AAMP and MTSS1 Are Novel Negative Regulators of Endothelial Barrier Function Identified in a Proteomics Screen.

Cell-cell adhesion in endothelial monolayers is tightly controlled and crucial for vascular integrity. Recently, we reported on the importance of fast protein turnover for maintenance of endothelial barrier function. Specifically, continuous ubiquitination and degradation of the Rho GTPase RhoB is crucial to preserve quiescent endothelial integrity. Here, we sought to identify other barrier regulators, which are characterized by a short half-life, using a proteomics approach. Following short-term inhibition of ubiquitination with E1 ligase inhibitor MLN7243 or Cullin E3 ligase inhibitor MLN4924 in primary human endothelial cells, we identified sixty significantly differentially expressed proteins. Intriguingly, our data showed that AAMP and MTSS1 are novel negative regulators of endothelial barrier function and that their turnover is tightly controlled by ubiquitination. Mechanistically, AAMP regulates the stability and activity of RhoA and RhoB, and colocalizes with F-actin and cortactin at membrane ruffles, possibly regulating F-actin dynamics. Taken together, these findings demonstrate the critical role of protein turnover of specific proteins in the regulation of endothelial barrier function, contributing to our options to target dysregulation of vascular permeability.

Insight in the (Phospho)proteome of Vascular Smooth Muscle Cells Derived From Patients With Abdominal Aortic Aneurysm Reveals Novel Disease Mechanisms.

BACKGROUND: Abdominal aortic aneurysm (AAA) is characterized by weakening and dilatation of the aortic wall in the abdomen. The aim of this study was to gain insight into cell-specific mechanisms involved in AAA pathophysiology by analyzing the (phospho)proteome of vascular smooth muscle cells derived from patients with AAA compared with those of healthy donors. METHODS: A (phospho)proteomics analysis based on tandem mass spectrometry was performed on vascular smooth muscle cells derived from patients with AAA (n=24) and healthy, control individuals (C-SMC, n=8). Following protein identification and quantification using MaxQuant, integrative inferred kinase activity analysis was used to calculate kinase activity scores. RESULTS: Expression differences between vascular smooth muscle cells derived from patients with AAA and healthy, control individuals were predominantly found in proteins involved in ECM (extracellular matrix) remodeling (THSD4 [thrombospondin type-1 domain-containing protein 4] and ADAMTS1 [A disintegrin and metalloproteinase with thrombospondin motifs 1]), energy metabolism (GYS1 [glycogen synthase 1] and PCK2 [phosphoenolpyruvate carboxykinase 2, mitochondrial]), and contractility (CACNA2D1 [calcium voltage-dependent channel subunit α-2/δ-1] and TPM1 [tropomyosin α-1 chain]). Phosphorylation patterns on proteins related to actin cytoskeleton organization dominated the phosphoproteome of vascular smooth muscle cells derived from patients with AAA . Besides, phosphorylation changes on proteins related to energy metabolism (GYS1), contractility (PARVA [α-parvin], PPP1R12A [protein phosphatase 1 regulatory subunit 12A], and CALD1 [caldesmon 1]), and intracellular communication (GJA1 [gap junction α-1 protein]) were seen. Kinase activity of NUAK1 (NUAK family SNF1-like kinase 1), FYN (tyrosine-protein kinase Fyn), MAPK7 (mitogen-activated protein kinase 7), and STK10 (serine/threonine kinase 10) was different in vascular smooth muscle cells derived from patients with AAA compared with those from healthy, control individuals. CONCLUSIONS: This study revealed changes in expression and phosphorylation levels of proteins involved in various processes responsible for AAA progression and development (eg, energy metabolism, ECM remodeling, actin cytoskeleton organization, contractility, intracellular communication, and cell adhesion). These newly identified proteins, phosphosites, and related kinases provide further insight into the underlying mechanism of vascular smooth muscle cell dysfunction within the aneurysmal wall. Our omics data thereby offer the opportunity to study the relevance, either as drug target or biomarker, of these proteins in AAA development.

IGF1R signaling induces epithelial-mesenchymal plasticity via ITGAV in cutaneous carcinoma.

