Targeted Proteomics Approach Research Articles

Regulation of NKCC2 Ubiquitination by baSubstrate Recognition Adaptor FBXL13 in Thick Ascending Limbs

NaCl reabsorption by the thick ascending limb (TAL) is mediated by the Na/K/2Cl cotransporter (NKCC2). Nitric Oxide and atrial natriuretic peptide decrease NaCl absorption in the TAL by increasing the second messenger cyclic guanosine monophosphate (cGMP). We recently showed that NKCC2 is constitutively ubiquitinated, and cGMP enhances the rate of ubiquitination. The cGMP-dependent increase in NKCC2 ubiquitination is mediated by the CRL (Cullin-Ring-E3 ubiquitin Ligase) complex, which is the largest family of E3 ubiquitin ligase in mammals. By using a targeted proteomics approach, we identified a substrate recognition adaptor F-Box leucine-rich repeat 13 (FBXL13) that bind the NH2-terminus portion of NKCC2. FBXL13 is expressed in rats TALs and co-immunoprecipitates with NKCC2. Therefore, we hypothesized that the substrate recognition adaptor FBXL13 mediates NKCC2 ubiquitination. To test our hypothesis, we generated a global FBXL13 knockout mice by crossing FBXL13-Flox with a global Cre mice. We confirmed the deletion of FBXL13 in tail by PCR and in TALs by Western blot. First, we studied whether deletion of FBXL13 affects total NKCC2 expression. For this, TALs from FBXL13-Flox and FBXL13-KO mice were lysed, and equal amount of protein resolved by SDS-PAGE and detected by Western blot. We found that total NKCC2 expression in FBXL13-KO mice is increased by 73% (FBXL13-Flox: 100% vs. FBXL13-KO mice: 173 ± 37%, p<0.05). These data suggest that NKCC2 has been accumulated and not degraded. We tested whether FBXL13 mediates the cGMP-dependent increase on NKCC2 ubiquitination. TALs from FBXL13-Flox control mice and FBXL13-KO mice were incubated at 37°C during proteasome inhibition with MG132 and treated with vehicle or db-cGMP 500μM. Ubiquitinated proteins were pulled down and NKCC2 measured by Western blot. We found that cGMP increased NKCC2 ubiquitination by 24 ± 10% in FBXL13-Flox mice, whereas this effect was completely absent in FBXL13-KO mice (-14 ± 12%, p<0.05). These data suggest that FBXL13 mediates the cGMP-dependent increase in NKCC2 ubiquitination in native TALs. Finally, we study the role of FBXL13 on renal function by measuring bumetanide-induced natriuresis. Bumetanide is a loop diuretic that specifically inhibits NKCC2. We observed that the FBXL13-KO mice has higher bumetanide-induced Na+ excretion (Flox: 88.8 ± 6.6 μmol Na+ vs. FBXL13-KO: 124.0 ± 14.6 μmol Na+, p<0.05). These data are in alignment with the results previous since higher bumetanide-induced natriuresis response would result with higher NKCC2 expression. We conclude that the cGMP-dependent increase in NKCC2 ubiquitination is mediated by the novel substrate recognition adaptor FBXL13. To our knowledge, this is the first evidence showing a substrate recognition adaptor FBXL13 that interacts with NKCC2 in native TALs. NIH - 1K01DK123192. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Quantitative Pattern of hPTMs by Mass Spectrometry-Based Proteomics with Implications for Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is known for its aggressive nature, and TNBC management is currently challenging due to the lack of effective targets. Despite the importance of histone post-translational modifications (hPTMs) in breast cancer, their associations with molecular subtypes of breast cancer, especially TNBC, are poorly understood. In this study, a combination of untargeted and targeted proteomics approaches, supplemented by a derivatization method, was applied to breast cancer cells and tissue samples. Untargeted proteomics of eight breast cancer cell lines belonging to different molecular subtypes revealed 36 modified peptides with 12 lysine modification sites in histone H3, and the most frequently reported top 5 histone H3 methylation and acetylation sites were covered. Then, targeted proteomics was carried out to quantify the total 20 target hPTMs at the covered modification sites (i.e., mono-, di-, trimethylation, and acetylation for each site), indicating the difficulty in distinguishing TNBC cells from normal cells. Subsequently, the analysis in TNBC patients revealed significant expression differences in 4 specific hPTMs (H3K14ac, H3K27me1, H3K36me2, and H3K36me3) between TNBC and adjacent normal tissue samples. These unique hPTM patterns allowed for the differentiation of TNBC from normal cases. This finding provides promising implications for advancing targeted treatment strategies for TNBC in the future.

