A high throughput immuno-affinity mass spectrometry method for detection and quantitation of SARS-CoV-2 nucleoprotein in human saliva and its comparison with RT-PCR, RT-LAMP, and lateral flow rapid antigen test.

OBJECTIVES: Many reverse transcription polymerase chain reaction (RT-PCR) methods exist that can detect SARS-CoV-2 RNA in different matrices. RT-PCR is highly sensitive, although viral RNA may be detected long after active infection has taken place. SARS-CoV-2 proteins have shorter detection windows hence their detection might be more meaningful. Given salivary droplets represent a main source of transmission, we explored the detection of viral RNA and protein using four different detection platforms including SISCAPA peptide immunoaffinity liquid chromatography-mass spectrometry (SISCAPA-LC-MS) using polyclonal capture antibodies. METHODS: The SISCAPA-LC MS method was compared to RT-PCR, RT-loop-mediated isothermal amplification (RT-LAMP), and a lateral flow rapid antigen test (RAT) for the detection of virus material in the drool saliva of 102 patients hospitalised after infection with SARS-CoV-2. Cycle thresholds (Ct) of RT-PCR (E gene) were compared to RT-LAMP time-to-positive (TTP) (NE and Orf1a genes), RAT optical densitometry measurements (test line/control line ratio) and to SISCAPA-LC-MS for measurements of viral protein. RESULTS: SISCAPA-LC-MS showed low sensitivity (37.7 %) but high specificity (89.8 %). RAT showed lower sensitivity (24.5 %) and high specificity (100 %). RT-LAMP had high sensitivity (83.0 %) and specificity (100.0 %). At high initial viral RNA loads (<20 Ct), results obtained using SISCAPA-LC-MS correlated with RT-PCR (R2 0.57, p-value 0.002). CONCLUSIONS: Detection of SARS-CoV-2 nucleoprotein in saliva was less frequent than the detection of viral RNA. The SISCAPA-LC-MS method allowed processing of multiple samples in <150 min and was scalable, enabling high throughput.

Virome-wide detection of natural infection events and the associated antibody dynamics using longitudinal highly-multiplexed serology.

Current methods for detecting infections either require a sample collected from an actively infected site, are limited in the number of agents they can query, and/or yield no information on the immune response. Here we present an approach that uses temporally coordinated changes in highly-multiplexed antibody measurements from longitudinal blood samples to monitor infection events at sub-species resolution across the human virome. In a longitudinally-sampled cohort of South African adolescents representing >100 person-years, we identify >650 events across 48 virus species and observe strong epidemic effects, including high-incidence waves of Aichivirus A and the D68 subtype of Enterovirus D earlier than their widespread circulation was appreciated. In separate cohorts of adults who were sampled at higher frequency using self-collected dried blood spots, we show that such events temporally correlate with symptoms and transient inflammatory biomarker elevations, and observe the responding antibodies to persist for periods ranging from ≤1 week to >5 years. Our approach generates a rich view of viral/host dynamics, supporting novel studies in immunology and epidemiology.

Operation Moonshot: rapid translation of a SARS-CoV-2 targeted peptide immunoaffinity liquid chromatography-tandem mass spectrometry test from research into routine clinical use.

OBJECTIVES: During 2020, the UK's Department of Health and Social Care (DHSC) established the Moonshot programme to fund various diagnostic approaches for the detection of SARS-CoV-2, the pathogen behind the COVID-19 pandemic. Mass spectrometry was one of the technologies proposed to increase testing capacity. METHODS: Moonshot funded a multi-phase development programme, bringing together experts from academia, industry and the NHS to develop a state-of-the-art targeted protein assay utilising enrichment and liquid chromatography tandem mass spectrometry (LC-MS/MS) to capture and detect low levels of tryptic peptides derived from SARS-CoV-2 virus. The assay relies on detection of target peptides, ADETQALPQRK (ADE) and AYNVTQAFGR (AYN), derived from the nucleocapsid protein of SARS-CoV-2, measurement of which allowed the specific, sensitive, and robust detection of the virus from nasopharyngeal (NP) swabs. The diagnostic sensitivity and specificity of LC-MS/MS was compared with reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) via a prospective study. RESULTS: Analysis of NP swabs (n=361) with a median RT-qPCR quantification cycle (Cq) of 27 (range 16.7-39.1) demonstrated diagnostic sensitivity of 92.4% (87.4-95.5), specificity of 97.4% (94.0-98.9) and near total concordance with RT-qPCR (Cohen's Kappa 0.90). Excluding Cq>32 samples, sensitivity was 97.9% (94.1-99.3), specificity 97.4% (94.0-98.9) and Cohen's Kappa 0.95. CONCLUSIONS: This unique collaboration between academia, industry and the NHS enabled development, translation, and validation of a SARS-CoV-2 method in NP swabs to be achieved in 5 months. This pilot provides a model and pipeline for future accelerated development and implementation of LC-MS/MS protein/peptide assays into the routine clinical laboratory.

