Multiplex technologies for the assessment of minimal residual disease and low-level mutation detection in leukaemia: mass spectrometry versus next-generation sequencing.

With the focus of leukaemia management shifting to the implications of low-level disease burden, increasing attention is being paid on the development of highly sensitive methodologies required for detection. There are various techniques capable of identification of measurable residual disease (MRD) either evidencing as relevant mutation detection [e.g. nucleophosmin 1 (NPM1) mutation] or trace levels of leukaemic clonal populations. The vast majority of these methods only permit detection of a single clone or mutation. However, mass spectrometry and next-generation sequencing enable the interrogation of multiple genes simultaneously, facilitating a more complete genomic profile. In the present review, we explore the methodologies of both techniques in conjunction with the important advantages and limitations associated with each assay. We also highlight the evidence and the various instances where either technique has been used and propose future strategies for MRD detection.

ICP-MS Determination of 23 Elements of Potential Health Concern in Liquids of e-Cigarettes. Method Development, Validation, and Application to 37 Real Samples.

The lack of interest in the determination of toxic elements in liquids for electronic cigarettes (e-liquids) has so far been reflected in the scarce number of accurate and validated analytical methods devoted to this aim. Since the strong matrix effects observed for e-liquids constitute an exciting analytical challenge, the main goal of this study was to develop and validate an ICP-MS method aimed to quantify 23 elements in 37 e-liquids of different flavors. Great attention has been paid to the critical phases of sample pre-treatment, as well as to the optimization of the ICP-MS conditions for each element and of the quantification. All samples exhibited a very low amount of the elements under investigation. Indeed, the sum of their average concentration was of ca. 0.6 mg kg-1. Toxic elements were always below a few tens of a µg per kg-1 and, very often, their amount was below the relevant quantification limits. Tobacco and tonic flavors showed the highest and the lowest concentration of elements, respectively. The most abundant elements came frequently from propylene glycol and vegetal glycerin, as confirmed by PCA. A proper choice of these substances could further decrease the elemental concentration in e-liquids, which are probably barely involved as potential sources of toxic elements inhaled by vapers.

Assessment of Breathomics Testing Using High-Pressure Photon Ionization Time-of-Flight Mass Spectrometry to Detect Esophageal Cancer.

Importance: A triage test is needed to increase the detection rate for esophageal cancer. Objective: To investigate whether breathomics can detect esophageal cancer among patients without a previous diagnosis of cancer using high-pressure photon ionization time-of-flight mass spectrometry (HPPI-TOFMS). Design, Setting, and Participants: This diagnostic study included participants who planned to receive an upper endoscopy or surgery of the esophagus at a single center in China. Exhaled breath was collected with a self-designed collector and air bags before participants underwent these procedures. Sample collection and analyses were performed by trained researchers following a standardized protocol. Participants were randomly divided into a discovery data set and a validation data set. Data were collected from December 2020 to March 2021. Exposures: Breath samples were analyzed by HPPI-TOFMS, and the support vector machine algorithm was used to construct a detection model. Main Outcomes and Measures: The accuracy of breathomics was measured by the sensitivity, specificity, accuracy, positive predictive value, negative predictive value, and area under the receiver operating characteristic curve. Results: Exhaled breath samples were obtained from 675 patients (216 [32%] with esophageal cancer; 459 [68%] with noncancer diseases). Of all patients, 206 (31%) were women, and the mean (SD) age was 64.0 (11.9) years. In the validation data set, esophageal cancer was detected with an accuracy of 93.33%, sensitivity of 97.83%, specificity of 83.72%, positive predictive value of 94.74%, negative predictive value of 92.78%, and area under the receiver operating characteristic curve of 0.89. Notably, for 16 patients with high-grade intraepithelial neoplasia, 12 (75%) were predicted to have esophageal cancer. Conclusions and Relevance: In this diagnostic study, testing breathomics using HPPI-TOFMS was feasible for esophageal cancer detection and totally noninvasive, which could help to improve the diagnosis of esophageal cancer.

A mass spectrometry-based targeted assay for detection of SARS-CoV-2 antigen from clinical specimens.

