National Cancer Institute Think-Tank Meeting Report on Proteomic Cartography and Biomarkers at the Single-Cell Level: Interrogation of Premalignant Lesions.

A Think-Tank Meeting was convened by the National Cancer Institute (NCI) to solicit experts' opinion on the development and application of multiomic single-cell analyses, and especially single-cell proteomics, to improve the development of a new generation of biomarkers for cancer risk, early detection, diagnosis, and prognosis as well as to discuss the discovery of new targets for prevention and therapy. It is anticipated that such markers and targets will be based on cellular, subcellular, molecular, and functional aberrations within the lesion and within individual cells. Single-cell proteomic data will be essential for the establishment of new tools with searchable and scalable features that include spatial and temporal cartographies of premalignant and malignant lesions. Challenges and potential solutions that were discussed included (i) The best way/s to analyze single-cells from fresh and preserved tissue; (ii) Detection and analysis of secreted molecules and from single cells, especially from a tissue slice; (iii) Detection of new, previously undocumented cell type/s in the premalignant and early stage cancer tissue microenvironment; (iv) Multiomic integration of data to support and inform proteomic measurements; (v) Subcellular organelles-identifying abnormal structure, function, distribution, and location within individual premalignant and malignant cells; (vi) How to improve the dynamic range of single-cell proteomic measurements for discovery of differentially expressed proteins and their post-translational modifications (PTM); (vii) The depth of coverage measured concurrently using single-cell techniques; (viii) Quantitation - absolute or semiquantitative? (ix) Single methodology or multiplexed combinations? (x) Application of analytical methods for identification of biologically significant subsets; (xi) Data visualization of N-dimensional data sets; (xii) How to construct intercellular signaling networks in individual cells within premalignant tumor microenvironments (TME); (xiii) Associations between intrinsic cellular processes and extrinsic stimuli; (xiv) How to predict cellular responses to stress-inducing stimuli; (xv) Identification of new markers for prediction of progression from precursor, benign, and localized lesions to invasive cancer, based on spatial and temporal changes within individual cells; (xvi) Identification of new targets for immunoprevention or immunotherapy-identification of neoantigens and surfactome of individual cells within a lesion.

Physician and Nurse Practitioner Attitudes on Generic Prescribing of Oral Contraceptive Pills and Antidepressants.

IMPORTANCE: As prescription drug costs rise, it is important to understand attitudes among primary care physicians and nurse practitioners (NPs) towards generic drugs. OBJECTIVE: We aimed to examine the generic skepticism index (GSI) among primary care clinicians, and their willingness to discuss and prescribe generic antidepressants (ADs) and generic oral contraceptives (OCPs). DESIGN: We used a factorial vignette design survey to test 4 factors: message source, message, brand preference, and drug class. Participants were randomized to different combinations of factors. SETTING: This was a cross-sectional study. PARTICIPANTS: Physicians registered with the American College of Physicians (ACP) and NPs registered with the American Association of Nurse Practitioners (AANP) participated in the study. MAIN MEASURES: The primary outcomes were generic skepticism as measured using the generic skepticism index (GSI), and clinician willingness to discuss and prescribe generics. RESULTS: Surveys were completed by 56% of physicians (n = 369/661) and 60% of NPs (n = 493/819). Compared with physicians, NPs were younger (p < 0.001), predominantly female (p < 0.001), and differed in the race (p < 0.001). According to the GSI, 16% (n = 138/862) were identified as generic skeptics (18.5% of NPs and 12.7% of physicians, p = 0.023). Generic skeptics had lower odds of willingness to discuss switching (OR 0.22, 95% CI (0.14-0.35), p < 0.001) or prescribe (OR 0.18, 95% CI (0.11-0.28), p < 0.001) generic OCPs. Participants had lower odds of willingness to prescribe generic drugs to patients with brand preference compared with brand-neutral patients (OR 0.64, 95% CI 0.50-0.82, p < 0.001). CONCLUSIONS AND RELEVANCE: Generic skepticism was associated with lower willingness to discuss or prescribe generic drugs. Clinicians reported lower willingness to discuss switching or prescribe generics for OCPs than for ADs. Patient brand preference hindered generic prescribing. Message source and message type were not significantly associated with outcomes.

Discovery of non-ETS gene fusions in human prostate cancer using next-generation RNA sequencing.