Cov2MS: An Automated and Quantitative Matrix-Independent Assay for Mass Spectrometric Measurement of SARS-CoV-2 Nucleocapsid Protein.

The pandemic readiness toolbox needs to be extended, targeting different biomolecules, using orthogonal experimental set-ups. Here, we build on our Cov-MS effort using LC-MS, adding SISCAPA technology to enrich proteotypic peptides of the SARS-CoV-2 nucleocapsid (N) protein from trypsin-digested patient samples. The Cov2MS assay is compatible with most matrices including nasopharyngeal swabs, saliva, and plasma and has increased sensitivity into the attomole range, a 1000-fold improvement compared to direct detection in a matrix. A strong positive correlation was observed with qPCR detection beyond a quantification cycle of 30-31, the level where no live virus can be cultured. The automatable sample preparation and reduced LC dependency allow analysis of up to 500 samples per day per instrument. Importantly, peptide enrichment allows detection of the N protein in pooled samples without sensitivity loss. Easily multiplexed, we detect variants and propose targets for Influenza A and B detection. Thus, the Cov2MS assay can be adapted to test for many different pathogens in pooled samples, providing longitudinal epidemiological monitoring of large numbers of pathogens within a population as an early warning system.

Rapid and sensitive detection of SARS-CoV-2 infection using quantitative peptide enrichment LC-MS analysis.

Reliable, robust, large-scale molecular testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is essential for monitoring the ongoing coronavirus disease 2019 (COVID-19) pandemic. We have developed a scalable analytical approach to detect viral proteins based on peptide immuno-affinity enrichment combined with liquid chromatography-mass spectrometry (LC-MS). This is a multiplexed strategy, based on targeted proteomics analysis and read-out by LC-MS, capable of precisely quantifying and confirming the presence of SARS-CoV-2 in phosphate-buffered saline (PBS) swab media from combined throat/nasopharynx/saliva samples. The results reveal that the levels of SARS-CoV-2 measured by LC-MS correlate well with their correspondingreal-time polymerase chain reaction (RT-PCR) read-out (r = 0.79). The analytical workflow shows similar turnaround times as regular RT-PCR instrumentation with a quantitative read-out of viral proteins corresponding to cycle thresholds (Ct) equivalents ranging from 21 to 34. Using RT-PCR as a reference, we demonstrate that the LC-MS-based method has 100% negative percent agreement (estimated specificity) and 95% positive percent agreement (estimated sensitivity) when analyzing clinical samples collected from asymptomatic individuals with a Ct within the limit of detection of the mass spectrometer (Ct ≤ 30). These results suggest that a scalable analytical method based on LC-MS has a place in future pandemic preparedness centers to complement current virus detection technologies.

Precision multiparameter tracking of inflammation on timescales of hours to years using serial dried blood spots.

Aim: High-frequency longitudinal tracking of inflammation using dried blood microsamples provides a new window for personalized monitoring of infections, chronic inflammatory disease and clinical trials of anti-inflammatory drugs. Results/methodology: Using 1662 dried blood spot samples collected by 16 subjects over periods of weeks to years, we studied the behavior of 12 acute phase response and related proteins in inflammation events correlated with infection, vaccination, surgery, intense exercise and Crohn's disease. Proteins were measured using SISCAPA mass spectrometry and normalized to constant plasma volume using low-variance proteins, generating high precision within-person biomarker trajectories with well-characterized personal baselines. Discussion/conclusion: The results shed new light on the dynamic regulation of APR responses, offering a new approach to visualization of multidimensional inflammation trajectories.

Development of an automated, interference-free, 2D-LC-MS/MS assay for quantification of a therapeutic mAb in human sera.

AIM: Hybrid LC-MS/MS assays are increasingly used to quantitate proteins in biological matrices. These assays involve analyte enrichment at the protein level. Although suitability has been demonstrated, they are limited by the lack of appropriate affinity reagents and may suffer from interferences caused by binding proteins or antibodies. RESULTS: An online stable isotope standards and capture by anti-peptide antibodies assay was developed, which involves tryptic digestion of a therapeutic monoclonal antibody in human serum to destroy interfering proteins followed by enrichment using high affinity peptide antibodies. The assay was validated and compared with a standard ligand-binding assay currently used for quantification. CONCLUSION: The data show that the stable isotope standards and capture by anti-peptide antibodies-2D-LC-MS/MS assay can be used as an alternative method for measurement of monoclonal antibodies in clinical samples.

Multiplexed longitudinal measurement of protein biomarkers in DBS using an automated SISCAPA workflow.

