A TRUSTED targeted mass spectrometry assay for pan-herpesvirus protein detection.
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ABSTRACT:
SUBMITTER: Kennedy MA
PROVIDER: S-EPMC9245836 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BackgroundThe 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.MethodsHere, 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.FindingsThe 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.InterpretationOur 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.FundingThis 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.
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Project description:Targeted proteomic mass spectrometry is emerging as a salient clinical diagnostic tool to track protein biomarkers. However, its strong analytical properties have not been exploited in the diagnosis and typing of flaviviruses. Here, we report the development of a sensitive and specific single-shot robust assay for flavivirus typing and diagnosis using targeted mass spectrometry technology. Our flavivirus parallel reaction monitoring assay (fvPRM) has the ability to track secreted flaviviral nonstructural protein 1 (NS1) over a broad diagnostic and typing window with high sensitivity, specificity, extendibility, and multiplexing capability. These features, pivotal and pertinent to efficient response toward flavivirus outbreaks, including newly emerging flavivirus strains, circumvent the limitations of current diagnostic assays. fvPRM thus carries high potential in positioning itself as a forerunner in delivering early and accurate diagnosis for disease management.
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Project description:We described a targeted mass spectrometry assay based on selected reaction monitoring (SRM) for five apolipoproteins (apoA1, apoB, apoJ, apoD, and apoE) in plasma lipoproteins isolated by anion exchange fast protein liquid chromatography using only 100 μL of plasma. We performed analytical characterization of the SRM assay and evaluated reproducibility of the entire workflow. The limit of detections for apoA1, apoB, apoD, apoJ, and apoE were 0.6, 4.6, 0.8, 1.2, and 0.7 nM, respectively; the limit of quantifications was 8.3 nM for all peptides except apoD (4.2 nM). The SRM assay was linear from 0.4 to 1667 nM. The intra-day and inter-day and total repeatability (CV%) of the assay ranged from 2.2% to 21.7% for all five peptides. The intra-day and inter-day and total reproducibility of the entire workflow ranged from 12.2% to 43.9% for all five peptides in fractionated high-density lipoprotein, low-density lipoprotein, and IDL. In the future, we will apply this workflow to investigate the association of fractionated plasma lipoproteins with amyloid deposition and cognitive changes in the context of Alzheimer's disease.
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Project description:Differential ion mobility spectrometry (DIMS) can be used as a filter to remove undesired background ions from reaching the mass spectrometer. The ability to use DIMS as a filter for known analytes makes DIMS coupled to tandem mass spectrometry (DIMS-MS/MS) a promising technique for the detection of cancer antigens that can be predicted by computational algorithms. In experiments using DIMS-MS/MS that were performed without the use of high-performance liquid chromatography (HPLC), a predicted model antigen, GLR (FLSSANEHL), was detected at a concentration of 10 pM (20 amol) in a mixture containing 94 competing model peptide antigens, each at a concentration of 1 ?M. Without DIMS filtering, the GLR peptide was undetectable in the mixture even at 100 nM. Again, without using HPLC, DIMS-MS/MS was used to detect 2 of 3 previously characterized antigens produced by the leukemia cell line U937.A2. Because of its sensitivity, a targeted DIMS-MS/MS methodology can likely be used to probe for predicted cancer antigens from cancer cell lines as well as human tumor samples.
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Project description:IntroductionColorectal cancer (CRC) testing programs reduce mortality; however, approximately 40% of the recommended population who should undergo CRC testing does not. Early colon cancer detection in patient populations ineligible for testing, such as the elderly or those with significant comorbidities, could have clinical benefit. Despite many attempts to identify individual protein markers of this disease, little progress has been made. Targeted mass spectrometry, using multiple reaction monitoring (MRM) technology, enables the simultaneous assessment of groups of candidates for improved detection performance.Materials and methodsA multiplex assay was developed for 187 candidate marker proteins, using 337 peptides monitored through 674 simultaneously measured MRM transitions in a 30-minute liquid chromatography-mass spectrometry analysis of immunodepleted blood plasma. To evaluate the combined candidate marker performance, the present study used 274 individual patient blood plasma samples, 137 with biopsy-confirmed colorectal cancer and 137 age- and gender-matched controls. Using 2 well-matched platforms running 5 days each week, all 274 samples were analyzed in 52 days.ResultsUsing one half of the data as a discovery set (69 disease cases and 69 control cases), the elastic net feature selection and random forest classifier assembly were used in cross-validation to identify a 15-transition classifier. The mean training receiver operating characteristic area under the curve was 0.82. After final classifier assembly using the entire discovery set, the 136-sample (68 disease cases and 68 control cases) validation set was evaluated. The validation area under the curve was 0.91. At the point of maximum accuracy (84%), the sensitivity was 87% and the specificity was 81%.ConclusionThese results have demonstrated the ability of simultaneous assessment of candidate marker proteins using high-multiplex, targeted-mass spectrometry to identify a subset group of CRC markers with significant and meaningful performance.
