Differences in the proteome within extracellular vesicles between premalignant and malignant plasma cell disorders.

BACKGROUND: Precursor plasma cell disorders such as monoclonal gammopathy of undetermined significance (MGUS) always precede the development of active malignancies such as multiple myeloma (MM). There is a need for novel biomarkers to identify those patients with such precursor plasma cell disorders who rapidly progress to MM. Plasma-derived extracellular vesicles (EVs) may serve as a reservoir of potential biomarkers that can shed light on the pathogenesis and disease biology of MM. METHODS: This study isolated small EVs (SEVs) and large EVs (LEVs) from the platelet-poor peripheral blood plasma of MGUS (n = 9) and MM (n = 12) patients using the size exclusion chromatography-based method and evaluated their proteome using a label-free proteomics workflow. RESULTS: In total, 2055 proteins were identified in SEVs, while 2794 proteins were identified in LEVs. The transferrin receptor (or CD71) protein was upregulated in both populations of EVs derived from MM patients compared to MGUS patients and was of prognostic significance. Similarly, three isoforms of serum amyloid A (SAA) protein, SAA1, SAA2, and SAA4, were also highly upregulated in SEVs within MM patients relative to MGUS patients. Finally, CD40 expression was also higher in the LEVs derived from MM patients than in MGUS patients. CONCLUSIONS: This study demonstrates the feasibility of successfully isolating both SEVs and LEVs from the peripheral blood of patients with plasma cell disorders and quantifying protein biomarkers within these EVs that could be of prognostic and diagnostic interest.

Proteomic Signature of Host Response to SARS-CoV-2 Infection in the Nasopharynx.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, has become a global health pandemic. COVID-19 severity ranges from an asymptomatic infection to a severe multiorgan disease. Although the inflammatory response has been implicated in the pathogenesis of COVID-19, the exact nature of dysregulation in signaling pathways has not yet been elucidated, underscoring the need for further molecular characterization of SARS-CoV-2 infection in humans. Here, we characterize the host response directly at the point of viral entry through analysis of nasopharyngeal swabs. Multiplexed high-resolution MS-based proteomic analysis of confirmed COVID-19 cases and negative controls identified 7582 proteins and revealed significant upregulation of interferon-mediated antiviral signaling in addition to multiple other proteins that are not encoded by interferon-stimulated genes or well characterized during viral infections. Downregulation of several proteasomal subunits, E3 ubiquitin ligases, and components of protein synthesis machinery was significant upon SARS-CoV-2 infection. Targeted proteomics to measure abundance levels of MX1, ISG15, STAT1, RIG-I, and CXCL10 detected proteomic signatures of interferon-mediated antiviral signaling that differentiated COVID-19-positive from COVID-19-negative cases. Phosphoproteomic analysis revealed increased phosphorylation of several proteins with known antiviral properties as well as several proteins involved in ciliary function (CEP131 and CFAP57) that have not previously been implicated in the context of coronavirus infections. In addition, decreased phosphorylation levels of AKT and PKC, which have been shown to play varying roles in different viral infections, were observed in infected individuals relative to controls. These data provide novel insights that add depth to our understanding of SARS-CoV-2 infection in the upper airway and establish a proteomic signature for this viral infection.

Machine Learning-Based Fragment Selection Improves the Performance of Qualitative PRM Assays.

Targeted mass spectrometry-based platforms have become a valuable tool for the sensitive and specific detection of protein biomarkers in clinical and research settings. Traditionally, developing a targeted assay for peptide quantification has involved manually preselecting several fragment ions and establishing a limit of detection (LOD) and a lower limit of quantitation (LLOQ) for confident detection of the target. Established thresholds such as LOD and LLOQ, however, inherently sacrifice sensitivity to afford specificity. Here, we demonstrate that machine learning can be applied to qualitative PRM assays to discriminate positive from negative samples more effectively than a traditional approach utilizing conventional methods. To demonstrate the utility of this method, we trained an ensemble machine learning model using 282 SARS-CoV-2 positive and 994 SARS-CoV-2 negative nasopharyngeal swabs (NP swab) analyzed using a targeted PRM method. This model was then validated using an independent set of 200 positive and 150 negative samples and achieved a sensitivity of 92% relative to results obtained by RT-PCR, which was superior to a traditional approach that resulted in 86.5% sensitivity when analyzing the same data. These results demonstrate that machine learning can be applied to qualitative PRM assays and results in superior performance relative to traditional methods.