BACKGROUND: Early cutaneous squamous cell carcinomas (cSCCs) generally show epithelial differentiation features and good prognosis, whereas advanced cSCCs present mesenchymal traits associated with tumor relapse, metastasis, and poor survival. Currently, the mechanisms involved in cSCC progression are unclear, and the established markers are suboptimal for accurately predicting the clinical course of the disease. METHODS: Using a mouse model of cSCC progression, expression microarray analysis, immunofluorescence and flow cytometry assays, we have identified a prognostic biomarker of tumor relapse, which has been evaluated in a cohort of cSCC patient samples. Phosphoproteomic analysis have revealed signaling pathways induced in epithelial plastic cancer cells that promote epithelial-mesenchymal plasticity (EMP) and tumor progression. These pathways have been validated by genetic and pharmacological inhibition assays. RESULTS: We show that the emergence of epithelial cancer cells expressing integrin αV (ITGAV) promotes cSCC progression to a mesenchymal state. Consistently, ITGAV expression allows the identification of patients at risk of cSCC relapse above the currently employed clinical histopathological parameters. We also demonstrate that activation of insulin-like growth factor-1 receptor (IGF1R) pathway in epithelial cancer cells is necessary to induce EMP and mesenchymal state acquisition in response to tumor microenvironment-derived factors, while promoting ITGAV expression. Likewise, ITGAV knockdown in epithelial plastic cancer cells also blocks EMP acquisition, generating epithelial tumors. CONCLUSIONS: Our results demonstrate that ITGAV is a prognostic biomarker of relapse in cSCCs that would allow improved patient stratification. ITGAV also collaborates with IGF1R to induce EMP in epithelial cancer cells and promotes cSCC progression, revealing a potential therapeutic strategy to block the generation of advanced mesenchymal cSCCs.

Integrating Clinical Phenotype With Multiomics Analyses of Human Cardiac Tissue Unveils Divergent Metabolic Remodeling in Genotype-Positive and Genotype-Negative Patients With Hypertrophic Cardiomyopathy.

BACKGROUND: Hypertrophic cardiomyopathy (HCM) is caused by sarcomere gene mutations (genotype-positive HCM) in ≈50% of patients and occurs in the absence of mutations (genotype-negative HCM) in the other half of patients. We explored how alterations in the metabolomic and lipidomic landscape are involved in cardiac remodeling in both patient groups. METHODS: We performed proteomics, metabolomics, and lipidomics on myectomy samples (genotype-positive N=19; genotype-negative N=22; and genotype unknown N=6) from clinically well-phenotyped patients with HCM and on cardiac tissue samples from sex- and age-matched and body mass index-matched nonfailing donors (N=20). These data sets were integrated to comprehensively map changes in lipid-handling and energy metabolism pathways. By linking metabolomic and lipidomic data to variability in clinical data, we explored patient group-specific associations between cardiac and metabolic remodeling. RESULTS: HCM myectomy samples exhibited (1) increased glucose and glycogen metabolism, (2) downregulation of fatty acid oxidation, and (3) reduced ceramide formation and lipid storage. In genotype-negative patients, septal hypertrophy and diastolic dysfunction correlated with lowering of acylcarnitines, redox metabolites, amino acids, pentose phosphate pathway intermediates, purines, and pyrimidines. In contrast, redox metabolites, amino acids, pentose phosphate pathway intermediates, purines, and pyrimidines were positively associated with septal hypertrophy and diastolic impairment in genotype-positive patients. CONCLUSIONS: We provide novel insights into both general and genotype-specific metabolic changes in HCM. Distinct metabolic alterations underlie cardiac disease progression in genotype-negative and genotype-positive patients with HCM.

msproteomics sitereport: reporting DIA-MS phosphoproteomics experiments at site level with ease.