Proteomics analysis of differentially abundant proteins in the rohu kidney infected with Edwardsiella tarda

Edwardsiella tarda (Et) is a zoonotic gram-negative pathogen with a diverse host range, including fish. However, the in-depth molecular mechanisms underlying the response of Labeo rohita (rohu) kidney to Et are poorly understood. A proteomic and histopathological analysis was performed for the rohu kidney after Et infection. The histopathology of the infected rohu kidney showed vacuolation and necrosis. After LC-MS/MS analysis, ~1240 proteins were identified with ≥2 unique peptides. A total of 96 differentially abundant proteins (DAPs) were observed between the control and Et infected group (ET). Metascape and STRING analysis were used for the gene ontology (GO), and protein-protein interaction network (PPI) for the significant pathways of DAPs. In PPI, low-abundant proteins were mapped to metabolic pathways and oxidative phosphorylation (cox5ab, uqcrfs1). High-abundance proteins were mapped to ribosomes (rplp2), protein process in the ER (hspa8), and immune system (ptgdsb.1, muc2). Our label-free proteomic approach in the rohu kidney revealed abundant enriched proteins involved in vesicle coat (ehd4), complement activation (c3a.1, c9, c7a), phagosome (thbs4, mapk1), metabolic reprogramming (hao1, glud1a), wound healing (vim, alox5), and the immune system (psap) after Et infection. A targeted proteomics approach of multiple reaction monitoring (MRM) validated the DAPs (nprl3, ambp, vmo1a, hspg2, muc2, hao1 and glud1a) between control and ET. Overall, the current analysis of histology and proteome in the rohu kidney provides comprehensive data on pathogenicity and the potential immune proteins against Et.

Absolute quantitation of human wild-type DNAI1 protein in lung tissue using a nanoLC-PRM-MS-based targeted proteomics approach coupled with immunoprecipitation

BackgroundDynein axonemal intermediate chain 1 protein (DNAI1) plays an essential role in cilia structure and function, while its mutations lead to primary ciliary dyskinesia (PCD). Accurate quantitation of DNAI1 in lung tissue is crucial for comprehensive understanding of its involvement in PCD, as well as for developing the potential PCD therapies. However, the current protein quantitation method is not sensitive enough to detect the endogenous level of DNAI1 in complex biological matrix such as lung tissue.MethodsIn this study, a quantitative method combining immunoprecipitation with nanoLC-MS/MS was developed to measure the expression level of human wild-type (WT) DNAI1 protein in lung tissue. To our understanding, it is the first immunoprecipitation (IP)-MS based method for absolute quantitation of DNAI1 protein in lung tissue. The DNAI1 quantitation was achieved through constructing a standard curve with recombinant human WT DNAI1 protein spiked into lung tissue matrix.ResultsThis method was qualified with high sensitivity and accuracy. The lower limit of quantitation of human DNAI1 was 4 pg/mg tissue. This assay was successfully applied to determine the endogenous level of WT DNAI1 in human lung tissue.ConclusionsThe results clearly demonstrate that the developed assay can accurately quantitate low-abundance WT DNAI1 protein in human lung tissue with high sensitivity, indicating its high potential use in the drug development for DNAI1 mutation-caused PCD therapy.

Assessing the Impacts of Cu and Mo Engineered Nanomaterials on Crop Plant Growth Using a Targeted Proteomics Approach.