Half of prostate cancers harbor gene fusions between TMPRSS2 and members of the ETS transcription factor family. To date, little is known about the presence of non-ETS fusion events in prostate cancer. We used next-generation transcriptome sequencing (RNA-seq) in order to explore the whole transcriptome of 25 human prostate cancer samples for the presence of chimeric fusion transcripts. We generated more than 1 billion sequence reads and used a novel computational approach (FusionSeq) in order to identify novel gene fusion candidates with high confidence. In total, we discovered and characterized seven new cancer-specific gene fusions, two involving the ETS genes ETV1 and ERG, and four involving non-ETS genes such as CDKN1A (p21), CD9, and IKBKB (IKK-beta), genes known to exhibit key biological roles in cellular homeostasis or assumed to be critical in tumorigenesis of other tumor entities, as well as the oncogene PIGU and the tumor suppressor gene RSRC2. The novel gene fusions are found to be of low frequency, but, interestingly, the non-ETS fusions were all present in prostate cancer harboring the TMPRSS2-ERG gene fusion. Future work will focus on determining if the ETS rearrangements in prostate cancer are associated or directly predispose to a rearrangement-prone phenotype.

A genome-wide scalable SNP genotyping assay using microarray technology.

Oligonucleotide probe arrays have enabled massively parallel analysis of gene expression levels from a single cDNA sample. Application of microarray technology to analyzing genomic DNA has been stymied by the sequence complexity of the entire human genome. A robust, single base-resolution direct genomic assay would extend the reach of microarray technology. We developed an array-based whole-genome genotyping assay that does not require PCR and enables effectively unlimited multiplexing. The assay achieves a high signal-to-noise ratio by combining specific hybridization of picomolar concentrations of whole genome-amplified DNA to arrayed probes with allele-specific primer extension and signal amplification. As proof of principle, we genotyped several hundred previously characterized SNPs. The conversion rate, call rate and accuracy were comparable to those of high-performance PCR-based genotyping assays.

B-cyclin/CDKs regulate mitotic spindle assembly by phosphorylating kinesins-5 in budding yeast.

Although it has been known for many years that B-cyclin/CDK complexes regulate the assembly of the mitotic spindle and entry into mitosis, the full complement of relevant CDK targets has not been identified. It has previously been shown in a variety of model systems that B-type cyclin/CDK complexes, kinesin-5 motors, and the SCF(Cdc4) ubiquitin ligase are required for the separation of spindle poles and assembly of a bipolar spindle. It has been suggested that, in budding yeast, B-type cyclin/CDK (Clb/Cdc28) complexes promote spindle pole separation by inhibiting the degradation of the kinesins-5 Kip1 and Cin8 by the anaphase-promoting complex (APC(Cdh1)). We have determined, however, that the Kip1 and Cin8 proteins are present at wild-type levels in the absence of Clb/Cdc28 kinase activity. Here, we show that Kip1 and Cin8 are in vitro targets of Clb2/Cdc28 and that the mutation of conserved CDK phosphorylation sites on Kip1 inhibits spindle pole separation without affecting the protein's in vivo localization or abundance. Mass spectrometry analysis confirms that two CDK sites in the tail domain of Kip1 are phosphorylated in vivo. In addition, we have determined that Sic1, a Clb/Cdc28-specific inhibitor, is the SCF(Cdc4) target that inhibits spindle pole separation in cells lacking functional Cdc4. Based on these findings, we propose that Clb/Cdc28 drives spindle pole separation by direct phosphorylation of kinesin-5 motors.

Artificial intelligence and machine learning in prehospital emergency care: A scoping review.

Our scoping review provides a comprehensive analysis of the landscape of artificial intelligence (AI) applications in prehospital emergency care (PEC). It contributes to the field by highlighting the most studied AI applications and identifying the most common methodological approaches across 106 included studies. The findings indicate a promising future for AI in PEC, with many unique use cases, such as prognostication, demand prediction, resource optimization, and the Internet of Things continuous monitoring systems. Comparisons with other approaches showed AI outperforming clinicians and non-AI algorithms in most cases. However, most studies were internally validated and retrospective, highlighting the need for rigorous prospective validation of AI applications before implementation in clinical settings. We identified knowledge and methodological gaps using an evidence map, offering a roadmap for future investigators. We also discussed the significance of explainable AI for establishing trust in AI systems among clinicians and facilitating real-world validation of AI models.