SUMMARY: Identification and quantification of phosphorylation sites are essential for biological interpretation of a phosphoproteomics experiment. For data independent acquisition mass spectrometry-based (DIA-MS) phosphoproteomics, extracting a site-level report from the output of current processing software is not straightforward as multiple peptides might contribute to a single site, multiple phosphorylation sites can occur on the same peptides, and protein isoforms complicate site specification. Currently only limited support is available from a commercial software package via a platform-specific solution with a rather simple site quantification method. Here, we present sitereport, a software tool implemented in an extendable Python package called msproteomics to report phosphosites and phosphopeptides from a DIA-MS phosphoproteomics experiment with a proven quantification method called MaxLFQ. We demonstrate the use of sitereport for downstream data analysis at site level, allowing benchmarking different DIA-MS processing software tools. AVAILABILITY AND IMPLEMENTATION: sitereport is available as a command line tool in the Python package msproteomics, released under the Apache License 2.0 and available from the Python Package Index (PyPI) at https://pypi.org/project/msproteomics and GitHub at https://github.com/tvpham/msproteomics.

VRK1 Regulates Sensitivity to Oxidative Stress by Altering Histone Epigenetic Modifications and the Nuclear Phosphoproteome in Tumor Cells.

The chromatin organization and its dynamic remodeling determine its accessibility and sensitivity to DNA damage oxidative stress, the main source of endogenous DNA damage. We studied the role of the VRK1 chromatin kinase in the response to oxidative stress. which alters the nuclear pattern of histone epigenetic modifications and phosphoproteome pathways. The early effect of oxidative stress on chromatin was studied by determining the levels of 8-oxoG lesions and the alteration of the epigenetic modification of histones. Oxidative stress caused an accumulation of 8-oxoG DNA lesions that were increased by VRK1 depletion, causing a significant accumulation of DNA strand breaks detected by labeling free 3'-DNA ends. In addition, oxidative stress altered the pattern of chromatin epigenetic marks and the nuclear phosphoproteome pathways that were impaired by VRK1 depletion. Oxidative stress induced the acetylation of H4K16ac and H3K9 and the loss of H3K4me3. The depletion of VRK1 altered all these modifications induced by oxidative stress and resulted in losses of H4K16ac and H3K9ac and increases in the H3K9me3 and H3K4me3 levels. All these changes were induced by the oxidative stress in the epigenetic pattern of histones and impaired by VRK1 depletion, indicating that VRK1 plays a major role in the functional reorganization of chromatin in the response to oxidative stress. The analysis of the nuclear phosphoproteome in response to oxidative stress detected an enrichment of the phosphorylated proteins associated with the chromosome organization and chromatin remodeling pathways, which were significantly decreased by VRK1 depletion. VRK1 depletion alters the histone epigenetic pattern and nuclear phosphoproteome pathways in response to oxidative stress. The enzymes performing post-translational epigenetic modifications are potential targets in synthetic lethality strategies for cancer therapies.

Protein homeostasis maintained by HOOK1 levels promotes the tumorigenic and stemness properties of ovarian cancer cells through reticulum stress and autophagy.

BACKGROUND: Ovarian cancer has a high mortality rate mainly due to its resistance to currently used therapies. This resistance has been associated with the presence of cancer stem cells (CSCs), interactions with the microenvironment, and intratumoral heterogeneity. Therefore, the search for new therapeutic targets, particularly those targeting CSCs, is important for improving patient prognosis. HOOK1 has been found to be transcriptionally altered in a substantial percentage of ovarian tumors, but its role in tumor initiation and development is still not fully understood. METHODS: The downregulation of HOOK1 was performed in ovarian cancer cell lines using CRISPR/Cas9 technology, followed by growth in vitro and in vivo assays. Subsequently, migration (Boyden chamber), cell death (Western-Blot and flow cytometry) and stemness properties (clonal heterogeneity analysis, tumorspheres assay and flow cytometry) of the downregulated cell lines were analysed. To gain insights into the specific mechanisms of action of HOOK1 in ovarian cancer, a proteomic analysis was performed, followed by Western-blot and cytotoxicity assays to confirm the results found within the mass spectrometry. Immunofluorescence staining, Western-blotting and flow cytometry were also employed to finish uncovering the role of HOOK1 in ovarian cancer. RESULTS: In this study, we observed that reducing the levels of HOOK1 in ovarian cancer cells reduced in vitro growth and migration and prevented tumor formation in vivo. Furthermore, HOOK1 reduction led to a decrease in stem-like capabilities in these cells, which, however, did not seem related to the expression of genes traditionally associated with this phenotype. A proteome study, along with other analysis, showed that the downregulation of HOOK1 also induced an increase in endoplasmic reticulum stress levels in these cells. Finally, the decrease in stem-like properties observed in cells with downregulated HOOK1 could be explained by an increase in cell death in the CSC population within the culture due to endoplasmic reticulum stress by the unfolded protein response. CONCLUSION: HOOK1 contributes to maintaining the tumorigenic and stemness properties of ovarian cancer cells by preserving protein homeostasis and could be considered an alternative therapeutic target, especially in combination with inducers of endoplasmic reticulum or proteotoxic stress such as proteasome inhibitors.