Targeted Detection of SARS-CoV-2 Nucleocapsid Sequence Variants by Mass Spectrometric Analysis of Tryptic Peptides.

COVID-19 vaccines are becoming more widely available, but accurate and rapid testing remains a crucial tool for slowing the spread of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) virus. Although the quantitative reverse transcription-polymerase chain reaction (qRT-PCR) remains the most prevalent testing methodology, numerous tests have been developed that are predicated on detection of the SARS-CoV-2 nucleocapsid protein, including liquid chromatography-tandem mass spectrometry (LC-MS/MS) and immunoassay-based approaches. The continuing emergence of SARS-CoV-2 variants has complicated these approaches, as both qRT-PCR and antigen detection methods can be prone to missing viral variants. In this study, we describe several COVID-19 cases where we were unable to detect the expected peptide targets from clinical nasopharyngeal swabs. Whole genome sequencing revealed that single nucleotide polymorphisms in the gene encoding the viral nucleocapsid protein led to sequence variants that were not monitored in the targeted assay. Minor modifications to the LC-MS/MS method ensured detection of the variants of the target peptide. Additional nucleocapsid variants could be detected by performing the bottom-up proteomic analysis of whole viral genome-sequenced samples. This study demonstrates the importance of considering variants of SARS-CoV-2 in the assay design and highlights the flexibility of mass spectrometry-based approaches to detect variants as they evolve.

13C15N: glucagon-based novel isotope dilution mass spectrometry method for measurement of glucagon metabolism in humans.

BACKGROUND: Glucagon serves as an important regulatory hormone for regulating blood glucose concentration with tight feedback control exerted by insulin and glucose. There are critical gaps in our understanding of glucagon kinetics, pancreatic α cell function and intra-islet feedback network that are disrupted in type 1 diabetes. This is important for translational research applications of evolving dual-hormone (insulin + glucagon) closed-loop artificial pancreas algorithms and their usage in type 1 diabetes. Thus, it is important to accurately measure glucagon kinetics in vivo and to develop robust models of glucose-insulin-glucagon interplay that could inform next generation of artificial pancreas algorithms. METHODS: Here, we describe the administration of novel 13C15N heavy isotope-containing glucagon tracers-FF glucagon [(Phe 6 13C9,15N; Phe 22 13C9,15N)] and FFLA glucagon [(Phe 6 13C9,15N; Phe 22 13C9,15N; Leu 14 13C6,15N; Ala 19 13C3)] followed by anti-glucagon antibody-based enrichment and LC-MS/MS based-targeted assays using high-resolution mass spectrometry to determine levels of infused glucagon in plasma samples. The optimized assay results were applied for measurement of glucagon turnover in subjects with and without type 1 diabetes infused with isotopically labeled glucagon tracers. RESULTS: The limit of quantitation was found to be 1.56 pg/ml using stable isotope-labeled glucagon as an internal standard. Intra and inter-assay variability was < 6% and < 16%, respectively, for FF glucagon while it was < 5% and < 23%, respectively, for FFLA glucagon. Further, we carried out a novel isotope dilution technique using glucagon tracers for studying glucagon kinetics in type 1 diabetes. CONCLUSIONS: The methods described in this study for simultaneous detection and quantitation of glucagon tracers have clinical utility for investigating glucagon kinetics in vivo in humans.

DIA-Based Proteome Profiling of Nasopharyngeal Swabs from COVID-19 Patients.