BACKGROUND: The COVID-19 pandemic caused by severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) has overwhelmed health systems worldwide and highlighted limitations of diagnostic testing. Several types of diagnostic tests including RT-PCR-based assays and antigen detection by lateral flow assays, each with their own strengths and weaknesses, have been developed and deployed in a short time. METHODS: Here, we describe an immunoaffinity purification approach followed a by high resolution mass spectrometry-based targeted qualitative assay capable of detecting SARS-CoV-2 viral antigen from nasopharyngeal swab samples. Based on our discovery experiments using purified virus, recombinant viral protein and nasopharyngeal swab samples from COVID-19 positive patients, nucleocapsid protein was selected as a target antigen. We then developed an automated antibody capture-based workflow coupled to targeted high-field asymmetric waveform ion mobility spectrometry (FAIMS) - parallel reaction monitoring (PRM) assay on an Orbitrap Exploris 480 mass spectrometer. An ensemble machine learning-based model for determining COVID-19 positive samples was developed using fragment ion intensities from the PRM data. FINDINGS: The optimized targeted assay, which was used to analyze 88 positive and 88 negative nasopharyngeal swab samples for validation, resulted in 98% (95% CI = 0.922-0.997) (86/88) sensitivity and 100% (95% CI = 0.958-1.000) (88/88) specificity using RT-PCR-based molecular testing as the reference method. INTERPRETATION: Our results demonstrate that direct detection of infectious agents from clinical samples by tandem mass spectrometry-based assays have potential to be deployed as diagnostic assays in clinical laboratories, which has hitherto been limited to analysis of pure microbial cultures. FUNDING: This study was supported by DBT/Wellcome Trust India Alliance Margdarshi Fellowship grant IA/M/15/1/502023 awarded to AP and the generosity of Eric and Wendy Schmidt.

Review of Methodological Approaches to Human Milk Small Extracellular Vesicle Proteomics.

Proteomics can map extracellular vesicles (EVs), including exosomes, across disease states between organisms and cell types. Due to the diverse origin and cargo of EVs, tailoring methodological and analytical techniques can support the reproducibility of results. Proteomics scans are sensitive to in-sample contaminants, which can be retained during EV isolation procedures. Contaminants can also arise from the biological origin of exosomes, such as the lipid-rich environment in human milk. Human milk (HM) EVs and exosomes are emerging as a research interest in health and disease, though the experimental characterization and functional assays remain varied. Past studies of HM EV proteomes have used data-dependent acquisition methods for protein detection, however, improvements in data independent acquisition could allow for previously undetected EV proteins to be identified by mass spectrometry. Depending on the research question, only a specific population of proteins can be compared and measured using isotope and other labelling techniques. In this review, we summarize published HM EV proteomics protocols and suggest a methodological workflow with the end-goal of effective and reproducible analysis of human milk EV proteomes.

Mass spectrometry-based metabolomics: a guide for annotation, quantification and best reporting practices.

Mass spectrometry-based metabolomics approaches can enable detection and quantification of many thousands of metabolite features simultaneously. However, compound identification and reliable quantification are greatly complicated owing to the chemical complexity and dynamic range of the metabolome. Simultaneous quantification of many metabolites within complex mixtures can additionally be complicated by ion suppression, fragmentation and the presence of isomers. Here we present guidelines covering sample preparation, replication and randomization, quantification, recovery and recombination, ion suppression and peak misidentification, as a means to enable high-quality reporting of liquid chromatography- and gas chromatography-mass spectrometry-based metabolomics-derived data.

Acoustic Ejection Mass Spectrometry: A Fully Automatable Technology for High-Throughput Screening in Drug Discovery.

Acoustic droplet ejection (ADE)-open port interface (OPI)-mass spectrometry (MS) has recently been introduced as a versatile analytical method that combines fast and contactless acoustic sampling with sensitive and accurate electrospray ionization (ESI)-MS-based analyte detection. The potential of the technology to provide label-free measurements in subsecond analytical cycle times makes it an attractive option for high-throughput screening (HTS). Here, we report the first implementation of ADE-OPI-MS in a fully automated HTS environment, based on the example of a biochemical assay aiming at the identification of small-molecule inhibitors of the cyclic guanosine monophosphate-adenosine monophosphate (GMP-AMP) synthase (cGAS). First, we describe the optimization of the method to enable sensitive and accurate determination of enzyme activity and inhibition in miniaturized 1536-well microtiter plate format. Then we show both results from a validation single-concentration screen using a test set of 5500 compounds, and the subsequent concentration-response testing of selected hits in direct comparison with a previously established matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) readout. Finally, we present the development of an in-line OPI cleaning procedure aiming to match the instrument robustness required for large-scale HTS campaigns. Overall, this work points to critical method development parameters and provides guidance for the establishment of integrated ADE-OPI-MS as HTS-compatible technology for early drug discovery.