In this study, we investigated the effects of molybdenum (Mo)-based nanofertilizer and copper (Cu)-based nanopesticide exposure on wheat through a multifaceted approach, including physiological measurements, metal uptake and translocation analysis, and targeted proteomics analysis. Wheat plants were grown under a 16 h photoperiod (light intensity 150 μmol·m-2·s-1) for 4 weeks at 22 °C and 60% humidity with 6 different treatments, including control, Mo, and Cu exposure through root and leaf. The exposure dose was 6.25 mg of element per plant through either root or leaf. An additional low-dose (0.6 mg Mo/plant) treatment of Mo through root was added after phytotoxicity was observed. Using targeted proteomics approach, 24 proteins involved in 12 metabolomic pathways were quantitated to understand the regulation at the protein level. Mo exposure, particularly through root uptake, induced significant upregulation of 16 proteins associated with 11 metabolic pathways, with the fold change (FC) ranging from 1.28 to 2.81. Notably, a dose-dependent response of Mo exposure through the roots highlighted the delicate balance between nutrient stimulation and toxicity as a high Mo dose led to robust protein upregulation but also resulted in depressed physiological measurements, while a low Mo dose resulted in no depression of physiological measurements but downregulations of proteins, especially in the first leaf (0.23 < FC < 0.68) and stem (0.13 < FC < 0.68) tissues. Conversely, Cu exposure exhibited tissue-specific effects, with pronounced downregulation (18 proteins involved in 11 metabolic pathways) particularly in the first leaf tissues (root exposure: 0.35 < FC < 0.74; leaf exposure: 0.49 < FC < 0.72), which indicated the quick response of plants to Cu-induced stress in the early stage of exposure. By revealing the complexities of plants' response to engineered nanomaterials at both physiological and molecular levels, this study provides insights for optimizing nutrient management practices in crop production and advancing toward sustainable agriculture.

Identification of CSF biomarkers for cerebral amyloid angiopathy using a targeted proteomics approach

AbstractBackgroundCerebral amyloid angiopathy (CAA) is characterized by progressive vascular deposition of amyloid‐β (Aβ), and can cause cognitive decline in the elderly. Currently, the diagnosis of CAA is based on neuroimaging and includes the identification of lobar (micro‐)bleeds and cortical superficial siderosis. There is, however, a need for alternative methods as these neuroimaging methods are not optimal for detection of CAA during life. Moreover, CAA is strongly associated with the risk to develop edematous or hemorrhagic amyloid‐related imaging abnormalities (ARIA) as a consequence of anti‐Aβ immunotherapy (e.g. aducanumab and lecanemab) for Alzheimer’s disease (AD). There is a need for biomarkers that can provide an accurate and early diagnosis of CAA, and additionally help in the identification of AD patients with a high burden of CAA, who are at risk of developing ARIA due to immunotherapy. We aimed to identify novel biomarker candidates for CAA in cerebrospinal fluid (CSF) using a targeted proteomics approach.MethodWe analyzed a panel of 367 proteins in CSF samples of 34 CAA patients and 51 age‐ and sex‐matched controls using the Olink Explore multiplex immunoassay platform (Neurology panel; Olink, Uppsala, Sweden). Proteins were considered measurable when their levels were above the limit of detection of the assay in at least 70% of the samples.ResultFor 15 of the 263 proteins that could be measured in CSF, we identified significantly different (p&lt;0.05) levels in CSF of CAA patients as compared to controls. The strongest difference was observed for neurofilament light chain (NFL). Among these 15 proteins, there were two proteins (milk fat globule‐epidermal growth factor 8 (MFG‐E8/lactadherin) and urokinase‐type plasminogen activator (uPA)) for which we previously observed a significant changed level in CSF of CAA patients compared to controls 1,2.ConclusionConfirmation of previously identified CAA biomarker candidates shows the validity and strength of our biomarker identification study using a targeted proteomics approach. The 15 candidates identified in our study, therefore, hold promise for development into biomarkers that may improve the detection of CAA.

