MALDI-TOF Mass Spectrometric Detection of SARS-CoV-2 Using Cellulose Sulfate Ester Enrichment and Hot Acid Treatment.
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ABSTRACT:
SUBMITTER: Chen D
PROVIDER: S-EPMC9305670 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Cyanobacteria represent a bacterial phyllum characteristic by the ability to photosynthesize. They are potentially applicable for the production of useful compounds but may also cause poisoning or at least health problems as they can produce cyanotoxins. The introduction of a fast methodology is important not only for fundamental taxonomic purposes, but also for reliable identifications in biological studies. In this work, we have used matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of intact cells to study Chroococcidiopsis strains. A library of the obtained reference mass spectra containing characteristic peptide/protein profiles was examined by software tools to characterize similarities and differences applicable for diagnostics and taxonomy. Both a similarity tree and heat map constructed from the mass spectrometric data proved consistent with 16S rRNA sequencing results. We show as novelty that a binary matrix combining ferulic and sinapinic acids performs well in acquiring reproducible mass spectra of cyanobacteria. Using the matrix solvent, a protein extraction from cells was done. After polyacrylamide gel electrophoresis, the separated protein fractions were in-gel digested and the resulting peptides analyzed by liquid chromatography coupled with tandem mass spectrometry. For the first time, photosystem protein components, phycobilisome proteins, electron transport proteins, nitrogen-metabolism and nucleic acids binding-proteins, cytochromes plus other enzymes and various uncharacterized proteins could be assigned to characteristic peaks in the mass spectrometric profiles and some of them suggested as markers in addition to 30S and 50S ribosomal proteins known from previous studies employing intact cell mass spectrometry of microorganisms.
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Project description:BackgroundOligosaccharidoses, which belong to the lysosomal storage diseases, are inherited metabolic disorders due to the absence or the loss of function of one of the enzymes involved in the catabolic pathway of glycoproteins and indirectly of glycosphingolipids. This enzymatic deficiency typically results in the abnormal accumulation of uncompletely degraded oligosaccharides in the urine. Since the clinical features of many of these disorders are not specific for a single enzyme deficiency, unambiguous screening is critical to limit the number of costly enzyme assays which otherwise must be performed.MethodsHere we provide evidence for the advantages of using a MALDI-TOF/TOF (matrix-assisted laser desorption ionization time-of-flight) mass spectrometric (MS) method for screening oligosaccharidoses. Urine samples from previously diagnosed patients or from unaffected subjects were randomly divided into a training set and a blind testing set. Samples were directly analyzed without prior treatment.ResultsThe characteristic MS and MS/MS molecular profiles obtained allowed us to identify fucosidosis, aspartylglucosaminuria, GM1 gangliosidosis, Sandhoff disease, α-mannosidosis, sialidosis and mucolipidoses type II and III.ConclusionsThis method, which is easily run in less than 30 minutes, is performed in a single step, and is sensitive and specific. Invaluable for clinical chemistry purposes this MALDI-TOF/TOF mass spectrometry procedure is semi-automatizable and suitable for the urinary screening of oligosacharidoses.
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Project description:As severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infections continue, there is a substantial need for cost-effective and large-scale testing that utilizes specimens that can be readily collected from both symptomatic and asymptomatic individuals in various community settings. Although multiple diagnostic methods utilize nasopharyngeal specimens, saliva specimens represent an attractive alternative as they can rapidly and safely be collected from different populations. While saliva has been described as an acceptable clinical matrix for the detection of SARS-CoV-2, evaluations of analytic performance across platforms for this specimen type are limited. Here, we used a novel sensitive RT-PCR/MALDI-TOF mass spectrometry-based assay (Agena MassARRAY®) to detect SARS-CoV-2 in saliva specimens. The platform demonstrated high diagnostic sensitivity and specificity when compared to matched patient upper respiratory specimens. We also evaluated the analytical sensitivity of the platform and determined the limit of detection of the assay to be 1562.5 copies/ml. Furthermore, across the five individual target components of this assay, there was a range in analytic sensitivities for each target with the N2 target being the most sensitive. Overall, this system also demonstrated comparable performance when compared to the detection of SARS-CoV-2 RNA in saliva by the cobas® 6800/8800 SARS-CoV-2 real-time RT-PCR Test (Roche). Together, we demonstrate that saliva represents an appropriate matrix for SARS-CoV-2 detection on the novel Agena system as well as on a conventional real-time RT-PCR assay. We conclude that the MassARRAY® system is a sensitive and reliable platform for SARS-CoV-2 detection in saliva, offering scalable throughput in a large variety of clinical laboratory settings.