Since the recent outbreak of COVID-19, there have been intense efforts to understand viral pathogenesis and host immune response to combat SARS-CoV-2. It has become evident that different host alterations can be identified in SARS-CoV-2 infection based on whether infected cells, animal models or clinical samples are studied. Although nasopharyngeal swabs are routinely collected for SARS-CoV-2 detection by RT-PCR testing, host alterations in the nasopharynx at the proteomic level have not been systematically investigated. Thus, we sought to characterize the host response through global proteome profiling of nasopharyngeal swab specimens. A mass spectrometer combining trapped ion mobility spectrometry (TIMS) and high-resolution QTOF mass spectrometer with parallel accumulation-serial fragmentation (PASEF) was deployed for unbiased proteome profiling. First, deep proteome profiling of pooled nasopharyngeal swab samples was performed in the PASEF enabled DDA mode, which identified 7723 proteins that were then used to generate a spectral library. This approach provided peptide level evidence of five missing proteins for which MS/MS spectrum and mobilograms were validated with synthetic peptides. Subsequently, quantitative proteomic profiling was carried out for 90 individual nasopharyngeal swab samples (45 positive and 45 negative) in DIA combined with PASEF, termed as diaPASEF mode, which resulted in a total of 5023 protein identifications. Of these, 577 proteins were found to be upregulated in SARS-CoV-2 positive samples. Functional analysis of these upregulated proteins revealed alterations in several biological processes including innate immune response, viral protein assembly, and exocytosis. To the best of our knowledge, this study is the first to deploy diaPASEF for quantitative proteomic profiling of clinical samples and shows the feasibility of adopting such an approach to understand mechanisms and pathways altered in diseases.

Mass Spectrometric Analysis of Urine from COVID-19 Patients for Detection of SARS-CoV-2 Viral Antigen and to Study Host Response.

SARS-CoV-2 infection has become a major public health burden and affects many organs including lungs, kidneys, the liver, and the brain. Although the virus is readily detected and diagnosed using nasopharyngeal swabs by reverse transcriptase polymerase chain reaction (RT-PCR), detection of its presence in body fluids is fraught with difficulties. A number of published studies have failed to detect viral RNA by RT-PCR methods in urine. Although microbial identification in clinical microbiology using mass spectrometry is undertaken after culture, here we undertook a mass spectrometry-based approach that employed an enrichment step to capture and detect SARS-CoV-2 nucleocapsid protein directly from urine of COVID-19 patients without any culture. We detected SARS-CoV-2 nucleocapsid protein-derived peptides from 13 out of 39 urine samples. Further, a subset of COVID-19 positive and COVID-19 negative urine samples validated by mass spectrometry were used for the quantitative proteomics analysis. Proteins with increased abundance in urine of SARS-CoV-2 positive individuals were enriched in the acute phase response, regulation of complement system, and immune response. Notably, a number of renal proteins such as podocin (NPHS2), an amino acid transporter (SLC36A2), and sodium/glucose cotransporter 5 (SLC5A10), which are intimately involved in normal kidney function, were decreased in the urine of COVID-19 patients. Overall, the detection of viral antigens in urine using mass spectrometry and alterations of the urinary proteome could provide insights into understanding the pathogenesis of COVID-19.

Analytical Sensitivity and Specificity of Four Point of Care Rapid Antigen Diagnostic Tests for SARS-CoV-2 Using Real-Time Quantitative PCR, Quantitative Droplet Digital PCR, and a Mass Spectrometric Antigen Assay as Comparator Methods.

