicted that gentamicin in saliva is 5-7 times that in plasma, which is in agreement with observed results. Saliva can be used as an alternative for TDM of gentamicin in preterm infant patients. Exposure to gentamicin in plasma and saliva can reliably be predicted using the developed PBPK model in patients.

Combining Mass Spectrometry with Paternò-Büchi Reaction to Determine Double-Bond Positions in Lipids at the Single-Cell Level.

Single cell MS (SCMS) techniques are under rapid development for molecular analysis of individual cells among heterogeneous populations. Lipids are basic cellular constituents playing essential functions in energy storage and the cellular signaling processes of cells. Unsaturated lipids are characterized with one or multiple carbon-carbon double (C═C) bonds, and they are critical for cell functions and human diseases. Characterizing unsaturated lipids in single cells allows for better understanding of metabolomic biomarkers and therapeutic targets of rare cells (e.g., cancer stem cells); however, these studies remain challenging. We developed a new technique using a micropipette needle, in which Paternò-Büchi (PB) reactions at C═C bond can be induced, to determine locations of C═C bonds in unsaturated lipids at the single-cell level. The micropipette needle is produced by combining a pulled glass capillary needle with a fused silica capillary. Cell lysis solvent and PB reagent (acetone or benzophenone) are delivered into the micropipette needle (tip size ≈ 15 um) through a fused silica capillary. The capillary needle plays multiple functions (i.e., single cell sampling probe, cell lysis container, microreactor, and nano-ESI emitter) in the experiments. Both regular (no reaction) and reactive (with PB reaction) SCMS analyses of the same cell can be achieved. C═C bond locations were determined from MS scan and MS/MS of PB products assisted by Python programs. This technique can potentially be used for other reactive SCMS studies to enhance molecular analysis for broad ranges of single cells.

Dynamic pH barrage junction focusing of amino acids, peptides, and digested monoclonal antibodies in capillary electrophoresis-mass spectrometry.

Dynamic pH barrage junction focusing in CE enables effective signal enhancement, quantitative capture efficiencies, and straightforward optimization. The method is a technical variant of dynamic pH junction focusing. CE separation with dynamic pH barrage junction focusing is compatible with both optical and mass spectrometric detection. We developed a CE-MS/MS method using hydrophilic polyethyleneimine-coated capillaries and validated it for the qualitative analysis of amino acids, peptides, and tryptic peptides of digested monoclonal antibodies. The S/N of extracted ion electropherograms of zwitterionic analytes were enhanced by approximately two orders of magnitude with a tradeoff of a shortened separation window. Online focusing improved the MS signal intensity of a diluted antibody digest, enabling more precursor ions to be analyzed with subsequent tandem mass spectrometric identification. It also broadened the concentration range of protein digest samples for which adequate sequence coverage data can be obtained. With only 0.9 ng of digested infliximab sample loaded into the capillary, 76% and 100% sequence coverage was realized for antibody heavy and light chains, respectively, after online focusing. Full coverage was achieved with 9 ng of injected digest.

Fully automated dried blood spot sample preparation enables the detection of lower molecular mass peptide and non-peptide doping agents by means of LC-HRMS.

The added value of dried blood spot (DBS) samples complementing the information obtained from commonly routine doping control matrices is continuously increasing in sports drug testing. In this project, a robotic-assisted non-destructive hematocrit measurement from dried blood spots by near-infrared spectroscopy followed by a fully automated sample preparation including strong cation exchange solid-phase extraction and evaporation enabled the detection of 46 lower molecular mass (< 2 kDa) peptide and non-peptide drugs and drug candidates by means of LC-HRMS. The target analytes included, amongst others, agonists of the gonadotropin-releasing hormone receptor, the ghrelin receptor, the human growth hormone receptor, and the antidiuretic hormone receptor. Furthermore, several glycine derivatives of growth hormone-releasing peptides (GHRPs), arguably designed to undermine current anti-doping testing approaches, were implemented to the presented detection method. The initial testing assay was validated according to the World Anti-Doping Agency guidelines with estimated LODs between 0.5 and 20 ng/mL. As a proof of concept, authentic post-administration specimens containing GHRP-2 and GHRP-6 were successfully analyzed. Furthermore, DBS obtained from a sampling device operating with microneedles for blood collection from the upper arm were analyzed and the matrix was cross-validated for selected parameters. The introduction of the hematocrit measurement method can be of great value for doping analysis as it allows for quantitative DBS applications by managing the well-recognized "hematocrit effect." Graphical abstract.

Evaluation of ultraviolet photodissociation tandem mass spectrometry for the structural assignment of unsaturated fatty acid double bond positional isomers.

Fatty acids are a major source of structural diversity within the lipidome due to variations in their acyl chain lengths, branching, and cyclization, as well as the number, position, and stereochemistry of double bonds within their mono- and poly-unsaturated species. Here, the utility of 193 nm UltraViolet PhotoDissociation tandem mass spectrometry (UVPD-MS/MS) has been evaluated for the detailed structural characterization of a series of unsaturated fatty acid lipid species. UVPD-MS/MS of unsaturated fatty acids is shown to yield pairs of unique diagnostic product ions resulting from cleavages adjacent to their C=C double bonds, enabling unambiguous localization of the site(s) of unsaturation within these lipids. The effect of several experimental variables on the observed fragmentation behaviour and UVPD-MS/MS efficiency, including the position and number of double bonds, the effect of conjugated versus non-conjugated double bonds, the number of laser pulses, and the influence of alkali metal cations (Li, Na, K) as the ionizing adducts, has been evaluated. Importantly, the abundance of the diagnostic ions is shown to enable relative quantitation of mixtures of fatty acid isomers across a range of molar ratios. Finally, the practical application of 193 nm UVPD-MS/MS is demonstrated via characterization of changes in the ratios of fatty acid double bond positional isomers in isogenic colorectal cancer cell lines. This study therefore demonstrates the practicality of UVPD-MS/MS for the structural characterization of fatty acid isomers in lipidome analysis workflows.