| S-EPMC8961700 | biostudies-literature
Project description:Human Norwalk viruses (HuNoVs), the most common etiological agents of acute gastroenteritis, are genetically diverse RNA viruses that frequently cause mass food poisoning internationally. Although nucleic acid detection methods, such as reverse transcription-quantitative polymerase chain reaction (RT-qPCR), are the gold standard for the diagnosis of norovirus infection, alternative methods are needed for the specific and sensitive viral protein detection for rapid diagnosis and surveillance. In this study, we developed a robust and high-throughput targeted proteomic assay workflow to directly detect the VP1 major capsid protein of HuNoVs. A parallel reaction monitoring (PRM) assay using a high-resolution mass spectrometer was used to detect representative peptides derived from VP1 in six different HuNoV genotypes. An optimized protocol using synthesized heavy isotope-labeled peptides as internal standards was also used to simultaneously genotype and quantify the VP1 protein in human stool specimens. This method is expected to become a new tool for studying the molecular epidemiology of HuNoV and to shed new light on targeted proteomics in clinical practice.
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Project description:The genus Orthopoxvirus contains several species of related viruses, including the causative agent of smallpox (Variola virus). In addition to smallpox, several other members of the genus are capable of causing human infection, including monkeypox, cowpox, and other zoonotic rodent-borne poxviruses. Therefore, a single assay that can accurately identify all orthopoxviruses could provide a valuable tool for rapid broad orthopovirus identification. We have developed a pan-Orthopoxvirus assay for identification of all members of the genus based on four PCR reactions targeting Orthopoxvirus DNA and RNA helicase and polymerase genes. The amplicons are detected using electrospray ionization-mass spectrometry (PCR/ESI-MS) on the Ibis T5000 system. We demonstrate that the assay can detect and identify a diverse collection of orthopoxviruses, provide sub-species information and characterize viruses from the blood of rabbitpox infected rabbits. The assay is sensitive at the stochastic limit of PCR and detected virus in blood containing approximately six plaque-forming units per milliliter from a rabbitpox virus-infected rabbit.
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Project description:BackgroundMounting evidence demonstrates a causal role for S100 proteins in tumourigenesis and several S100 isoforms have shown utility as biomarkers of several types of cancer. The S100 family is comprised of 21 small isoforms, many of them implicated in important cellular functions such as proliferation, motility and survival. Furthermore, in vivo experiments have proven the role of S100 proteins in tumour growth and disease progression, while other studies have shown their prognostic value and involvement in resistance to chemotherapy drugs. Taken together, all these aspects highlight S100 proteins as potential therapeutic targets and as a promising panel of cancer biomarkers. In this work, we have developed a mass spectrometry (MS)-based method for the multiplexed and specific analysis of the entire S100 protein family in tumour tissues and have applied it to investigate the expression of S100 isoforms in the context of thyroid cancer, the main endocrine malignancy.MethodsSelected Reaction Monitoring (SRM)-MS and stable isotope labelling/label-free analysis were employed to investigate the expression of the 21 S100 protein isoforms in thyroid tissue samples. Specimens included 9 normal thyroid tissues and 27 tumour tissues consisting of 9 follicular adenomas (FA), 8 follicular carcinomas (FTC) and 10 papillary carcinomas (PTC).ResultsThe multiplexed and targeted mass spectrometry method led to the detection of eleven S100 protein isoforms across all tissues. Label- and label-free analyses showed the same significant differences and results were confirmed by western blot. S100A6, S100A11 and its putative interaction partner annexin A1 showed the highest overexpression in PTC compared to normal thyroid. S100A13 was also elevated in PTC. Reduced S100A4 expression was observed in FA compared to all other tissues. FA and FTC showed reduction of S100A10 and annexin A2 expression.ConclusionsTargeted mass spectrometry allows the multiplexed and specific analysis of S100 protein isoforms in tumour tissue specimens. It revealed S100A13 as a novel candidate PTC biomarker. Results show that S100A6, S100A11 and Annexin A1 could help discriminate follicular and papillary tumours. The diagnostic and functional significance of S100A4 and S100A10 reduction in follicular tumours requires further investigation.
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Project description:Rising population density and global mobility are among the reasons why pathogens such as SARS-CoV-2, the virus that causes COVID-19, spread so rapidly across the globe. The policy response to such pandemics will always have to include accurate monitoring of the spread, as this provides one of the few alternatives to total lockdown. However, COVID-19 diagnosis is currently performed almost exclusively by reverse transcription polymerase chain reaction (RT-PCR). Although this is efficient, automatable, and acceptably cheap, reliance on one type of technology comes with serious caveats, as illustrated by recurring reagent and test shortages. We therefore developed an alternative diagnostic test that detects proteolytically digested SARS-CoV-2 proteins using mass spectrometry (MS). We established the Cov-MS consortium, consisting of 15 academic laboratories and several industrial partners to increase applicability, accessibility, sensitivity, and robustness of this kind of SARS-CoV-2 detection. This, in turn, gave rise to the Cov-MS Digital Incubator that allows other laboratories to join the effort, navigate, and share their optimizations and translate the assay into their clinic. As this test relies on viral proteins instead of RNA, it provides an orthogonal and complementary approach to RT-PCR using other reagents that are relatively inexpensive and widely available, as well as orthogonally skilled personnel and different instruments. Data are available via ProteomeXchange with identifier PXD022550.
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