BACKGROUND: We evaluated the analytical sensitivity and specificity of 4 rapid antigen diagnostic tests (Ag RDTs) for severe acute respiratory syndrome coronavirus 2, using reverse transcription quantitative PCR (RT-qPCR) as the reference method and further characterizing samples using droplet digital quantitative PCR (ddPCR) and a mass spectrometric antigen test. METHODS: Three hundred fifty (150 negative and 200 RT-qPCR positive) residual PBS samples were tested for antigen using the BD Veritor lateral flow (LF), ACON LF, ACON fluorescence immunoassay (FIA), and LumiraDx FIA. ddPCR was performed on RT-qPCR-positive samples to quantitate the viral load in copies/mL applied to each Ag RDT. Mass spectrometric antigen testing was performed on PBS samples to obtain a set of RT-qPCR-positive, antigen-positive samples for further analysis. RESULTS: All Ag RDTs had nearly 100% specificity compared to RT-qPCR. Overall analytical sensitivity varied from 66.5% to 88.3%. All methods detected antigen in samples with viral load >1 500 000 copies/mL RNA, and detected ≥75% of samples with viral load of 500 000 to 1 500 000 copies/mL. The BD Veritor LF detected only 25% of samples with viral load between 50 000 to 500 000 copies/mL, compared to 75% for the ACON LF device and >80% for LumiraDx and ACON FIA. The ACON FIA detected significantly more samples with viral load <50 000 copies/mL compared to the BD Veritor. Among samples with detectable antigen and viral load <50 000 copies/mL, sensitivity of the Ag RDT varied between 13.0% (BD Veritor) and 78.3% (ACON FIA). CONCLUSIONS: Ag RDTs differ significantly in analytical sensitivity, particularly at viral load <500 000 copies/mL.

A SISCAPA-based approach for detection of SARS-CoV-2 viral antigens from clinical samples.

SARS-CoV-2, a novel human coronavirus, has created a global disease burden infecting > 100 million humans in just over a year. RT-PCR is currently the predominant method of diagnosing this viral infection although a variety of tests to detect viral antigens have also been developed. In this study, we adopted a SISCAPA-based enrichment approach using anti-peptide antibodies generated against peptides from the nucleocapsid protein of SARS-CoV-2. We developed a targeted workflow in which nasopharyngeal swab samples were digested followed by enrichment of viral peptides using the anti-peptide antibodies and targeted parallel reaction monitoring (PRM) analysis using a high-resolution mass spectrometer. This workflow was applied to 41 RT-PCR-confirmed clinical SARS-CoV-2 positive nasopharyngeal swab samples and 30 negative samples. The workflow employed was highly specific as none of the target peptides were detected in negative samples. Further, the detected peptides showed a positive correlation with the viral loads as measured by RT-PCR Ct values. The SISCAPA-based platform described in the current study can serve as an alternative method for SARS-CoV-2 viral detection and can also be applied for detecting other microbial pathogens directly from clinical samples.

Insulin deficiency and intranasal insulin alter brain mitochondrial function: a potential factor for dementia in diabetes.

Despite the strong association between diabetes and dementia, it remains to be fully elucidated how insulin deficiency adversely affects brain functions. We show that insulin deficiency in streptozotocin-induced diabetic mice decreased mitochondrial ATP production and/or citrate synthase and cytochrome oxidase activities in the cerebrum, hypothalamus, and hippocampus. Concomitant decrease in mitochondrial fusion proteins and increased fission proteins in these brain regions likely contributed to altered mitochondrial function. Although insulin deficiency did not cause any detectable increase in reactive oxygen species (ROS) emission, inhibition of monocarboxylate transporters increased ROS emission and further reduced ATP production, indicating the causative roles of elevated ketones and lactate in counteracting oxidative stress and as a fuel source for ATP production during insulin deficiency. Moreover, in healthy mice, intranasal insulin administration increased mitochondrial ATP production, demonstrating a direct regulatory role of insulin on brain mitochondrial function. Proteomics analysis of the cerebrum showed that although insulin deficiency led to oxidative post-translational modification of several proteins that cause tau phosphorylation and neurofibrillary degeneration, insulin administration enhanced neuronal development and neurotransmission pathways. Together these results render support for the critical role of insulin to maintain brain mitochondrial homeostasis and provide mechanistic insight into the potential therapeutic benefits of intranasal insulin.-Ruegsegger, G. N., Manjunatha, S., Summer, P., Gopala, S., Zabeilski, P., Dasari, S., Vanderboom, P. M., Lanza, I. R., Klaus, K. A., Nair, K. S. Insulin deficiency and intranasal insulin alter brain mitochondrial function: a potential factor for dementia in diabetes.