BACKGROUND: The use of DBS for quantitative protein biomarker measurement has been hindered by issues associated with blood hematocrit variations and lack of detection sensitivity, particularly when multiple biomarkers are measured. MATERIALS & METHODS: An automated, multiplexed SISCAPA analysis was used to normalize blood volume variations in DBS and quantify proteins of varying abundance in longitudinal specimens. CONCLUSION: The results showed that after normalizing the spot-to-spot hematocrit variations, peptide surrogates of protein biomarkers could be accurately quantitated in DBS. This allowed the establishment of baselines for a variety of biomarkers in multiple individuals and enabled detection of changes over time, thus offering an effective solution for longitudinal personal monitoring of biomarkers relevant in health and disease.

High precision quantification of human plasma proteins using the automated SISCAPA Immuno-MS workflow.

Efficient robotic workflows for trypsin digestion of human plasma and subsequent antibody-mediated peptide enrichment (the SISCAPA method) were developed with the goal of improving assay precision and throughput for multiplexed protein biomarker quantification. First, an 'addition only' tryptic digestion protocol was simplified from classical methods, eliminating the need for sample cleanup, while improving reproducibility, scalability and cost. Second, methods were developed to allow multiplexed enrichment and quantification of peptide surrogates of protein biomarkers representing a very broad range of concentrations and widely different molecular masses in human plasma. The total workflow coefficients of variation (including the 3 sequential steps of digestion, SISCAPA peptide enrichment and mass spectrometric analysis) for 5 proteotypic peptides measured in 6 replicates of each of 6 different samples repeated over 6 days averaged 3.4% within-run and 4.3% across all runs. An experiment to identify sources of variation in the workflow demonstrated that MRM measurement and tryptic digestion steps each had average CVs of ∼2.7%. Because of the high purity of the peptide analytes enriched by antibody capture, the liquid chromatography step is minimized and in some cases eliminated altogether, enabling throughput levels consistent with requirements of large biomarker and clinical studies.

Quantification of serum apolipoproteins A-I and B-100 in clinical samples using an automated SISCAPA-MALDI-TOF-MS workflow.

A fully automated workflow was developed and validated for simultaneous quantification of the cardiovascular disease risk markers apolipoproteins A-I (apoA-I) and B-100 (apoB-100) in clinical sera. By coupling of stable-isotope standards and capture by anti-peptide antibodies (SISCAPA) for enrichment of proteotypic peptides from serum digests to matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS detection, the standardized platform enabled rapid, liquid chromatography-free quantification at a relatively high throughput of 96 samples in 12h. The average imprecision in normo- and triglyceridemic serum pools was 3.8% for apoA-I and 4.2% for apoB-100 (4 replicates over 5 days). If stored properly, the MALDI target containing enriched apoA-1 and apoB-100 peptides could be re-analyzed without any effect on bias or imprecision for at least 7 days after initial analysis. Validation of the workflow revealed excellent linearity for daily calibration with external, serum-based calibrators (R(2) of 0.984 for apoA-I and 0.976 for apoB-100 as average over five days), and absence of matrix effects or interference from triglycerides, protein content, hemolysates, or bilirubins. Quantification of apoA-I in 93 normo- and hypertriglyceridemic clinical sera showed good agreement with immunoturbidimetric analysis (slope = 1.01, R(2) = 0.95, mean bias = 4.0%). Measurement of apoB-100 in the same clinical sera using both methods, however, revealed several outliers in SISCAPA-MALDI-TOF-MS measurements, possibly as a result of the lower MALDI-TOF-MS signal intensity (slope = 1.09, R(2) = 0.91, mean bias = 2.0%). The combination of analytical performance, rapid cycle time and automation potential validate the SISCAPA-MALDI-TOF-MS platform as a valuable approach for standardized and high-throughput quantification of apoA-I and apoB-100 in large sample cohorts.

AroER tri-screen is a biologically relevant assay for endocrine disrupting chemicals modulating the activity of aromatase and/or the estrogen receptor.

Endocrine disrupting chemicals (EDCs) interfere with the biosynthesis, metabolism, and functions of steroid hormones, including estrogens and androgens. Aromatase enzyme converts androgen to estrogen. Thus, EDCs against aromatase significantly impact estrogen- and/or androgen-dependent functions, including the development of breast cancer. The current study aimed to develop a biologically relevant cell-based high-throughput screening assay to identify EDCs that act as aromatase inhibitors (AIs), estrogen receptor (ER) agonists, and/or ER antagonists. The AroER tri-screen assay was developed by stable transfection of ER-positive, aromatase-expressing MCF-7 breast cancer cells with an estrogen responsive element (ERE) driven luciferase reporter plasmid. The AroER tri-screen can identify: estrogenic EDCs, which increase luciferase signal without 17ß-estradiol (E2); anti-estrogenic EDCs, which inhibit the E2-induced luciferase signal; and AI-like EDCs, which suppress a testosterone-induced luciferase signal. The assay was first optimized in a 96-well plate format and then miniaturized into a 1536-well plate format. The AroER tri-screen was demonstrated to be suitable for high-throughput screening in the 1536-well plate format, with a 6.9-fold signal-to-background ratio, a 5.4% coefficient of variation, and a screening window coefficient (Z-factor) of 0.78. The assay suggested that bisphenol A (BPA) functions mainly as an ER agonist. Results from screening the 446 drugs in the National Institutes of Health Clinical Collection revealed 106 compounds that modulated ER and/or aromatase activities. Among these, two AIs (bifonazole and oxiconazole) and one ER agonist (paroxetine) were confirmed through alternative aromatase and ER activity assays. These findings indicate that AroER tri-screen is a useful high-throughput screening system for identifying ER ligands and aromatase-inhibiting chemicals.