Kinase activities in pancreatic ductal adenocarcinoma with prognostic and therapeutic avenues.

Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease with a limited number of known driver mutations but considerable cancer cell heterogeneity. Phosphoproteomics provides a direct read-out of aberrant signaling and the resultant clinically relevant phenotype. Mass spectrometry (MS)-based proteomics and phosphoproteomics were applied to 42 PDAC tumors. Data encompassed over 19 936 phosphoserine or phosphothreonine (pS/T; in 5412 phosphoproteins) and 1208 phosphotyrosine (pY; in 501 phosphoproteins) sites and a total of 3756 proteins. Proteome data identified three distinct subtypes with tumor intrinsic and stromal features. Subsequently, three phospho-subtypes were apparent: two tumor intrinsic (Phos1/2) and one stromal (Phos3), resembling known PDAC molecular subtypes. Kinase activity was analyzed by the Integrative iNferred Kinase Activity (INKA) scoring. Phospho-subtypes displayed differential phosphorylation signals and kinase activity, such as FGR and GSK3 activation in Phos1, SRC kinase family and EPHA2 in Phos2, and EGFR, INSR, MET, ABL1, HIPK1, JAK, and PRKCD in Phos3. Kinase activity analysis of an external PDAC cohort supported our findings and underscored the importance of PI3K/AKT and ERK pathways, among others. Interestingly, unfavorable patient prognosis correlated with higher RTK, PAK2, STK10, and CDK7 activity and high proliferation, whereas long survival was associated with MYLK and PTK6 activity, which was previously unknown. Subtype-associated activity profiles can guide therapeutic combination approaches in tumor and stroma-enriched tissues, and emphasize the critical role of parallel signaling pathways. In addition, kinase activity profiling identifies potential disease markers with prognostic significance.

Loss of VRK1 alters the nuclear phosphoproteome in the DNA damage response to doxorubicin.

Dynamic chromatin remodeling requires regulatory mechanisms for its adaptation to different nuclear function, which are likely to be mediated by signaling proteins. In this context, VRK1 is a nuclear Ser-Thr kinase that regulates pathways associated with transcription, replication, recombination, and DNA repair. Therefore, VRK1 is a potential regulatory, or coordinator, molecule in these processes. In this work we studied the effect that VRK1 depletion has on the basal nuclear and chromatin phosphoproteome, and their associated pathways. VRK1 depletion caused an alteration in the pattern of the nuclear phosphoproteome, which is mainly associated with nucleoproteins, ribonucleoproteins, RNA splicing and processing. Next, it was determined the changes in proteins associated with DNA damage that was induced by doxorubicin treatment. Doxorubicin alters the nuclear phosphoproteome affecting proteins implicated in DDR, including DSB repair proteins NBN and 53BP1, cellular response to stress and chromatin organization proteins. In VRK1-depleted cells, the effect of doxorubicin on protein phosphorylation was reverted to basal levels. The nuclear phosphoproteome patterns induced by doxorubicin are altered by VRK1 depletion, and is enriched in histone modification proteins and chromatin associated proteins. These results indicate that VRK1 plays a major role in processes requiring chromatin remodeling in its adaptation to different biological contexts.

Protein kinase D drives the secretion of invasion mediators in triple-negative breast cancer cell lines.

