The detection of viral RNA by polymerase chain reaction (PCR) is currently the main diagnostic tool for COVID-19 ( Eurosurveillance 2019, 25 (3), 1 ). The PCR-based test, however, shows limited ...sensitivity, especially in the early and late stages of disease development ( Nature 2020, 581, 465−469 ; J. Formosan Med. Assoc. 2020, 119 (6) 1123 ), and is relatively time-consuming. Fast and reliable complementary methods for detecting the viral infection would be of help in the current pandemic conditions. Mass spectrometry is one of such possibilities. We have developed a mass-spectrometry-based method for the detection of the SARS CoV-2 virus in nasopharynx epithelial swabs based on the detection of the viral nucleocapsid N protein. Our approach shows confident identification of the N protein in patient samples, even those with the lowest viral loads, and a much simpler preparation procedure. Our main protocol consists of virus inactivation by heating and the addition of isopropanol and tryptic digestion of the proteins sedimented from the swabs followed by MS analysis. A set of unique peptides, produced as a result of proteolysis of the nucleocapsid phosphoprotein of SARS-CoV-2, is detected. The obtained results can further be used to create fast parallel mass-spectrometric approaches for the detection of the virus in the nasopharyngeal mucosa, saliva, sputum and other physiological fluids.
The goal of this study was to establish a relationship between the optical properties of soil dissolved organic matter (DOM) and acidic functions carried out by its individual constituents. We ...obtained 12 fractions of DOM samples using sequential solid phase extraction on nonionic sorbent at steadily lowered pH values: 7, 5, 3, 2, which correspond to low bounds of pK a values of phenols, aliphatic, and aromatic carboxylic acids, and ketoacids. The structural studies were conducted with the use of NMR and selective deuteromethylation of isolated fractions coupled to ultrahigh resolution mass spectrometry. First, a gradual shift of molecular compositions was observed from reduced components to aromatic oxidized compounds isolated at pH 7 and 2, respectively. Changes in molecular compositions were accompanied by a red shift of fluorescence spectra. Further application of deuteromethylation enabled us to distinguish DOM constituents with different amounts of carboxylic groups. Moreover, identification of structural isomers in a single DOM sample was achieved. Statistical analysis revealed that red shift of fluorescence is facilitated by the increase of a contribution of aromatic poly(carboxylic acid)s with high conjugation lengths. Additionally, analysis of the labeled fractionated permafrost thaw DOM directly showed carboxyl-rich alicyclic molecules, while the same components from lower-latitude DOM were assigned to lignin-like species.
Direct comparison of high-resolution mass spectrometry (HRMS) data acquired with different instrumentation or parameters remains problematic as the derived lists of molecular species via HRMS, even ...for the same sample, appear distinct. This inconsistency is caused by inherent inaccuracies associated with instrumental limitations and sample conditions. Hence, experimental data may not reflect a corresponding sample. We propose a method that classifies HRMS data based on the differences in the number of elements between each pair of molecular formulae within the formulae list to preserve the essence of the given sample. The novel metric, formulae difference chains expected length (FDCEL), allowed for comparing and classifying samples measured by different instruments. We also demonstrate a web application and a prototype for a uniform database for HRMS data serving as a benchmark for future biogeochemical and environmental applications. FDCEL metric was successfully employed for both spectrum quality control and examination of samples of various nature.
LC–MS is a key technique for the identification of small molecules in complex samples. Accurate mass, retention time, and fragmentation spectra from LC–MS experiments are compared to reference values ...for pure chemical standards. However, this information is often unavailable or insufficient, leading to an assignment to a list of candidates instead of a single hit; therefore, additional features are desired to filter candidates. One such promising feature is the number of specific functional groups of a molecule that can be counted via derivatization or isotope-exchange techniques. Hydrogen/deuterium exchange (HDX) is the most widespread implementation of isotope exchange for mass spectrometry, while oxygen 16O/18O exchange is not applied as frequently as HDX. Nevertheless, it is known that some functional groups may be selectively exchanged in 18O enriched media. Here, we propose an implementation of 16O/18O isotope exchange to highlight various functional groups. We evaluated the possibility of using the number of exchanged oxygen atoms as a descriptor to filter database candidates in untargeted LC–MS-based workflows. It was shown that 16O/18O exchange provides 62% (median, n = 45) search space reduction for a panel of drug molecules. Additionally, it was demonstrated that studying the fragmentation spectra after 16O/18O can aid in eliminating false positives and, in some cases, help to annotate fragments formed with water traces in the collisional cell.