Alkylation of cysteine 468 in Stat3 defines a novel site for therapeutic development.

Stat3 is a latent transcription factor that promotes cell survival and proliferation and is often constitutively active in multiple cancers. Inhibition of Stat3 signaling pathways suppresses cell survival signals and leads to apoptosis in cancer cells, suggesting direct inhibition of Stat3 function is a viable therapeutic approach. Herein, we identify a small molecule, C48, as a selective Stat3-family member inhibitor. To determine its mechanism of action, we used site-directed mutagenesis and multiple biochemical techniques to show that C48 alkylates Cys468 in Stat3, a residue at the DNA-binding interface. We further demonstrate that C48 blocks accumulation of activated Stat3 in the nucleus in tumor cell lines that overexpress active Stat3, leading to impressive inhibition of tumor growth in mouse models. Collectively, these findings suggest Cys468 in Stat3 represents a novel site for therapeutic intervention and demonstrates the promise of alkylation as a potentially effective chemical approach for Stat3-dependent cancers.

Molecular dynamics simulations of the conformational changes in signal transducers and activators of transcription, Stat1 and Stat3.

All signal transducers and activators of transcription (STAT) factors are a family of cytoplasmic transcription factors that mediate the signal response to cytokines, growth factors, and hormonal factors. The phosphorylation and subsequent activation of Stat3, a member of the STAT family, has been found to be elevated in a large number of diverse human cancers. Understanding of the dynamics of the Stat3 dimer interface is pertinent to designing small molecule inhibitors to activated Stat3 dimer. To this end, we have performed molecular dynamics simulations in explicit water of the activated Stat3 homodimer, and also its closely related member of the STAT family, activated Stat1 homodimer. We observed a large-scale domain motion in the Stat3 dimer while the structure of the monomer remains intact. The driving force for this conformational change is enhanced binding of the Stat3 dimer to the DNA, thereby regulating gene expression. Our model shows that the carboxy terminus of one monomer wraps around the core of the SH2 domain of the other monomer, and this region that makes up the dimer interface remains intact during the dynamics. Water diffuses into a cavity under this dimer interface, thus expanding a pre-existing cavity that gets gated and closed by the loops in the SH2 domain. This cavity could serve as a potential binding pocket for inhibitor design.

Discovery of a novel proteasome inhibitor selective for cancer cells over non-transformed cells.

Numerous proteins controlling cell cycle progression, apoptosis and angiogenesis are degraded by the ubiquitin/proteasome system, which has become the subject for intense investigations for cancer therapeutics. Therefore, we used in silico and experimental approaches to screen compounds from the NCI chemical libraries for inhibitors against the chymotrypsin-like (CT-L) activity of the proteasome and discovered PI-083. Molecular docking indicates that PI-083 interacts with the Thr21, Gly47 and Ala49 residues of the beta5 subunit and Asp114 of the beta6 subunit of the proteasome. PI-083 inhibits CT-L activity and cell proliferation and induces apoptosis selectively in cancer cells (ovarian T80-Hras, pancreatic C7-Kras and breast MCF-7) as compared to their normal/immortalized counterparts (T80, C7 and MCF-10A, respectively). In contrast, Bortezomib, the only proteasome inhibitor approved by the Food and Drug Administration (FDA), did not exhibit this selectivity for cancer over non-transformed cells. In addition, in all cancer cells tested, including Multiple Myeloma (MM), breast, pancreatic, ovarian, lung, prostate cancer cell lines as well as fresh MM cells from patients, PI-083 required less time than Bortezomib to induce its antitumor effects. Furthermore, in nude mouse xenografts in vivo, PI-083, but not Bortezomib, suppressed the growth of human breast and lung tumors. Finally, following in vivo treatment of mice, PI-083 inhibited tumor, but not hepatic liver CT-L activity, whereas Bortezomib inhibited both tumor and liver CT-L activities. These results suggest that PI-083 is more selective for cancer cells and may have broader antitumor activity and therefore warrants further advanced preclinical studies.