A design of experiments concept for the minimization of nonspecific peptide adsorption in the mass spectrometric determination of substance P and related hemokinin-1.

Substance P and hemokinin-1 were predominantly examined by immunoassays with their limitation to differentiate appropriately between both peptides. The use of liquid chromatography coupled with tandem mass spectrometry is a promising, highly selective alternative. Adsorption processes have been identified in preliminary experiments to play a crucial role in the loss of mass spectrometry intensity of both peptides. Therefore, a design of experiments concept was created to minimize nonspecific peptide adsorption. For this purpose, the most critical influencing parameters-(1) the composition of the injection solvent as well as (2) the most suitable container material-were systematically and concordantly investigated. The addition of modifiers, such as formic acid, dimethyl sulfoxide, and organic solvents, to the injection solvent led to a substantial gain of intensity of substance P and hemokinin-1 compared to the start gradient as an injection solvent. Furthermore, the systematic investigation underlined the high impact of the container material, demonstrating polypropylene as the most favorable material. A conjoint injection solvent optimum was found to determine both peptides simultaneously by the conduction of a sweet-spot analysis. The experimental design substantially reduced nonspecific peptide adsorption and enabled the simultaneous and selective determination of endogenous substance P and hemokinin-1 plasma levels.

Intraoperative detection of isocitrate dehydrogenase mutations in human gliomas using a miniature mass spectrometer.

Knowledge of the isocitrate dehydrogenase (IDH) mutation status of glioma patients could provide insights for decision-making during brain surgery. However, pathology is not able to provide such information intraoperatively. Here we describe the first application of a miniature mass spectrometer (MS) to the determination of IDH mutation status in gliomas intraoperatively. The instrumentation was modified to be compatible with use in the operating room. Tandem MS was performed on the oncometabolite, 2-hydroxyglutarate, and a reference metabolite, glutamate, which is not involved in the IDH mutation. Ratios of fragment ion intensities were measured to calculate an IDH mutation score, which was used to differentiate IDH mutant and wild-type tissues. The results of analyzing 25 biopsies from 13 patients indicate that reliable determination of IDH mutation status was achieved (p = 0.0001, using the Kruskal-Wallis non-parametric test). With its small footprint and low power consumption and noise level, this application of miniature mass spectrometers represents a simple and cost-effective platform for an important intraoperative measurement. Graphical abstract.

Determination of antipsychotic drugs in oral fluid using dried saliva spots by gas chromatography-tandem mass spectrometry.

The present work describes the optimization and validation of an analytical method for the determination of six antipsychotic drugs (chlorpromazine, levomepromazine, cyamemazine, clozapine, haloperidol, and quetiapine) in oral fluid samples after solvent extraction from dried saliva spots, by gas chromatography coupled to tandem mass spectrometry. The method was fully validated, and the included parameters were selectivity, linearity, limits of quantification, precision and accuracy, stability, and recovery. The method was linear for all compounds from 10 to 400 ng/mL, except for haloperidol (5-100 ng/mL), presenting coefficients of determination higher than 0.99. Inter- and intra-day precision and accuracy were in conformity with the criteria usually seen in bioanalytical method validation; i.e., coefficients of variation were lower than 15% and an accuracy of 15% or better for all studied drugs. The recoveries obtained with this miniaturized technique ranged from 63 to 97%. The herein described method is the first to be reported using the dried saliva spots approach for the analysis of these antypshychotic drugs, proving great sensitivity apart from its simple and fast procedure. The method was considered a good alternative to the conventional techniques to be applied in clinical and toxicological analyses, even more taking into account the extremely low sample volume used (50 µL). Graphical abstract.

The utilisation of ion chromatography and tandem mass spectrometry (IC-MS/MS) for the multi-residue simultaneous determination of highly polar anionic pesticides in fruit and vegetables.

A robust and sensitive method utilising a hybrid ion chromatography tandem mass spectrometry system (IC-MS/MS) for the simultaneous determination of nine (9) highly polar anionic pesticides (chlorate, ethephon, fosetyl aluminium, glufosinate, glyphosate, N-acetyl AMPA, N-acetyl glyphosate, perchlorate and phosphonic acid) in fruit and vegetables is described. Mean recoveries (n = 6) at two fortification levels ranged from 83 to 112% with %CVs in the range 3-14%. The linearity range was 0.005-0.4 mg kg-1 and R2 values were >0.99 and the sensitivity of the method allowed (20× or 30×) dilution of samples. Provision of qualitative determination of aminomethylphosphonic acid (AMPA) was also facilitated via minor modification of the chromatographic conditions. Compliance with method validation criteria, survey results from the statutory UK/EU Pesticide Residues in Food 2018 programmes i.e. pea, pineapple, melon and successful z-scores for a UK proficiency testing scheme sample (ethephon in pineapple) demonstrate successful application of this IC-MS/MS method.