Serum levels of DNAJB9 are elevated in fibrillary glomerulonephritis patients.

Fibrillary glomerulonephritis (FGN) is a rare glomerular disease. Kidney biopsy is required to establish the diagnosis. Recent studies have identified abundant glomerular deposition of DNAJB9 as a unique histological marker of FGN. We developed an immunoprecipitation-based multiple reaction monitoring method to measure serum levels of DNAJB9. We detected a 4-fold higher abundance of serum DNAJB9 in FGN patients when compared to controls, including patients with other glomerular diseases. Serum DNAJB9 levels were also negatively associated with estimated glomerular filtration rate in patients with FGN. Serum DNAJB9 levels accurately predicted FGN with moderate sensitivity (67%) and with high specificity (98%) and positive and negative predictive value (89% and 95%, respectively). A receiver operating curve analysis demonstrated an AUC of 0.958. These results suggest that serum levels of DNAJB9 could be a valuable marker to predict FGN, with the potential to complement kidney biopsy for the diagnosis of FGN.

Hepatic adenomas with synchronous or metachronous fibrolamellar carcinomas: both are characterized by LFABP loss.

Rare hepatic adenomas are associated with synchronous or metachronous fibrolamellar carcinomas. The morphology of these adenomas has not been well described and they have not been subclassifed using the current molecular classification schema. We examined four hepatic adenomas co-occurring with or preceding a diagnosis of fibrolamellar carcinoma in three patients. On histological examination, three of the adenomas showed the typical morphology of HNF1-α inactivated adenomas, whereas one showed a myxoid adenoma morphology. All of the adenomas were negative for PRKACA rearrangements by Fluorescence in situ Hybridization (FISH) analysis. All four of the adenomas showed complete loss or significant reduction of liver fatty acid binding protein (LFABP) expression by immunohistochemistry. Interestingly, the fibrolamellar carcinomas in each case also showed loss of LFABP by immunohistochemistry. One of the fibrolamellar carcinomas was negative for PRKACA rearrangements by FISH, whereas the others were positive. To investigate if LFBAP loss is typical of fibrolamellar carcinomas in general, an additional cohort of tumors was studied (n=19). All 19 fibrolamellar carcinomas showed the expected PRKACA rearrangements and immunostains showed loss of LFABP in each case, consistent with HNF1-α inactivation. To validate this observation, mass spectrometry-based proteomics was performed on tumor-normal pairs of six fibrolamellar carcinomas and showed an average 10-fold reduction in LFABP protein levels, compared with matched normal liver tissue. In conclusion, hepatic adenomas co-occurring with fibrolamellar carcinomas show LFABP loss and are negative for PRKACA rearrangements, indicating they are genetically distinct lesions. These data also demonstrate that LFABP loss, which characterizes HNF1-α inactivation, is a consistent feature of fibrolamellar carcinoma, indicating HNF1-α inactivation is an important event in fibrolamellar carcinoma pathogenesis.

Comprehensive Assessment of M-Proteins Using Nanobody Enrichment Coupled to MALDI-TOF Mass Spectrometry.