Analytical quality by design methodology for botanical raw material analysis: a case study of flavonoids in Genkwa Flos.

The present study introduces a systematic approach using analytical quality by design (AQbD) methodology for the development of a qualified liquid chromatographic analytical method, which is a challenge in herbal medicinal products due to the intrinsic complex components of botanical sources. The ultra-high-performance liquid chromatography-photodiode array-mass spectrometry (UHPLC-PDA-MS) technique for 11 flavonoids in Genkwa Flos was utilized through the entire analytical processes, from the risk assessment study to the factor screening test, and finally in method optimization employing central composite design (CCD). In this approach, column temperature and mobile solvent slope were found to be critical method parameters (CMPs) and each of the eleven flavonoid peaks' resolution values were used as critical method attributes (CMAs) through data mining conversion formulas. An optimum chromatographic method in the design space was calculated by mathematical and response surface methodology (RSM). The established chromatographic condition is as follows: acetonitrile and 0.1% formic acid gradient elution (0-13 min, 10-45%; 13-13.5 min, 45-100%; 13.5-14 min, 100-10%; 14-15 min, 10% acetonitrile), column temperature 28℃, detection wavelength 335 nm, and flow rate 0.35 mL/min using C18 (50 × 2.1 mm, 1.7 µm) column. A validation study was also performed successfully for apigenin 7-O-glucuronide, apigenin, and genkwanin. A few important validation results were as follows: linearity over 0.999 coefficient of correlation, detection limit of 2.87-22.41, quantitation limit of 8.70-67.92, relative standard deviation of precision less than 0.22%, and accuracy between 100.13 and 102.49% for apigenin, genkwanin, and apigenin 7-O-glucuronide. In conclusion, the present design-based approach provide a systematic platform that can be effectively applied to ensure pharmaceutically qualified analytical data from complex natural products based botanical drug.

Bioanalysis in the Age of New Drug Modalities.

In the absence of regulatory guidelines for the bioanalysis of new drug modalities, many of which contain multiple functional domains, bioanalytical strategies have been carefully designed to characterize the intact drug and each functional domain in terms of quantity, functionality, biotransformation, and immunogenicity. The present review focuses on the bioanalytical challenges and considerations for RNA-based drugs, bispecific antibodies and multi-domain protein therapeutics, prodrugs, gene and cell therapies, and fusion proteins. Methods ranging from the conventional ligand binding assays and liquid chromatography-mass spectrometry assays to quantitative polymerase chain reaction or flow cytometry often used for oligonucleotides and cell and gene therapies are discussed. Best practices for method selection and validation are proposed as well as a future perspective to address the bioanalytical needs of complex modalities.

Combining Isotope Dilution and Standard Addition-Elemental Analysis in Complex Samples.

A new method combining isotope dilution mass spectrometry (IDMS) and standard addition has been developed to determine the mass fractions w of different elements in complex matrices: (a) silicon in aqueous tetramethylammonium hydroxide (TMAH), (b) sulfur in biodiesel fuel, and (c) iron bound to transferrin in human serum. All measurements were carried out using inductively coupled plasma mass spectrometry (ICP-MS). The method requires the gravimetric preparation of several blends (bi)-each consisting of roughly the same masses (mx,i) of the sample solution (x) and my,i of a spike solution (y) plus different masses (mz,i) of a reference solution (z). Only these masses and the isotope ratios (Rb,i) in the blends and reference and spike solutions have to be measured. The derivation of the underlying equations based on linear regression is presented and compared to a related concept reported by Pagliano and Meija. The uncertainties achievable, e.g., in the case of the Si blank in extremely pure TMAH of urel (w(Si)) = 90% (linear regression method, this work) and urel (w(Si)) = 150% (the method reported by Pagliano and Meija) seem to suggest better applicability of the new method in practical use due to the higher robustness of regression analysis.