Microarray-based multicycle-enrichment of genomic subsets for targeted next-generation sequencing.

The lack of efficient high-throughput methods for enrichment of specific sequences from genomic DNA represents a key bottleneck in exploiting the enormous potential of next-generation sequencers. Such methods would allow for a systematic and targeted analysis of relevant genomic regions. Recent studies reported sequence enrichment using a hybridization step to specific DNA capture probes as a possible solution to the problem. However, so far no method has provided sufficient depths of coverage for reliable base calling over the entire target regions. We report a strategy to multiply the enrichment performance and consequently improve depth and breadth of coverage for desired target sequences by applying two iterative cycles of hybridization with microfluidic Geniom biochips. Using this strategy, we enriched and then sequenced the cancer-related genes BRCA1 and TP53 and a set of 1000 individual dbSNP regions of 500 bp using Illumina technology. We achieved overall enrichment factors of up to 1062-fold and average coverage depths of 470-fold. Combined with high coverage uniformity, this resulted in nearly complete consensus coverages with >86% of target region covered at 20-fold or higher. Analysis of SNP calling accuracies after enrichment revealed excellent concordance, with the reference sequence closely mirroring the previously reported performance of Illumina sequencing conducted without sequence enrichment.

Quantitative phenotyping via deep barcode sequencing.

Next-generation DNA sequencing technologies have revolutionized diverse genomics applications, including de novo genome sequencing, SNP detection, chromatin immunoprecipitation, and transcriptome analysis. Here we apply deep sequencing to genome-scale fitness profiling to evaluate yeast strain collections in parallel. This method, Barcode analysis by Sequencing, or "Bar-seq," outperforms the current benchmark barcode microarray assay in terms of both dynamic range and throughput. When applied to a complex chemogenomic assay, Bar-seq quantitatively identifies drug targets, with performance superior to the benchmark microarray assay. We also show that Bar-seq is well-suited for a multiplex format. We completely re-sequenced and re-annotated the yeast deletion collection using deep sequencing, found that approximately 20% of the barcodes and common priming sequences varied from expectation, and used this revised list of barcode sequences to improve data quality. Together, this new assay and analysis routine provide a deep-sequencing-based toolkit for identifying gene-environment interactions on a genome-wide scale.

Epitope-resolved profiling of the SARS-CoV-2 antibody response identifies cross-reactivity with endemic human coronaviruses.

The SARS-CoV-2 proteome shares regions of conservation with endemic human coronaviruses (CoVs), but it remains unknown to what extent these may be cross-recognized by the antibody response. Here, we study cross-reactivity using a highly multiplexed peptide assay (PepSeq) to generate an epitope-resolved view of IgG reactivity across all human CoVs in both COVID-19 convalescent and negative donors. PepSeq resolves epitopes across the SARS-CoV-2 Spike and Nucleocapsid proteins that are commonly targeted in convalescent donors, including several sites also recognized in some uninfected controls. By comparing patterns of homologous reactivity between CoVs and using targeted antibody-depletion experiments, we demonstrate that SARS-CoV-2 elicits antibodies that cross-recognize pandemic and endemic CoV antigens at two Spike S2 subunit epitopes. We further show that these cross-reactive antibodies preferentially bind endemic homologs. Our findings highlight sites at which the SARS-CoV-2 response appears to be shaped by previous CoV exposures and which have the potential to raise broadly neutralizing responses.

Development and testing of a module to promote generic oral contraceptive prescribing among nurse practitioners.

Although generic oral contraceptives (OCPs) can improve adherence and reduce health care expenditures, use of generic OCPs remains low, and the factors that affect generic prescribing are not well understood. We aimed to understand the barriers and facilitators of generic OCP prescribing and potential solutions to increase generic OCP prescribing, as well as pilot an educational module to address clinician misconceptions about generic OCPs. We developed focus group scripts using the 4D model of appreciative inquiry. A total of four focus groups occurred, two at the American Association of Nurse Practitioners (AANP) national conference and two at the American College of Physicians (ACP) Internal Medicine meeting. Focus group transcripts were analyzed using a constant comparative method with no a priori hypothesis to generate emerging and reoccurring themes. Findings from these focus groups were used to develop an educational module promoting generic OCP prescribing. Participants were recruited from the AANP Network for Research and the ACP Research Panel. This study demonstrates that health system factors, workflow factors, clinician factors, and patient factors were the main barriers to and facilitators of generic OCP prescribing. Nurse practitioners were responsive to an educational module and reported increased willingness to discuss and prescribe generic OCPs after completing the module. Interventions to increase generic OCP prescribing must address clinician and patient factors within the context of workflow and larger health system factors.