BACKGROUND: Electrophoretic separation of serum and urine proteins has played a central role in diagnosing and monitoring plasma cell disorders. Despite limitations in resolution and analytical sensitivity, plus the necessity for adjunct methods, protein gel electrophoresis and immunofixation electrophoresis (IFE) remain front-line tests. METHODS: We developed a MALDI mass spectrometry-based assay that was simple to perform, automatable, analytically sensitive, and applicable to analyzing the wide variety of monoclonal proteins (M-proteins) encountered clinically. This assay, called MASS-FIX, used the unique molecular mass signatures of the different Ig isotypes in combination with nanobody immunoenrichment to generate information-rich mass spectra from which M-proteins could be identified, isotyped, and quantified. The performance of MASS-FIX was compared to current gel-based electrophoresis assays. RESULTS: MASS-FIX detected all M-proteins that were detectable by urine or serum protein electrophoresis. In serial dilution studies, MASS-FIX was more analytically sensitive than IFE. For patient samples, MASS-FIX provided the same primary isotype information for 98% of serum M-proteins (n = 152) and 95% of urine M-proteins (n = 55). MASS-FIX accurately quantified M-protein to <1 g/dL, with reduced bias as compared to protein electrophoresis. Intraassay and interassay CVs were <20% across all samples having M-protein concentrations >0.045 g/dL, with the ability to detect M-proteins <0.01 g/dL. In addition, MASS-FIX could simultaneously measure κ:λ light chain ratios for IgG, IgA, and IgM. Retrospective serial monitoring of patients with myeloma posttreatment demonstrated that MASS-FIX provided equivalent quantitative information to either protein electrophoresis or the Hevylite(™) assay. CONCLUSIONS: MASS-FIX can advance how plasma cell disorders are screened, diagnosed, and monitored.

Screening Method for M-Proteins in Serum Using Nanobody Enrichment Coupled to MALDI-TOF Mass Spectrometry.

BACKGROUND: Current recommendations for screening for monoclonal gammopathies include serum protein electrophoresis (PEL), imunofixation electrophoresis (IFE), and free light chain (FLC) ratios to identify or rule out an M-protein. The aim of this study was to examine the feasibility of an assay based on immunoenrichment and MALDI-TOF-MS (MASS-SCREEN) to qualitatively screen for M-proteins. METHODS: Serum from 556 patients previously screened for M-proteins by PEL and IFE were immunopurified using a κ/λ-specific nanobody bead mixture. Following purification, light chains (LC) were released from their heavy chains by reduction. MALDI-TOF analysis was performed and the mass-to-charge LC distributions were visually examined for the presence of an M-protein by both unblinded and blinded analysts. RESULTS: In unblinded analysis, MASS-SCREEN detected 100% of the PEL-positive samples with an analytical sensitivity and specificity of 96% and 81% using IFE positivity as the standard. In a blinded analysis using 6 different laboratory personnel, consensus was reached in 92% of the samples. Overall analytical sensitivity and specificity were reduced to 92% and 80%, respectively. FLC ratios were found to be abnormal in 28% of MASS-SCREEN-negative samples, suggesting FLC measurements need to be considered in screening. CONCLUSIONS: MASS-SCREEN could replace PEL in a panel that would include FLC measurements. Further studies and method development should be performed to validate the clinical sensitivity and specificity and to determine if this panel will suffice as a general screen for monoclonal proteins.

Detection and quantification of Babesia species intraerythrocytic parasites by flow cytometry.

OBJECTIVES: Recent work has demonstrated that automated fluorescence flow cytometry (FLC) is a potential alternative for the detection and quantification of Plasmodium parasites. The objective of this study was to apply this novel FLC method to detect and quantify Babesia parasites in venous blood and compare results to light microscopy and polymerase chain reaction methods. METHODS: An automated hematology/malaria analyzer (XN-31; Sysmex) was used to detect and quantify B microti-infected red blood cells from residual venous blood samples (n = 250: Babesia positive, n = 170; Babesia negative, n = 80). As no instrument software currently exists for Babesia, qualitative and quantitative machine learning (ML) algorithms were developed to facilitate analysis. RESULTS: Performance of the ML models was verified against the XN-31 software using P falciparum-infected samples. When applied to Babesia-infected samples, the qualitative ML model demonstrated an area under the curve (AUC) of 0.956 (sensitivity, 95.9%; specificity, 83.3%) relative to polymerase chain reaction. For valid scattergrams, the qualitive model achieved an AUC of 1.0 (sensitivity and specificity, 100%), while the quantitative model demonstrated an AUC of 0.986 (sensitivity, 94.4%; specificity, 100%). CONCLUSIONS: This investigation demonstrates that Babesia parasites can be detected and quantified directly from venous blood using FLC. Although promising, opportunities remain to improve the general applicability of the method.

GADD45A is a mediator of mitochondrial loss, atrophy, and weakness in skeletal muscle.