Absolute Quantification of Pan-Cancer Plasma Proteomes Reveals Unique Signature in Multiple Myeloma.

Mass spectrometry based on data-independent acquisition (DIA) has developed into a powerful quantitative tool with a variety of implications, including precision medicine. Combined with stable isotope recombinant protein standards, this strategy provides confident protein identification and precise quantification on an absolute scale. Here, we describe a comprehensive targeted proteomics approach to profile a pan-cancer cohort consisting of 1800 blood plasma samples representing 15 different cancer types. We successfully performed an absolute quantification of 253 proteins in multiplex. The assay had low intra-assay variability with a coefficient of variation below 20% (CV = 17.2%) for a total of 1013 peptides quantified across almost two thousand injections. This study identified a potential biomarker panel of seven protein targets for the diagnosis of multiple myeloma patients using differential expression analysis and machine learning. The combination of markers, including the complement C1 complex, JCHAIN, and CD5L, resulted in a prediction model with an AUC of 0.96 for the identification of multiple myeloma patients across various cancer patients. All these proteins are known to interact with immunoglobulins.

Quantitative Proteomic Analysis of MCM3 in ThinPrep Samples of Patients with Cervical Preinvasive Cancer

Triage methods for cervical cancer detection show moderate accuracy and present considerable false-negative and false-positive result rates. A complementary diagnostic parameter could help improve the accuracy of identifying patients who need treatment. A pilot study was performed using a targeted proteomics approach with opportunistic ThinPrep samples obtained from women collected at the hospital’s outpatient clinic to determine the concentration levels of minichromosome maintenance-3 (MCM3) and envoplakin (EVPL) proteins. Forty samples with ‘negative for intraepithelial lesion or malignancy’ (NILM), 21 samples with ‘atypical squamous cells of undetermined significance’ (ASC-US), and 33 samples with ‘low-grade squamous intraepithelial lesion and worse’ (≥LSIL) were analyzed, using cytology and the patients’ histology reports. Highly accurate concordance was obtained for gold-standard-confirmed samples, demonstrating that the MCM3/EVPL ratio can discriminate between non-dysplastic and dysplastic samples. On that account, we propose that MCM3 and EVPL are promising candidate protein biomarkers for population-based cervical cancer screening.

CAMP stimulated NKCC2 phosphorylation at Thr-96,101 is in part independent from SPAK: a Role for TNIK