The protein kinase D (PKD) family members regulate the fission of cargo vesicles at the Golgi complex and play a pro-oncogenic role in triple-negative breast cancer (TNBC). Whether PKD facilitates the secretion of tumor-promoting factors in TNBC, however, is still unknown. Using the pharmacological inhibition of PKD activity and siRNA-mediated depletion of PKD2 and PKD3, we identified the PKD-dependent secretome of the TNBC cell lines MDA-MB-231 and MDA-MB-468. Mass spectrometry-based proteomics and antibody-based assays revealed a significant downregulation of extracellular matrix related proteins and pro-invasive factors such as LIF, MMP-1, MMP-13, IL-11, M-CSF and GM-CSF in PKD-perturbed cells. Notably, secretion of these proteins in MDA-MB-231 cells was predominantly controlled by PKD2 and enhanced spheroid invasion. Consistently, PKD-dependent secretion of pro-invasive factors was more pronounced in metastatic TNBC cell lines. Our study thus uncovers a novel role of PKD2 in releasing a pro-invasive secretome.

Secretome processing for proteomics: A methods comparison.

The cancer cell secretome comprises a treasure-trove for biomarkers since it reflects cross-talk between tumor cells and their surrounding environment with high detectability in biofluids. In this study, we evaluated six secretome sample processing workflows coupled to single-shot mass spectrometry: (1) Protein concentration by ultrafiltration with a molecular weight cut-off (MWCO) filter and sample preparation through in-gel digestion (IGD); (2) Acetone protein precipitation coupled to IGD; (3) MWCO filter-based protein concentration followed by to in-solution digestion (ISD); (4) Acetone protein precipitation coupled to ISD; (5) Direct ISD; (6) Secretome lyophilization and ISD. To this end, we assessed workflow triplicates in terms of total number of protein identifications, unique identifications, reproducibility of protein identification and quantification and detectability of small proteins with important functions in cancer biology such as cytokines, chemokines, and growth factors. Our findings revealed that acetone protein precipitation coupled to ISD outperformed the other methods in terms of the number of identified proteins (2246) and method reproducibility (correlation coefficient between replicates (r = 0.94, CV = 19%). Overall, especially small proteins such as those from the classes mentioned above were better identified using ISD workflows. Concluding, herein we report that secretome protein precipitation coupled to ISD is the method of choice for high-throughput secretome proteomics via single shot nanoLC-MS/MS.

Candidate biomarkers for treatment benefit from sunitinib in patients with advanced renal cell carcinoma using mass spectrometry-based (phospho)proteomics.

The tyrosine kinase inhibitor sunitinib is an effective first-line treatment for patients with advanced renal cell carcinoma (RCC). Hypothesizing that a functional read-out by mass spectrometry-based (phospho, p-)proteomics will identify predictive biomarkers for treatment outcome of sunitinib, tumor tissues of 26 RCC patients were analyzed. Eight patients had primary resistant (RES) and 18 sensitive (SENS) RCC. A 78 phosphosite signature (p < 0.05, fold-change > 2) was identified; 22 p-sites were upregulated in RES (unique in RES: BCAR3, NOP58, EIF4A2, GDI1) and 56 in SENS (35 unique). EIF4A1/EIF4A2 were differentially expressed in RES at the (p-)proteome and, in an independent cohort, transcriptome level. Inferred kinase activity of MAPK3 (p = 0.026) and EGFR (p = 0.045) as determined by INKA was higher in SENS. Posttranslational modifications signature enrichment analysis showed that different p-site-centric signatures were enriched (p < 0.05), of which FGF1 and prolactin pathways in RES and, in SENS, vanadate and thrombin treatment pathways, were most significant. In conclusion, the RCC (phospho)proteome revealed differential p-sites and kinase activities associated with sunitinib resistance and sensitivity. Independent validation is warranted to develop an assay for upfront identification of patients who are intrinsically resistant to sunitinib.

Identification of protein biomarkers for prediction of response to platinum-based treatment regimens in patients with non-small cell lung cancer.

