Proteomics of prostate cancer serum and plasma using low and high throughput approaches.

Despite progress, MS-based proteomics in biofluids, especially blood, faces challenges such as dynamic range and throughput limitations in biomarker and disease studies. In this work, we used cutting-edge proteomics technologies to construct label-based and label-free workflows, capable of quantifying approximately 2,000 proteins in biofluids. With 70µL of blood and a single depletion strategy, we conducted an analysis of a homogenous cohort (n = 32), comparing medium-grade prostate cancer patients (Gleason score: 7(3 + 4); TNM stage: T2cN0M0, stage IIB) to healthy donors. The results revealed dozens of differentially expressed proteins in both plasma and serum. We identified the upregulation of Prostate Specific Antigen (PSA), a well-known biomarker for prostate cancer, in the serum of cancer cohort. Further bioinformatics analysis highlighted noteworthy proteins which appear to be differentially secreted into the bloodstream, making them good candidates for further exploration.

Mechanistic safety assessment via multi-omic characterisation of systemic pathway perturbations following in vivo MAT2A inhibition.

The tumour suppressor p16/CDKN2A and the metabolic gene, methyl-thio-adenosine phosphorylase (MTAP), are frequently co-deleted in some of the most aggressive and currently untreatable cancers. Cells with MTAP deletion are vulnerable to inhibition of the metabolic enzyme, methionine-adenosyl transferase 2A (MAT2A), and the protein arginine methyl transferase (PRMT5). This synthetic lethality has paved the way for the rapid development of drugs targeting the MAT2A/PRMT5 axis. MAT2A and its liver- and pancreas-specific isoform, MAT1A, generate the universal methyl donor S-adenosylmethionine (SAM) from ATP and methionine. Given the pleiotropic role SAM plays in methylation of diverse substrates, characterising the extent of SAM depletion and downstream perturbations following MAT2A/MAT1A inhibition (MATi) is critical for safety assessment. We have assessed in vivo target engagement and the resultant systemic phenotype using multi-omic tools to characterise response to a MAT2A inhibitor (AZ'9567). We observed significant SAM depletion and extensive methionine accumulation in the plasma, liver, brain and heart of treated rats, providing the first assessment of both global SAM depletion and evidence of hepatic MAT1A target engagement. An integrative analysis of multi-omic data from liver tissue identified broad perturbations in pathways covering one-carbon metabolism, trans-sulfuration and lipid metabolism. We infer that these pathway-wide perturbations represent adaptive responses to SAM depletion and confer a risk of oxidative stress, hepatic steatosis and an associated disturbance in plasma and cellular lipid homeostasis. The alterations also explain the dramatic increase in plasma and tissue methionine, which could be used as a safety and PD biomarker going forward to the clinic.

Characterization of the Secretome, Transcriptome, and Proteome of Human ß Cell Line EndoC-ßH1.

Early diabetes research is hampered by limited availability, variable quality, and instability of human pancreatic islets in culture. Little is known about the human ß cell secretome, and recent studies question translatability of rodent ß cell secretory profiles. Here, we verify representativeness of EndoC-ßH1, one of the most widely used human ß cell lines, as a translational human ß cell model based on omics and characterize the EndoC-ßH1 secretome. We profiled EndoC-ßH1 cells using RNA-seq, data-independent acquisition, and tandem mass tag proteomics of cell lysate. Omics profiles of EndoC-ßH1 cells were compared to human ß cells and insulinomas. Secretome composition was assessed by data-independent acquisition proteomics. Agreement between EndoC-ßH1 cells and primary adult human ß cells was ∼90% for global omics profiles as well as for ß cell markers, transcription factors, and enzymes. Discrepancies in expression were due to elevated proliferation rate of EndoC-ßH1 cells compared to adult ß cells. Consistently, similarity was slightly higher with benign nonmetastatic insulinomas. EndoC-ßH1 secreted 783 proteins in untreated baseline state and 3135 proteins when stressed with nontargeting control siRNA, including known ß cell hormones INS, IAPP, and IGF2. Further, EndoC-ßH1 secreted proteins known to generate bioactive peptides such as granins and enzymes required for production of bioactive peptides. EndoC-ßH1 secretome contained an unexpectedly high proportion of predicted extracellular vesicle proteins. We believe that secretion of extracellular vesicles and bioactive peptides warrant further investigation with specialized proteomics workflows in future studies.

Proprotein convertase PCSK9 affects expression of key surface proteins in human pancreatic beta cells via intracellular and extracellular regulatory circuits.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is involved in the degradation of the low-density lipoprotein receptor. PCSK9 also targets proteins involved in lipid metabolism (very low-density lipoprotein receptor), immunity (major histocompatibility complex I), and viral infection (cluster of differentiation 81). Recent studies have also indicated that PCSK9 loss-of-function mutations are associated with an increased incidence of diabetes; however, the expression and function of PCSK9 in insulin-producing pancreatic beta cells remain unclear. Here, we studied PCSK9 regulation and function by performing loss- and gain-of-function experiments in the human beta cell line EndoC-ßH1. We demonstrate that PCSK9 is expressed and secreted by EndoC-ßH1 cells. We also found that PCSK9 expression is regulated by cholesterol and sterol regulatory element-binding protein transcription factors, as previously demonstrated in other cell types such as hepatocytes. Importantly, we show that PCSK9 knockdown using siRNA results in deregulation of various elements of the transcriptome, proteome, and secretome, and increases insulin secretion. We also observed that PCSK9 decreases low-density lipoprotein receptor and very low-density lipoprotein receptor levels via an extracellular signaling mechanism involving exogenous PCSK9, as well as levels of cluster of differentiation 36, a fatty acid transporter, through an intracellular signaling mechanism. Finally, we found that PCSK9 regulates the cell surface expression of PDL1 and HLA-ABC, proteins involved in cell-lymphocyte interaction, also via an intracellular mechanism. Collectively, these results highlight PCSK9 as a regulator of multiple cell surface receptors in pancreatic beta cells.