Epitope-resolved profiling of the SARS-CoV-2 antibody response identifies cross-reactivity with an endemic human CoV.

A high-resolution understanding of the antibody response to SARS-CoV-2 is important for the design of effective diagnostics, vaccines and therapeutics. However, SARS-CoV-2 antibody epitopes remain largely uncharacterized, and it is unknown whether and how the response may cross-react with related viruses. Here, we use a multiplexed peptide assay ('PepSeq') to generate an epitope-resolved view of reactivity across all human coronaviruses. PepSeq accurately detects SARS-CoV-2 exposure and resolves epitopes across the Spike and Nucleocapsid proteins. Two of these represent recurrent reactivities to conserved, functionally-important sites in the Spike S2 subunit, regions that we show are also targeted for the endemic coronaviruses in pre-pandemic controls. At one of these sites, we demonstrate that the SARS-CoV-2 response strongly and recurrently cross-reacts with the endemic virus hCoV-OC43. Our analyses reveal new diagnostic and therapeutic targets, including a site at which SARS-CoV-2 may recruit common pre-existing antibodies and with the potential for broadly-neutralizing responses.

Risk Factors for Severe Acute Respiratory Syndrome Coronavirus 2 Infection in Homeless Shelters in Chicago, Illinois-March-May, 2020.

BACKGROUND: People experiencing homelessness are at increased risk of coronavirus disease 2019 (COVID-19), but little is known about specific risk factors for infection within homeless shelters. METHODS: We performed widespread severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) polymerase chain reaction testing and collected risk factor information at all homeless shelters in Chicago with at least 1 reported case of COVID-19 (n = 21). Multivariable, mixed-effects log-binomial models were built to estimate adjusted prevalence ratios (aPRs) for SARS-CoV-2 infection for both individual- and facility-level risk factors. RESULTS: During March 1 to May 1, 2020, 1717 shelter residents and staff were tested for SARS-CoV-2; 472 (27%) persons tested positive. Prevalence of infection was higher for residents (431 of 1435, 30%) than for staff (41 of 282, 15%) (prevalence ratio = 2.52; 95% confidence interval [CI], 1.78-3.58). The majority of residents with SARS-CoV-2 infection (293 of 406 with available information about symptoms, 72%) reported no symptoms at the time of specimen collection or within the following 2 weeks. Among residents, sharing a room with a large number of people was associated with increased likelihood of infection (aPR for sharing with >20 people compared with single rooms = 1.76; 95% CI, 1.11-2.80), and current smoking was associated with reduced likelihood of infection (aPR = 0.71; 95% CI, 0.60-0.85). At the facility level, a higher proportion of residents leaving and returning each day was associated with increased prevalence (aPR = 1.08; 95% CI, 1.01-1.16), whereas an increase in the number of private bathrooms was associated with reduced prevalence (aPR for 1 additional private bathroom per 100 people = 0.92; 95% CI, 0.87-0.98). CONCLUSIONS: We identified a high prevalence of SARS-CoV-2 infections in homeless shelters. Reducing the number of residents sharing dormitories might reduce the likelihood of SARS-CoV-2 infection. When community transmission is high, limiting movement of persons experiencing homelessness into and out of shelters might also be beneficial.

Profiling alternative splicing on fiber-optic arrays.

The human transcriptome is marked by extensive alternative mRNA splicing and the expression of many closely related genes, which may be difficult to distinguish using standard microarray techniques. Here we describe a sensitive and specific assay for parallel analysis of mRNA isoforms on a fiber-optic microarray platform. The method permits analysis of mRNA transcripts without prior RNA purification or cDNA synthesis. Using an endogenously expressed viral transcript as a model, we demonstrated that the assay readily detects mRNA isoforms from as little as 10-100 pg of total cellular RNA or directly from a few cells. Multiplexed analysis of human cancer cell lines revealed differences in mRNA splicing and suggested a potential autocrine mechanism in the development of choriocarcinomas. Our approach may be useful in the large-scale analysis of the role of alternative splicing in development and disease.