Interlaboratory Comparison of Untargeted Mass Spectrometry Data Uncovers Underlying Causes for Variability.

Despite the value of mass spectrometry in modern natural products discovery workflows, it remains very difficult to compare data sets between laboratories. In this study we compared mass spectrometry data for the same sample set from two different laboratories (quadrupole time-of-flight and quadrupole-Orbitrap) and evaluated the similarity between these two data sets in terms of both mass spectrometry features and their ability to describe the chemical composition of the sample set. Somewhat surprisingly, the two data sets, collected with appropriate controls and replication, had very low feature overlap (25.7% of Laboratory A features overlapping 21.8% of Laboratory B features). Our data clearly demonstrate that differences in fragmentation, charge state, and adduct formation in the ionization source are a major underlying cause for these differences. Consistent with other recent literature, these findings challenge the conventional wisdom that electrospray ionization mass spectrometry (ESI-MS) yields a simple one-to-one correspondence between analytes in solution and features in the data set. Importantly, despite low overlap in feature lists, principal component analysis (PCA) generated qualitatively similar PCA plots. Overall, our findings demonstrate that comparing untargeted metabolomics data between laboratories is challenging, but that data sets with low feature overlap can yield the same qualitative description of a sample set using PCA.

Iodine determination in animal feed by inductively coupled plasma mass spectrometry - results of a collaborative study.

A collaborative study was conducted in order to fully validate the performance characteristics and to evaluate the suitability of a method for determination of iodine in animal feed. The method consists of an alkaline extraction in tetramethylammonium hydroxide (TMAH) solution followed by the determination of iodine by inductively coupled plasma-mass spectrometry (ICP-MS). The method was validated for different types of feed and feed materials with a broad concentration range of 0.65-622 mg I/kg. Good agreement was found between the overall mean mass fraction values from the collaborative trial (13.8 ± 1.3 mg I/kg and 0.657 ± 0.228 mg I/kg) and the values previously determined in proficiency tests for two of the test materials (12.65 ± 2.47 mg I/kg and 0.72 ± 0.22 mg I/kg) indicating satisfactory accuracy of the method. Reproducibility standard deviations were between 7.85% and 34.65% and the HorRat values were under the acceptable limit of 2 so the between-laboratory precision was considered acceptable. Based on the statistical evaluation of the results it was concluded that the method is suitable for its intended purpose; it has been accepted as European Standard EN17050:2017 by the European Committee for Standardisation (CEN).

A perspective on the standards describing mass spectrometry-based metabolic phenotyping (metabolomics/metabonomics) studies in publications.

As metabolic phenotyping (metabolomics, metabonomics and also lipidomics) gains in popularity and new investigators enter the field, the need to maintain and improve standards in publication is ever more pressing. In this perspective the requirements for information that should be included in manuscripts published in the Journal of Chromatography B, to ensure that the work is both credible and repeatable, are discussed. These include aspects such as study design, ethics, quality assurance (QA), quality control (QC) and data processing. In addition, aspects such as the level of confidence required for reporting metabolite identification (to a level where they could be subsequently used to develop hypotheses) are discussed.

An approach for calibrating laser-based N2 O isotopic analyzers for soil biogeochemistry research.