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Project description:Chondroitin sulfate proteoglycans (CSPGs) are extracellular matrix components composed of linear glycosaminoglycan (GAG) side chains attached to a core protein. CSPGs play a vital role in neurodevelopment, signal transduction, cellular proliferation and differentiation and tumor metastasis through interaction with growth factors and signaling proteins. These pleiotropic functions of proteoglycans are regulated spatiotemporally by the GAG chains attached to the core protein. There are over 70 chondroitin sulfate-linked proteoglycans reported in cells, cerebrospinal fluid and urine. A core glycan linker of 3-6 monosaccharides attached to specific serine residues can be extended by 20-200 disaccharide repeating units making intact CSPGs very large and impractical to analyze. The current paradigm of CSPG analysis involves digesting the GAG chains by chondroitinase enzymes and analyzing either the protein part, the disaccharide repeats, or both by mass spectrometry. This method, however, provides no information about the site of attachment or the composition of linker oligosaccharides and the degree of sulfation and/or phosphorylation. Further, the analysis by mass spectrometry and subsequent identification of novel CSPGs is hampered by technical challenges in their isolation, less optimal ionization and data analysis. Unknown identity of the linker oligosaccharide also makes it more difficult to identify the glycan composition using database searching approaches. Following chondroitinase digestion of long GAG chains linked to tryptic peptides, we identified intact GAG-linked peptides in clinically relevant samples including plasma, urine and dermal fibroblasts. These intact glycopeptides including their core linker glycans were identified by mass spectrometry using optimized stepped higher energy collision dissociation and electron-transfer/higher energy collision dissociation combined with hybrid database search/de novo glycan composition search. We identified 25 CSPGs including three novel CSPGs that have not been described earlier. Our findings demonstrate the utility of combining enrichment strategies and optimized high-resolution mass spectrometry analysis including alternative fragmentation methods for the characterization of CSPGs.Supplementary informationThe online version contains supplementary material available at 10.1007/s42485-022-00092-3.
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Project description:BACKGROUND:Daratumumab, a therapeutic IgG kappa monoclonal antibody, can cause a false positive interference on electrophoretic assays that are routinely used to monitor patients with monoclonal gammopathies. In this study, we evaluate the ability of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to distinguish daratumumab from disease-related IgG kappa monoclonal proteins (M-protein). METHODS:Waste clinical samples from 31 patients who were receiving daratumumab and had a history of IgG kappa monoclonal gammopathy were collected. Immunoglobulins were purified from serum and analyzed by MALDI-TOF MS. Mass spectra were assessed for the presence of distinct monoclonal proteins. For samples in which only one monoclonal peak was identified near the expected m/z of daratumumab, the Hydrashift 2/4 Daratumumab Assay was used to confirm the presence of an M-protein. RESULTS:Using MALDI-TOF MS, daratumumab could be distinguished from M-proteins in 26 out of 31 samples (84%). Results from 2 samples were inconclusive since the M-protein was not detected by the Hydrashift assay and may also be undetectable by MALDI-TOF MS. Comparatively, daratumumab was distinguishable from M-proteins in 14 out of 31 samples (45%) by immunofixation. CONCLUSIONS:MALDI-TOF MS offers greater specificity compared to immunofixation for distinguishing daratumumab from M-proteins.
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