Mass spectrometry (MS)-based quantitative proteomic methods have become some of the major tools for protein biomarker discovery and validation. The recently developed parallel reaction ...monitoring-parallel accumulation–serial fragmentation (prm-PASEF) approach on a Bruker timsTOF Pro mass spectrometer allows the addition of ion mobility as a new dimension to LC-MS-based proteomics and increases proteome coverage at a reduced analysis time. In this study, a prm-PASEF approach was used for the multiplexed absolute quantitation of proteins in human plasma using isotope-labeled peptide standards for 125 plasma proteins, over a broad (104–106) dynamic range. Optimization of LC and MS parameters, such as accumulation time and collision energy, resulted in improved sensitivity for more than half of the targets (73 out of 125 peptides) by increasing the signal-to-noise ratio by a factor of up to 10. Overall, 41 peptides showed up to a 2-fold increase in sensitivity, 25 peptides showed up to a 5-fold increase in sensitivity, and 7 peptides showed up to a 10-fold increase in sensitivity. Implementation of the prm-PASEF method allowed absolute protein quantitation (down to 1.13 fmol) in human plasma samples. A comparison of the concentration values of plasma proteins determined by MRM on a QTRAP instrument and by prm-PASEF on a timsTOF Pro revealed an excellent correlation (R 2 = 0.97) with a slope of close to 1 (0.99), demonstrating that prm-PASEF is well suited for “absolute” quantitative proteomics.
Natural organic matter (NOM) components measured with ultrahigh-resolution mass spectrometry (UHRMS) are often assessed by molecular formula-based indices, particularly related to their aromaticity, ...which are further used as proxies to explain biogeochemical reactivity. An aromaticity index (AI) is calculated mostly with respect to carboxylic groups abundant in NOM. Here, we propose a new constrained AI
based on the measured distribution of carboxylic groups among individual NOM components obtained by deuteromethylation and UHRMS. Applied to samples from diverse sources (coal, marine, peat, permafrost, blackwater river, and soil), the method revealed that the most probable number of carboxylic groups was two, which enabled to set a reference point
= 2 for carboxyl-accounted AI
calculation. The examination of the proposed AI
showed the smallest deviation to the experimentally determined index for all NOM samples under study as well as for individual natural compounds obtained from the Coconut database. In particular, AI
performed better than AI
for all compound classes in which aromatic moieties are expected: aromatics, condensed aromatics, and unsaturated compounds. Therefore, AI
referenced with two carboxyl groups is preferred over conventional AI and AI
for biogeochemical studies where the aromaticity of compounds is important to understand the transformations and fate of NOM compounds.
Primary focal segmental glomerulosclerosis (FSGS), along with minimal change disease (MCD), are diseases with primary podocyte damage that are clinically manifested by the nephrotic syndrome. The ...pathogenesis of these podocytopathies is still unknown, and therefore, the search for biomarkers of these diseases is ongoing. Our aim was to determine of the proteomic profile of urine from patients with FSGS and MCD. Patients with a confirmed diagnosis of FSGS (n = 30) and MCD (n = 9) were recruited for the study. For a comprehensive assessment of the severity of FSGS a special index was introduced, which was calculated as follows: the first score was assigned depending on the level of eGFR, the second score—depending on the proteinuria level, the third score—resistance to steroid therapy. Patients with the sum of these scores of less than 3 were included in group 1, with 3 or more—in group 2. The urinary proteome was analyzed using liquid chromatography/mass spectrometry. The proteome profiles of patients with severe progressive FSGS from group 2, mild FSGS from group 1 and MCD were compared. Results of the label free analysis were validated using targeted LC-MS based on multiple reaction monitoring (MRM) with stable isotope labelled peptide standards (SIS) available for 47 of the 76 proteins identified as differentiating between at least one pair of groups. Quantitative MRM SIS validation measurements for these 47 proteins revealed 22 proteins with significant differences between at least one of the two group pairs and 14 proteins were validated for both comparisons. In addition, all of the 22 proteins validated by MRM SIS analysis showed the same direction of change as at the discovery stage with label-free LC-MS analysis, i.e., up or down regulation in MCD and FSGS1 against FSGS2. Patients from the FSGS group 2 showed a significantly different profile from both FSGS group 1 and MCD. Among the 47 significantly differentiating proteins, the most significant were apolipoprotein A-IV, hemopexin, vitronectin, gelsolin, components of the complement system (C4b, factors B and I), retinol- and vitamin D-binding proteins. Patients with mild form of FSGS and MCD showed lower levels of Cystatin C, gelsolin and complement factor I.