RATIONALE: Technological advances have motivated researchers to transition from traditional gas chromatography/isotope ratio mass spectrometry to rapid, high-throughput, laser-based instrumentation for N2 O isotopic research. However, calibrating laser-based instruments to yield accurate and precise isotope ratios has been an ongoing challenge. To streamline the N2 O isotope research pipeline, we developed the calibration protocol for laser-based analyzers described here. While our approach is targeted at laboratory soil incubations, we anticipate that it will be broadly applicable for diverse types of stable isotope research. METHODS: We prepared standards diluted from USGS52 and from a commercial cylinder to develop a calibration curve spanning from 0.3 to 300 ppm N2 O. To calibrate over this broad range, we binned each isotopocule (N2 O, N15 NO, 15 NNO, and NN18 O) into low, medium, and high concentration ranges and then used mathematically similar polynomial functions to calibrate the isotopocules within each concentration range. We also assessed the temporal stability of the instrument and the capacity for our calibration approach to work with isotopically enriched gas samples. RESULTS: Our calibration approach yielded generally accurate and precise data when isotopocules were calibrated in concentration ranges, and the measurements appeared to be temporally stable. For all isotopocules at natural abundance, the residual percentage error was smallest in the medium N2 O range. There was more noise in the corrected isotopomers and isotopologue at natural abundance in samples with the lowest and highest N2 O concentrations. Corrected isotopomer results from isotopically enriched samples were very precise. CONCLUSIONS: Developing our calibration strategy involved learning several key lessons: (1) calibrate isotopocules in distinct concentration ranges, (2) use mathematically similar models to calibrate the isotopocules in each range, (3) calibrated N2 O concentrations and δ values tend to be most accurate and precise in the medium N2 O range, and (4) we encourage users to take advantage of isotopic enrichment to capitalize on laser-based instrument strengths.

Total and Exchangeable Copper Assay Using Inductively Coupled Plasma Mass Spectrometry and Establishment of a Pediatric Reference Interval.

CONTEXT.­: Recently, an exchangeable copper (CuEXC) assay has been suggested as a robust and feasible diagnostic tool for Wilson disease (WD). Although WD is a disorder that requires lifelong treatment and monitoring, few data are currently available regarding the status of copper levels in children. OBJECTIVE.­: To evaluate the performance of copper assays and establish a reference interval for total copper and CuEXC in the pediatric population. DESIGN.­: Serum samples from children aged 1-5 (n = 122), 6-12 (n = 125), and 13-18 years (n = 120) were analyzed. Total copper and CuEXC concentrations were directly measured using inductively coupled plasma mass spectrometry, and relative CuEXC levels were calculated. Total copper reference intervals, CuEXC levels, and relative CuEXC levels were determined based on the 2.5th and 97.5th percentiles of the data with 90% confidence intervals. RESULTS.­: There were significant differences in the median concentrations of total copper and relative CuEXC among the age groups. Reference intervals determined for total copper were 82 to 167, 75 to 139, and 64 to 133 µg/dL for children aged 1 to 5, 6 to 12, and 13 to 18 years, respectively. The reference intervals for CuEXC were 4.29 to 9.79, 4.02 to 9.09, and 3.55 to 8.25 µg/dL for children aged 1 to 5, 6 to 12, and 13 to 18 years, respectively. Among 11 patients with suspected WD, relative CuEXC values were elevated in all 3 diagnosed with WD. CONCLUSIONS.­: The pediatric reference intervals derived in this study are expected to be useful for the diagnosis, differential diagnosis, treatment, and monitoring of pediatric patients with WD.

Optimisation of the use of sliding window deconvolution for comprehensive characterisation of trastuzumab and adalimumab charge variants by native high resolution mass spectrometry.

The biopharmaceutical industry continues to develop mAb-based biotherapeutics in increasing numbers. Due to their complexity, there are several critical quality attributes (CQAs) that need to be measured and controlled to guarantee product safety and efficacy. Charge variant analysis is a widely used method to monitor changes in product quality during the manufacturing process of monoclonal antibodies (mAbs) and, together with a bottom-up peptide centred approach, acts as a key analytical platform to fulfil regulatory requirements. Native MS measures biomolecules under conditions that preserve most aspects of protein tertiary and quaternary structure, enabling direct characterization of large intact proteins such as mAbs. The resulting native mass spectrum of a mAb is characterized by a narrower charge-state envelope that simplifies the spectra and also condenses the ion signals into fewer peaks, increasing the signal-to-noise ratio. Algorithmic spectral deconvolution is needed for routine accurate and rapid molecular weight determination, and consequently, multiple deconvolution algorithms have evolved over the past decade. Here, we demonstrate the utility of the sliding window algorithm as a robust and powerful deconvolution tool for comprehensive characterisation of charge variant analysis data for mAbs. Optimum performance is evaluated by studying the impact of critical software parameters on detection, identification and relative quantitation of protein isoforms. By combining molecular mass and retention time information, it was possible to identify multiple modifications on adalimumab and trastuzumab, both IgG1 mAbs, including lysine truncation, deamidation and succinimide formation, along with the N-glycan distribution of each of the identified charge variants. Sliding window deconvolution also provides a key benefit of low abundant variant detection in a single analysis and the ability to detect co-eluting components with different relative abundances. The studied mAbs demonstrate the algoritms applicability for efficient data processing of both simple and complex mAbs analysed using pH gradient cation exchange chromatography coupled to native mass spectrometry.