Drosophila crinkled, mutations of which disrupt morphogenesis and cause lethality, encodes fly myosin VIIA.

Myosin VIIs provide motor function for a wide range of eukaryotic processes. We demonstrate that mutations in crinkled (ck) disrupt the Drosophila myosin VIIA heavy chain. The ck/myoVIIA protein is present at a low level throughout fly development and at the same level in heads, thoraxes, and abdomens. Severe ck alleles, likely to be molecular nulls, die as embryos or larvae, but all allelic combinations tested thus far yield a small fraction of adult "escapers" that are weak and infertile. Scanning electron microscopy shows that escapers have defects in bristles and hairs, indicating that this motor protein plays a role in the structure of the actin cytoskeleton. We generate a homology model for the structure of the ck/myosin VIIA head that indicates myosin VIIAs, like myosin IIs, have a spectrin-like, SH3 subdomain fronting their N terminus. In addition, we establish that the two myosin VIIA FERM repeats share high sequence similarity with only the first two subdomains of the three-lobed structure that is typical of canonical FERM domains. Nevertheless, the approximately 100 and approximately 75 amino acids that follow the first two lobes of the first and second FERM domains are highly conserved among myosin VIIs, suggesting that they compose a conserved myosin tail homology 7 (MyTH7) domain that may be an integral part of the FERM domain or may function independently of it. Together, our data suggest a key role for ck/myoVIIA in the formation of cellular projections and other actin-based functions required for viability.

BeadArray technology: enabling an accurate, cost-effective approach to high-throughput genotyping.

The Human Genome Project has opened the door to personalized medicine, provided that human genetic diversity can be analyzed in a high-throughput and cost-effective way Illumina has developed a genotyping system that combines very high throughput and accuracy with low cost per SNP analysis. The system uses our BeadArray platform, a high level of multiplexing, and modular, scalable automation to meet the requirements for cost-effective, genome-wide linkage disequilibrium studies. As implemented in a high-throughput genotyping service facility at Illumina, the system has a current capacity of one million SNP assays per day and is easily expandable. Each SNP call is associated with a quality score that correlates with accuracy

Personalized ovarian cancer disease surveillance and detection of candidate therapeutic drug target in circulating tumor DNA.

Retrospective studies have demonstrated that nearly 50% of patients with ovarian cancer with normal cancer antigen 125 (CA125) levels have persistent disease; however, prospectively distinguishing between patients is currently impossible. Here, we demonstrate that for one patient, with the first reported fibroblast growth factor receptor 2 (FGFR2) fusion transcript in ovarian cancer, circulating tumor DNA (ctDNA) is a more sensitive and specific biomarker than CA125, and it can also inform on a candidate therapeutic. For a 4-year period, during which the patient underwent primary debulking surgery and chemotherapy, tumor recurrences, and multiple chemotherapeutic regimens, blood samples were longitudinally collected and stored. Whereas postsurgical CA125 levels were elevated only three times for 28 measurements, the FGFR2 fusion ctDNA biomarker was readily detectable by quantitative real-time reverse transcription-polymerase chain reaction (PCR) in all of these same blood samples and in the tumor recurrences. Given the persistence of the FGFR2 fusion, we treated tumor cells derived from this patient and others with the FGFR2 inhibitor BGJ398. Only tumor cells derived from this patient were sensitive to FGFR2 inhibitor treatment. Using the same methodologic approach, we demonstrate in a second patient with a different fusion that PCR and agarose gel electrophoresis can also be used to identify tumor-specific DNA in the circulation. Taken together, we demonstrate that a relatively inexpensive, PCR-based ctDNA surveillance assay can outperform CA125 in identifying occult disease.

High-resolution analysis and functional mapping of cleavage sites and substrate proteins of furin in the human proteome.