Aging and many illnesses and injuries impair skeletal muscle mass and function, but the molecular mechanisms are not well understood. To better understand the mechanisms, we generated and studied transgenic mice with skeletal muscle-specific expression of growth arrest and DNA damage inducible α (GADD45A), a signaling protein whose expression in skeletal muscle rises during aging and a wide range of illnesses and injuries. We found that GADD45A induced several cellular changes that are characteristic of skeletal muscle atrophy, including a reduction in skeletal muscle mitochondria and oxidative capacity, selective atrophy of glycolytic muscle fibers, and paradoxical expression of oxidative myosin heavy chains despite mitochondrial loss. These cellular changes were at least partly mediated by MAP kinase kinase kinase 4, a protein kinase that is directly activated by GADD45A. By inducing these changes, GADD45A decreased the mass of muscles that are enriched in glycolytic fibers, and it impaired strength, specific force, and endurance exercise capacity. Furthermore, as predicted by data from mouse models, we found that GADD45A expression in skeletal muscle was associated with muscle weakness in humans. Collectively, these findings identify GADD45A as a mediator of mitochondrial loss, atrophy, and weakness in mouse skeletal muscle and a potential target for muscle weakness in humans.

A size-exclusion-based approach for purifying extracellular vesicles from human plasma.

Extracellular vesicles (EVs) are released into blood from multiple organs and carry molecular cargo that facilitates inter-organ communication and an integrated response to physiological and pathological stimuli. Interrogation of the protein cargo of EVs is currently limited by the absence of optimal and reproducible approaches for purifying plasma EVs that are suitable for downstream proteomic analyses. We describe a size-exclusion chromatography (SEC)-based method to purify EVs from platelet-poor plasma (PPP) for proteomics profiling via high-resolution mass spectrometry (SEC-MS). The SEC-MS method identifies more proteins with higher precision than several conventional EV isolation approaches. We apply the SEC-MS method to identify the unique proteomic signatures of EVs released from platelets, adipocytes, muscle cells, and hepatocytes, with the goal of identifying tissue-specific EV markers. Furthermore, we apply the SEC-MS approach to evaluate the effects of a single bout of exercise on EV proteomic cargo in human plasma.

A Comparative Proteomic Analysis of Extracellular Vesicles Associated With Lipotoxicity.

Extracellular vesicles (EVs) are emerging mediators of intercellular communication in nonalcoholic steatohepatitis (NASH). Palmitate, a lipotoxic saturated fatty acid, activates hepatocellular endoplasmic reticulum stress, which has been demonstrated to be important in NASH pathogenesis, including in the release of EVs. We have previously demonstrated that the release of palmitate-stimulated EVs is dependent on the de novo synthesis of ceramide, which is trafficked by the ceramide transport protein, STARD11. The trafficking of ceramide is a critical step in the release of lipotoxic EVs, as cells deficient in STARD11 do not release palmitate-stimulated EVs. Here, we examined the hypothesis that protein cargoes are trafficked to lipotoxic EVs in a ceramide-dependent manner. We performed quantitative proteomic analysis of palmitate-stimulated EVs in control and STARD11 knockout hepatocyte cell lines. Proteomics was performed on EVs isolated by size exclusion chromatography, ultracentrifugation, and density gradient separation, and EV proteins were measured by mass spectrometry. We also performed human EV proteomics from a control and a NASH plasma sample, for comparative analyses with hepatocyte-derived lipotoxic EVs. Size exclusion chromatography yielded most unique EV proteins. Ceramide-dependent lipotoxic EVs contain damage-associated molecular patterns and adhesion molecules. Haptoglobin, vascular non-inflammatory molecule-1, and insulin-like growth factor-binding protein complex acid labile subunit were commonly detected in NASH and hepatocyte-derived ceramide-dependent EVs. Lipotoxic EV proteomics provides novel candidate proteins to investigate in NASH pathogenesis and as diagnostic biomarkers for hepatocyte-derived EVs in NASH patients.

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.