Chronic kidney disease (CKD) is a non-specific type of kidney disease that causes a gradual decline in kidney function (from months to years). CKD is a significant risk factor for death, ...cardiovascular disease, and end-stage renal disease. CKDs of different origins may have the same clinical and laboratory manifestations but different progression rates, which requires early diagnosis to determine. This review focuses on protein/peptide biomarkers of the leading causes of CKD: diabetic nephropathy, IgA nephropathy, lupus nephritis, focal segmental glomerulosclerosis, and membranous nephropathy. Mass spectrometry (MS) approaches provided the most information about urinary peptide and protein contents in different nephropathies. New analytical approaches allow urinary proteomic-peptide profiles to be used as early non-invasive diagnostic tools for specific morphological forms of kidney disease and may become a safe alternative to renal biopsy. MS studies of the key pathogenetic mechanisms of renal disease progression may also contribute to developing new approaches for targeted therapy.
In land-based recirculating aquaculture systems (RAS), the accumulation of dissolved organic matter (DOM) can have detrimental effects on water quality impacting the system performance, microbial ...community, and consequently fish health and welfare. Ozone is used in the RAS water treatment process to improve water quality and remove DOM. However, little is known about the molecular composition of DOM in RAS and its transformation when exposed to ozone. In this study, we performed a detailed molecular characterization of DOM in RAS and explored its transformation induced by ozonation of RAS waters. Ultra-high resolution (UHR) Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS) was used to characterize the DOM matrix of RAS waters (pump-sump and tanks) and to evaluate its transformation by ozonation. The analysis of DOM extracted from makeup water and feed samples allowed for the determination of DOM sources in RAS prior to ozonation. The CHO and unsaturated group of compounds were the most abundant class found in water samples. On the contrary, the DOM from feed samples was unique and consisted mainly of CHO, CHON and unsaturated group of compounds. After the ozonation of RAS waters, humic-like and unsaturated compounds positive oxygen subtracted double bond equivalent per carbon (DBE–O)/C) were decomposed, particularly the CHO-DOM that contained fewer -CH2- features. Fulvic-like compounds and several hundred saturated compounds negative (DBE–O)/C) were formed post ozonation, particularly the CHON and CHONS group of compounds that were associated with fish diets, makeup waters and transformation products from the ozonation of the RAS waters. This study showed that the high accuracy of the ultra-high resolution FTICR MS can be applied to characterize and monitor the changes of DOM at a molecular level in RAS waters. To our knowledge, this is the first study where FTICR MS was incorporated for the characterization of DOM and its sources in RAS.
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•First characterization of DOM in recirculating aquaculture systems (RAS) by FTICR MS.•Makeup, pump-sump and tank water samples were rich in CHO and unsaturated compounds prior to ozonation.•Humic-like and unsaturated CHO group of compounds, particularly the -CH2- homologs of CHO-DOM, were decomposed by ozone.•Post ozonation of RAS waters, new fulvic-like and saturated compounds were formed.•CHON and CHONS compounds were produced during ozonation at high abundance.
The recent surge of coronavirus disease 2019 (COVID-19) hospitalizations severely challenges healthcare systems around the globe and has increased the demand for reliable tests predictive of disease ...severity and mortality. Using multiplexed targeted mass spectrometry assays on a robust triple quadrupole MS setup which is available in many clinical laboratories, we determined the precise concentrations of hundreds of proteins and metabolites in plasma from hospitalized COVID-19 patients. We observed a clear distinction between COVID-19 patients and controls and, strikingly, a significant difference between survivors and nonsurvivors. With increasing length of hospitalization, the survivors’ samples showed a trend toward normal concentrations, indicating a potential sensitive readout of treatment success. Building a machine learning multi-omic model that considers the concentrations of 10 proteins and five metabolites, we could predict patient survival with 92% accuracy (area under the receiver operating characteristic curve: 0.97) on the day of hospitalization. Hence, our standardized assays represent a unique opportunity for the early stratification of hospitalized COVID-19 patients.
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•Plasma samples were collected from 120 COVID-19 patients 0, 2, and 7 days after admission to the ICU.•Hundreds of proteins and metabolites were quantitated in these patient plasma samples.•Significant differences were found between COVID-19 survivors and nonsurvivors.•Day-0 expression levels of 10 proteins + 5 metabolites predicted 92% accurate survival.•Stratification of newly admitted COVID-19 patients by chance of survival was achievable.
During times of hospital admission overload, triage may be required to maximize the number of survivors. Mass spectometry–based proteomic and metabolomic analysis of COVID patients’ blood, collected at the time of admission to the ICU, enabled a prediction of survival versus nonsurvival with 92% accuracy. These analyses, which can be performed on widely available mass spectrometers, have the potential to assist physicians with these difficult decisions.
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