The majority of patients with resected stage II-IIIA non-small cell lung cancer (NSCLC) are treated with platinum-based adjuvant chemotherapy (ACT) in a one-size-fits-all approach. However, a significant number of patients do not derive clinical benefit, and no predictive patient selection biomarker is currently available. Using mass spectrometry-based proteomics, we have profiled tumour resection material of 2 independent, multi-centre cohorts of in total 67 patients with NSCLC who underwent ACT. Unsupervised cluster analysis of both cohorts revealed a poor response/survival sub-cluster composed of ~ 25% of the patients, that displayed a strong epithelial-mesenchymal transition signature and stromal phenotype. Beyond this stromal sub-population, we identified and validated platinum response prediction biomarker candidates involved in pathways relevant to the mechanism of action of platinum drugs, such as DNA damage repair, as well as less anticipated processes such as those related to the regulation of actin cytoskeleton. Integration with pre-clinical proteomics data supported a role for several of these candidate proteins in platinum response prediction. Validation of one of the candidates (HMGB1) in a third independent patient cohort using immunohistochemistry highlights the potential of translating these proteomics results to clinical practice.

Phosphoproteomics of patient-derived xenografts identifies targets and markers associated with sensitivity and resistance to EGFR blockade in colorectal cancer.

Epidermal growth factor receptor (EGFR) is a well-exploited therapeutic target in metastatic colorectal cancer (mCRC). Unfortunately, not all patients benefit from current EGFR inhibitors. Mass spectrometry-based proteomics and phosphoproteomics were performed on 30 genomically and pharmacologically characterized mCRC patient-derived xenografts (PDXs) to investigate the molecular basis of response to EGFR blockade and identify alternative drug targets to overcome resistance. Both the tyrosine and global phosphoproteome as well as the proteome harbored distinctive response signatures. We found that increased pathway activity related to mitogen-activated protein kinase (MAPK) inhibition and abundant tyrosine phosphorylation of cell junction proteins, such as CXADR and CLDN1/3, in sensitive tumors, whereas epithelial-mesenchymal transition and increased MAPK and AKT signaling were more prevalent in resistant tumors. Furthermore, the ranking of kinase activities in single samples confirmed the driver activity of ERBB2, EGFR, and MET in cetuximab-resistant tumors. This analysis also revealed high kinase activity of several members of the Src and ephrin kinase family in 2 CRC PDX models with genomically unexplained resistance. Inhibition of these hyperactive kinases, alone or in combination with cetuximab, resulted in growth inhibition of ex vivo PDX-derived organoids and in vivo PDXs. Together, these findings highlight the potential value of phosphoproteomics to improve our understanding of anti-EGFR treatment and response prediction in mCRC and bring to the forefront alternative drug targets in cetuximab-resistant tumors.

Potential diagnostic value of CSF metabolism-related proteins across the Alzheimer's disease continuum.

BACKGROUND: Alzheimer's disease (AD) cerebrospinal fluid (CSF) core biomarkers (Aß42/40 ratio, p-tau, and t-tau) provide high diagnostic accuracy, even at the earliest stage of disease. However, these markers do not fully reflect the complex AD pathophysiology. Recent large scale CSF proteomic studies revealed several new AD candidate biomarkers related to metabolic pathways. In this study we measured the CSF levels of four metabolism-related proteins not directly linked to amyloid- and tau-pathways (i.e., pyruvate kinase, PKM; aldolase, ALDO; ubiquitin C-terminal hydrolase L1, UCHL1, and fatty acid-binding protein 3, FABP3) across the AD continuum. We aimed at validating the potential value of these proteins as new CSF biomarkers for AD and their possible involvement in AD pathogenesis, with specific interest on the preclinical phase of the disease. METHODS: CSF PKM and ALDO activities were measured with specific enzyme assays while UCHL1 and FABP3 levels were measured with immunoassays in a cohort of patients composed as follows: preclinical AD (pre-AD, n = 19, cognitively unimpaired), mild cognitive impairment due to AD (MCI-AD, n = 50), dementia due to AD (ADdem, n = 45), and patients with frontotemporal dementia (FTD, n = 37). Individuals with MCI not due to AD (MCI, n = 30) and subjective cognitive decline (SCD, n = 52) with negative CSF AD-profile, were enrolled as control groups. RESULTS: CSF UCHL1 and FABP3 levels, and PKM activity were significantly increased in AD patients, already at the pre-clinical stage. CSF PKM activity was also increased in FTD patients compared with control groups, being similar between AD and FTD patients. No difference was found in ALDO activity among the groups. UCHL1 showed good performance in discriminating early AD patients (pre-AD and MCI-AD) from controls (AUC ~ 0.83), as assessed by ROC analysis. Similar results were obtained for FABP3. Conversely, PKM provided the best performance when comparing FTD vs. MCI (AUC = 0.80). Combination of PKM, FABP3, and UCHL1 improved the diagnostic accuracy for the detection of patients within the AD continuum when compared with single biomarkers. CONCLUSIONS: Our study confirmed the potential role of UCHL1 and FABP3 as neurodegenerative biomarkers for AD. Furthermore, our results validated the increase of PKM activity in CSF of AD patients, already at the preclinical phase of the disease. Increased PKM activity was observed also in FTD patients, possibly underlining similar alterations in energy metabolism in AD and FTD.