Affi-BAMS™: A Robust Targeted Proteomics Microarray Platform to Measure Histone Post-Translational Modifications.

For targeted protein panels, the ability to specifically assay post-translational modifications (PTMs) in a quantitative, sensitive, and straightforward manner would substantially advance biological and pharmacological studies. The present study highlights the effectiveness of the Affi-BAMS™ epitope-directed affinity bead capture/MALDI MS platform for quantitatively defining complex PTM marks of H3 and H4 histones. Using H3 and H4 histone peptides and isotopically labelled derivatives, this affinity bead and MALDI MS platform achieves a range of >3 orders of magnitude with a technical precision CV of <5%. Using nuclear cellular lysates, Affi-BAMS PTM-peptide capture resolves heterogeneous histone N-terminal PTMs with as little as 100 µg of starting material. In an HDAC inhibitor and MCF7 cell line model, the ability to monitor dynamic histone H3 acetylation and methylation events is further demonstrated (including SILAC quantification). Affi-BAMS (and its capacity for the multiplexing of samples and target PTM-proteins) thus provides a uniquely efficient and effective approach for analyzing dynamic epigenetic histone marks, which is critical for the regulation of chromatin structure and gene expression.

Chemical biology approaches to study histone interactors.

Protein-protein interactions (PPIs) in the nucleus play key roles in transcriptional regulation and ensure genomic stability. Critical to this are histone-mediated PPI networks, which are further fine-tuned through dynamic post-translational modification. Perturbation to these networks leads to genomic instability and disease, presenting epigenetic proteins as key therapeutic targets. This mini-review will describe progress in mapping the combinatorial histone PTM landscape, and recent chemical biology approaches to map histone interactors. Recent advances in mapping direct interactors of histone PTMs as well as local chromatin interactomes will be highlighted, with a focus on mass-spectrometry based workflows that continue to illuminate histone-mediated PPIs in unprecedented detail.

Affinity-Bead Assisted Mass Spectrometry (Affi-BAMS): A Multiplexed Microarray Platform for Targeted Proteomics.

The ability to quantitatively probe diverse panels of proteins and their post-translational modifications (PTMs) across multiple samples would aid a broad spectrum of biological, biochemical and pharmacological studies. We report a novel, microarray analytical technology that combines immuno-affinity capture with Matrix Assisted Laser Desorption Ionization Mass Spectrometry (MALDI MS), which is capable of supporting highly multiplexed, targeted proteomic assays. Termed "Affinity-Bead Assisted Mass Spectrometry" (Affi-BAMS), this LC-free technology enables development of highly specific and customizable assay panels for simultaneous profiling of multiple proteins and PTMs. While affinity beads have been used previously in combination with MS, the Affi-BAMS workflow uses enrichment on a single bead that contains one type of antibody, generally capturing a single analyte (protein or PTM) while having enough binding capacity to enable quantification within approximately 3 orders of magnitude. The multiplexing capability is achieved by combining Affi-BAMS beads with different protein specificities. To enable screening of bead-captured analytes by MS, we further developed a novel method of performing spatially localized elution of targets from individual beads arrayed on a microscope slide. The resulting arrays of micro spots contain highly concentrated analytes localized within 0.5 mm diameter spots that can be directly measured using MALDI MS. While both intact proteins and protein fragments can be monitored by Affi-BAMS, we initially focused on applying this technology for bottom-up proteomics to enable screening of hundreds of samples per day by combining the robust magnetic bead-based workflow with the high throughput nature of MALDI MS acquisition. To demonstrate the variety of applications and robustness of Affi-BAMS, several studies are presented that focus on the response of 4EBP1, RPS6, ERK1/ERK2, mTOR, Histone H3 and C-MET to stimuli including rapamycin, H2O2, EPO, SU11274, Staurosporine and Vorinostat.

Mechanistic Insights into a CDK9 Inhibitor Via Orthogonal Proteomics Methods.

Cyclin-dependent-kinases (CDKs) are members of the serine/threonine kinase family and are highly regulated by cyclins, a family of regulatory subunits that bind to CDKs. CDK9 represents one of the most studied examples of these transcriptional CDKs. CDK9 forms a heterodimeric complex with its regulatory subunit cyclins T1, T2 and K to form the positive transcription elongation factor b (P-TEFb). This complex regulates transcription via the phosphorylation of RNA polymerase II (RNAPolII) on Ser-2, facilitating promoter clearance and transcription elongation and thus remains an attractive therapeutic target. Herein, we have utilized classical affinity purification chemical proteomics, kinobeads assay, compressed CEllular Thermal Shift Assay (CETSA)-MS and Limited Proteolysis (LiP) to study the selectivity, target engagement and downstream mechanistic insights of a CDK9 tool compound. The above experiments highlight the value of quantitative mass spectrometry approaches to drug discovery, specifically proteome wide target identification and selectivity profiling. The approaches utilized in this study unanimously indicated that the CDK family of kinases are the main target of the compound of interest, with CDK9, showing the highest target affinity with remarkable consistency across approaches. We aim to provide guidance to the scientific community on the available chemical biology/proteomic tools to study advanced lead molecules and to highlight pros and cons of each technology while describing our findings in the context of the CDKs biology.