GlycoPOST realizes FAIR principles for glycomics mass spectrometry data.

For the reproducibility and sustainability of scientific research, FAIRness (Findable, Accessible, Interoperable and Re-usable), with respect to the release of raw data obtained by researchers, is one of the most important principles underpinning the future of open science. In genomics and transcriptomics, the sharing of raw data from next-generation sequencers is made possible through public repositories. In addition, in proteomics, the deposition of raw data from mass spectrometry (MS) experiments into repositories is becoming standardized. However, a standard repository for such MS data had not yet been established in glycomics. With the increasing number of glycomics MS data, therefore, we have developed GlycoPOST (https://glycopost.glycosmos.org/), a repository for raw MS data generated from glycomics experiments. In just the first year since the release of GlycoPOST, 73 projects have already been registered by researchers around the world, and the number of registered projects is continuously growing, making a significant contribution to the future FAIRness of the glycomics field. GlycoPOST is a free resource to the community and accepts (and will continue to accept in the future) raw data regardless of vendor-specific formats.

Comparison of Two Analytical Methods for Busulfan Therapeutic Drug Monitoring.

BACKGROUND AND OBJECTIVES: Busulfan (Bu) is an old drug, but is still well recommended as an alkylating agent during conditioning therapy, before hematopoietic stem cell transplantation. Although its dose administration is standardized and based on patient weight, therapeutic drug monitoring is required in order to maintain its exposure [as area under the concentration-time curve (AUC) from 0 to infinity AUC0-∞] within a narrow therapeutic range and, if necessary, to adjust the dose with as short a lead time as possible. The aim of the study is to evaluate the agreement (as calculated AUC) between a gold standard analytical method and a new one that is faster and easier. METHODS: We analyzed 221 plasma samples from 37 children (0.25-16 years; 4-62.5 kg) and 11 adults (21-59 years; 45-80 kg), corresponding to 52 AUC values (ng h/mL). The drug exposure was calculated, simultaneously, by two validated analytical methods. The reference method was a high-performance liquid chromatography (HPLC) assay combined with an ultraviolet detector (UV). The test method had a triple quadrupole mass spectrometer (MS) as detector; the clean-up procedures of the samples were different and faster. RESULTS: The agreement between the two methods (reference and test) was evaluated in terms of Bu exposure differences based on Lin's concordance correlation coefficient (CCC) and represented by the Bland-Altman plot. The CCC between the AUC of the two methods was excellent (0.868; 95% CI: 0.802-0.935). The precision of the measures (expressed by Pearson's italic "r") was 0.872, and the accuracy (accounted by the bias correction factor) was 0.996. CONCLUSIONS: We can conclude that the HPLC-MS/MS assay represents a very good alternative to the reference.

Spectral quality overrides software score-A brief tutorial on the analysis of peptide fragmentation data for mass spectrometry laymen.

The use of mass spectrometry has dramatically increased the research pace in the life sciences. The influence of the technique is enormous and its results can have far-reaching consequences such as jail time when applied in forensics. Therefore, analytical chemists trained in proper procedure know that they must validate their experiments. However, those quality measures have not been adopted in a similar manner in the omics technologies even though the stakes are equally high. Reasons are, among others, the segregation of the data generation and data mining functions and an undue belief in software capabilities. In this article, problematic issues such as false or overinterpretation of data are discussed, and assistance is provided for mass spectrometry laymen to evaluate the quality of their results; a quick guide to mass spectral data interpretation of peptide fragmentation experiments, the basis of bottom-up proteomics, is offered. Good science can only be generated in tight collaboration of principal investigator, analytical chemist, and bioinformatician so that the limits and the potential of each method and approach can be responsibly communicated.