DPHL v.2: An updated and comprehensive DIA pan-human assay library for quantifying more than 14,000 proteins.

A comprehensive pan-human spectral library is critical for biomarker discovery using mass spectrometry (MS)-based proteomics. DPHL v.1, a previous pan-human library built from 1,096 data-dependent acquisition (DDA) MS data of 16 human tissue types, allows quantifying of 10,943 proteins. Here, we generated DPHL v.2 from 1,608 DDA-MS data. The data included 586 DDA-MS data acquired from 18 tissue types, while 1,022 files were derived from DPHL v.1. DPHL v.2 thus comprises data from 24 sample types, including several cancer types (lung, breast, kidney, and prostate cancer, among others). We generated four variants of DPHL v.2 to include semi-tryptic peptides and protein isoforms. DPHL v.2 was then applied to two colorectal cancer cohorts. The numbers of identified and significantly dysregulated proteins increased by at least 21.7% and 14.2%, respectively, compared with DPHL v.1. Our findings show that the increased human proteome coverage of DPHL v.2 provides larger pools of potential protein biomarkers.

Atrial epicardial adipose tissue abundantly secretes myeloperoxidase and activates atrial fibroblasts in patients with atrial fibrillation.

BACKGROUND: Epicardial adipose tissue (EAT) secretome induces fibrosis. Fibrosis, primarily extracellular matrix (ECM) produced by fibroblasts, creates a substrate for atrial fibrillation (AF). Whether the EAT secretome from patients with AF activates human atrial fibroblasts and through which components, remains unexplored. RESEARCH AIMS: (a) To investigate if the EAT secretome from patients with versus without AF increases ECM production in atrial fibroblasts. (b) To identify profibrotic proteins and processes in the EAT secretome and EAT from patients with, who will develop (future onset), and without AF. METHODS: Atrial EAT was obtainded during thoracoscopic ablation (AF, n = 20), or open-heart surgery (future onset and non-AF, n = 35). ECM gene expression of human atrial fibroblasts exposed to the EAT secretome and the proteomes of EAT secretome and EAT were assessed in patients with and without AF. Myeloperoxidase and neutrophil extracellular traps (NETs) were assessed immunohistochemically in patients with paroxysmal, persistent, future onset, and those who remain free of AF (non-AF). RESULTS: The expression of COL1A1 and FN1 in fibroblasts exposed to secretome from patients with AF was 3.7 and 4.7 times higher than in patients without AF (p < 0.05). Myeloperoxidase was the most increased protein in the EAT secretome and EAT from patients with versus without AF (FC 18.07 and 21.57, p < 0.005), as was the gene-set neutrophil degranulation. Immunohistochemically, myeloperoxidase was highest in persistent (FC 13.3, p < 0.0001) and increased in future onset AF (FC 2.4, p = 0.02) versus non-AF. Myeloperoxidase aggregated subepicardially and around fibrofatty infiltrates. NETs were increased in patients with persistent versus non-AF (p = 0.03). CONCLUSION: In AF, the EAT secretome induces ECM gene expression in atrial fibroblasts and contains abundant myeloperoxidase. EAT myeloperoxidase was increased prior to AF onset, and both myeloperoxidase and NETs were highest in persistent AF, highlighting the role of EAT neutrophils in the pathophysiology of AF.