BACKGROUND: There is a growing appreciation of the role of proteolytic processes in human health and disease, but tools for analysis of such processes on a proteome-wide scale are limited. Furin is a ubiquitous proprotein convertase that cleaves after basic residues and transforms secretory proproteins into biologically active proteins. Despite this important role, many furin substrates remain unknown in the human proteome. METHODOLOGY/PRINCIPAL FINDINGS: We devised an approach for proteinase target identification that combines an in silico discovery pipeline with highly multiplexed proteinase activity assays. We performed in silico analysis of the human proteome and identified over 1,050 secretory proteins as potential furin substrates. We then used a multiplexed protease assay to validate these tentative targets. The assay was carried out on over 3,260 overlapping peptides designed to represent P7-P1' and P4-P4' positions of furin cleavage sites in the candidate proteins. The obtained results greatly increased our knowledge of the unique cleavage preferences of furin, revealed the importance of both short-range (P4-P1) and long-range (P7-P6) interactions in defining furin cleavage specificity, demonstrated that the R-X-R/K/X-R ↓ motif alone is insufficient for predicting furin proteolysis of the substrate, and identified ≈ 490 potential protein substrates of furin in the human proteome. CONCLUSIONS/SIGNIFICANCE: The assignment of these substrates to cellular pathways suggests an important role of furin in development, including axonal guidance, cardiogenesis, and maintenance of stem cell pluripotency. The novel approach proposed in this study can be readily applied to other proteinases.

New and Redesigned pRS Plasmid Shuttle Vectors for Genetic Manipulation of Saccharomycescerevisiae.

We have constructed a set of 42 plasmid shuttle vectors based on the widely used pRS series for use in the budding yeast Saccharomyces cerevisiae and the bacterium Escherichia coli. This set of pRSII plasmids includes new shuttle vectors that can be used with histidine and adenine auxotrophic laboratory yeast strains carrying mutations in the genes HIS2 and ADE1, respectively. Our pRSII plasmids also include updated versions of commonly used pRS plasmids from which common restriction sites that occur within their yeast-selectable biosynthetic marker genes have been removed to increase the availability of unique restriction sites within their polylinker regions. Hence, our pRSII plasmids are a complete set of integrating, centromere and 2µ episomal plasmids with the biosynthetic marker genes ADE2, HIS3, TRP1, LEU2, URA3, HIS2, and ADE1 and a standardized selection of at least 16 unique restriction sites in their polylinkers. Additionally, we have expanded the range of drug selection options that can be used for PCR-mediated homologous replacement using pRS plasmid templates by replacing the G418-resistance kanMX4 cassette of pRS400 with MX4 cassettes encoding resistance to phleomycin, hygromycin B, nourseothricin, and bialaphos. Finally, in the process of generating the new plasmids, we have determined several errors in existing publicly available sequences for several commonly used yeast plasmids. Using our updated sequences, we constructed pRS plasmid backbones with a unique restriction site for inserting new markers to facilitate future expansion of the pRS series.

New details of HCV NS3/4A proteinase functionality revealed by a high-throughput cleavage assay.

BACKGROUND: The hepatitis C virus (HCV) genome encodes a long polyprotein, which is processed by host cell and viral proteases to the individual structural and non-structural (NS) proteins. HCV NS3/4A serine proteinase (NS3/4A) is a non-covalent heterodimer of the N-terminal, ∼180-residue portion of the 631-residue NS3 protein with the NS4A co-factor. NS3/4A cleaves the polyprotein sequence at four specific regions. NS3/4A is essential for viral replication and has been considered an attractive drug target. METHODOLOGY/PRINCIPAL FINDINGS: Using a novel multiplex cleavage assay and over 2,660 peptide sequences derived from the polyprotein and from introducing mutations into the known NS3/4A cleavage sites, we obtained the first detailed fingerprint of NS3/4A cleavage preferences. Our data identified structural requirements illuminating the importance of both the short-range (P1-P1') and long-range (P6-P5) interactions in defining the NS3/4A substrate cleavage specificity. A newly observed feature of NS3/4A was a high frequency of either Asp or Glu at both P5 and P6 positions in a subset of the most efficient NS3/4A substrates. In turn, aberrations of this negatively charged sequence such as an insertion of a positively charged or hydrophobic residue between the negatively charged residues resulted in inefficient substrates. Because NS5B misincorporates bases at a high rate, HCV constantly mutates as it replicates. Our analysis revealed that mutations do not interfere with polyprotein processing in over 5,000 HCV isolates indicating a pivotal role of NS3/4A proteolysis in the virus life cycle. CONCLUSIONS/SIGNIFICANCE: Our multiplex assay technology in light of the growing appreciation of the role of proteolytic processes in human health and disease will likely have widespread applications in the proteolysis research field and provide new therapeutic opportunities.