Phosphoproteomics guides effective low-dose drug combinations against pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease with a limited set of known driver mutations but considerable cancer cell heterogeneity. Phosphoproteomics provides a readout of aberrant signaling and has the potential to identify new targets and guide treatment decisions. Using two-step sequential phosphopeptide enrichment, we generate a comprehensive phosphoproteome and proteome of nine PDAC cell lines, encompassing more than 20,000 phosphosites on 5,763 phospho-proteins, including 316 protein kinases. By using integrative inferred kinase activity (INKA) scoring, we identify multiple (parallel) activated kinases that are subsequently matched to kinase inhibitors. Compared with high-dose single-drug treatments, INKA-tailored low-dose 3-drug combinations against multiple targets demonstrate superior efficacy against PDAC cell lines, organoid cultures, and patient-derived xenografts. Overall, this approach is particularly more effective against the aggressive mesenchymal PDAC model compared with the epithelial model in both preclinical settings and may contribute to improved treatment outcomes in PDAC patients.

C1q is increased in cerebrospinal fluid-derived extracellular vesicles in Alzheimer's disease: A multi-cohort proteomics and immuno-assay validation study.

INTRODUCTION: Extracellular vesicles (EVs) may propagate and modulate Alzheimer's disease (AD) pathology. We aimed to comprehensively characterize the proteome of cerebrospinal fluid (CSF) EVs to identify proteins and pathways altered in AD. METHODS: CSF EVs were isolated by ultracentrifugation (Cohort 1) or Vn96 peptide (Cohort 2) from non-neurodegenerative controls (n = 15, 16) and AD patients (n = 22, 20, respectively). EVs were subjected to untargeted quantitative mass spectrometry-based proteomics. Results were validated by enzyme-linked immunosorbent assay (ELISA) in Cohorts 3 and 4, consisting of controls (n = 16, n = 43, (Cohort3, Cohort4)), and patients with AD (n = 24, n = 100). RESULTS: We found > 30 differentially expressed proteins in AD CSF EVs involved in immune-regulation. Increase of C1q levels in AD compared to non-demented controls was validated by ELISA (∼ 1.5 fold, p (Cohort 3) = 0.03, p (Cohort 4) = 0.005). DISCUSSION: EVs may be utilized as a potential biomarker and may play a so far unprecedented role in immune-regulation in AD.

Advancing wide implementation of precision oncology: A liquid nitrogen-free snap freezer preserves molecular profiles of biological samples.

PURPOSE: In precision oncology, tumor molecular profiles guide selection of therapy. Standardized snap freezing of tissue biospecimens is necessary to ensure reproducible, high-quality samples that preserve tumor biology for adequate molecular profiling. Quenching in liquid nitrogen (LN2 ) is the golden standard method, but LN2 has several limitations. We developed a LN2 -independent snap freezer with adjustable cold sink temperature. To benchmark this device against the golden standard, we compared molecular profiles of biospecimens. METHODS: Cancer cell lines and core needle normal tissue biopsies from five patients' liver resection specimens were used to compare mass spectrometry (MS)-based global phosphoproteomic and RNA sequencing profiles and RNA integrity obtained by both freezing methods. RESULTS: Unsupervised cluster analysis of phosphoproteomic and transcriptomic profiles of snap freezer versus LN2 -frozen K562 samples and liver biopsies showed no separation based on freezing method (with Pearson's r 0.96 (range 0.92-0.98) and >0.99 for K562 profiles, respectively), while samples with +2 h bench-time formed a separate cluster. RNA integrity was also similar for both snap freezing methods. Molecular profiles of liver biopsies were clearly identified per individual patient regardless of the applied freezing method. Two to 25 s freezing time variations did not induce profiling differences in HCT116 samples. CONCLUSION: The novel snap freezer preserves high-quality biospecimen and allows identification of individual patients' molecular profiles, while overcoming important limitations of the use of LN2 . This snap freezer may provide a useful tool in clinical cancer research and practice, enabling a wider implementation of (multi-)omics analyses for precision oncology.