Abnormally enhanced NaCl reabsorption by the thick ascending limb of the loop of Henle (TAL) contributes to the development of salt-sensitivity. NaCl reabsorption by the TAL is mediated by the apical Na/K/2Cl cotransporter NKCC2. NKCC2 activity can be stimulated by phosphorylation at Thr 96,101 by upstream kinases SPAK or OSR1. We found that Thr 96,101 phosphorylation is enhanced in TALs of Dahl Salt Sensitive rats (Dahl SS) on normal or high salt diet. Genetic deletion of SPAK in Dahl SS lowers NKCC2 phosphorylation by 40% and blunts salt-sensitive hypertension but does not completely restore these to baseline. Other kinases may be involved in NKCC2 phosphorylation. Using a targeted proteomics approach, we identified TNIK (Traf2 and NCK interacting kinase) as a kinase that binds and phosphorylates Thr 96,101 in NKCC2 in rats and mice (manuscript under revision). In addition, we and others found that cAMP is a potent stimulus for NKCC2 Thr-96,101 phosphorylation. We hypothesize that TNIK is in part responsible for cAMP-stimulated NKCC2 phosphorylation at Thr-96,101. First, we tested the ability of a novel TNIK inhibitor (NCB-0846) to decrease baseline NKCC2 phosphorylation in Dahl SS rats. Treating TALs from Dahl SS with NCB-0846 (0.1 μM) for 25 min decreased NKCC2 Thr 96,101 phosphorylation by 21 ± 2% compared to the vehicle group (p&lt;0.0001, n=3). Next, we measured NKCC2 phosphorylation in rats with genetic deletion of SPAK in a Dahl SS background. In SPAK KO rats, the cAMP analogue db-cAMP (500 μM) increased NKCC2 phosphorylation at Thr 96,101 by 626 ± 46% (p&lt;0.0001, n=3) and the TNIK inhibitor NCB-0846, blunted cAMP-stimulated NKCC2 phosphorylation by 56 ± 4.3% (p=0.0013, n=3). There was no significant decrease in phosphorylation at phospho-Ser 126 NKCC2 (cAMP=46 ± 7-fold vs cAMP+NCB= 31±5-fold, n.s). These data indicate that in the absence of SPAK, TNIK mediates cAMP-stimulated NKCC2 phosphorylation at Thr-96,101. To further support this, we studied TALs from whole animal TNIK knock-out mice (TNIK KO). db-cAMP (500 μM) increased NKCC2 phosphorylation by 1330 ± 268% in wild-type mice (p&lt;0.01) whereas cAMP-stimulated NKCC2 phosphorylation was blunted by 49 ± 15% in TNIK KO TALs (p&lt;0.05, n=3). No significant difference was found in cAMP-stimulated NKCC2-Ser 126 phosphorylation in TNIK KO (WT=23 ± 12-fold vs TNIK KO=15 ± 4-fold, n=3). We conclude that enhanced NKCC2 phosphorylation at baseline in Dahl SS TALs is in part caused by TNIK and that cAMP-stimulated NKCC2 Thr-96,101 phosphorylation in TALs is in part independent from SPAK and involves TNIK. Our data point to potential role for TNIK in renal ion transport, blood pressure regulation and urine concentration. AHA15GRNT25710369 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

High throughput proteomic analysis of Labeo rohita liver infected with Edwardsiella tarda

The liver is a multifunctional organ and very crucial for pathogenicity and toxicological analysis in fish. In this study, histopathological and proteomic analysis of liver tissue was performed to overview the changes in tissue anatomy and proteome profile following Edwardsiella tarda (Et) infection. The differential proteomic response against Et infection was investigated using label free quantitative proteomics. Compared to the control tissue, the histopathological changes in bacterial challenged group (ET) include hepatocyte enlargement and melanomacrophage centre (MMC) aggregation in response to disease. With the proteomic analysis, a total of 1275 proteins were identified of which 257 were found to be differentially expressed proteins (DEP) between the control and ET group. Functional analysis of DEPs using STRING and metascape tools revealed expression of proteins belonging to different pathways like ribosome, protein processing in the endoplasmic reticulum (rpl5a), metabolic pathways (acss2, prodha, cyp51a), oxidative phosphorylation (uqcrfs1, ndufb10, atp6v0d1), proteasome (psmd3), and N-glycan biosynthesis (dpm1) pathways. Proteins involved in response to the Et infection activate downstream processes of apoptosis, inflammatory cytokines (TIR, chp1, apoa1), MAC (c9, c5, c8a), PPAR signalling (acsl4), APR (a2m1, cp), and T-cell activation (Ilf2). A targeted proteomics approach (MRM) was used in rohu livers to validate differentially expressed proteins (itln2, steap4, gst, psap) involved in Et pathogenesis. Our results showed the putative histopathological effects of Et infection on liver tissue and insights into the metabolic and immune pathway-associated proteins that play a role during Et infection, thereby highlighting a potential way of enhancing fish immunity against bacteria. Our comprehensive liver proteome findings would contribute to understanding pathogenesis and revealing novel disease markers for rohu liver in Et infection.

Integrative Proteomic and Pharmacological Analysis of Colon Cancer Reveals the Classical Lipogenic Pathway with Prognostic and Therapeutic Opportunities.

Despite recent advancements, the high mortality rate remains a concern in colon cancer (CAC). Identification of therapeutic markers could prove to be a great asset in CAC management. Multiple studies have reported hyperactivation of de novo lipogenesis (DNL), but its association with the pathology is unclear. This study aims to establish the importance as well as the prognostic and therapeutic potential of DNL in CAC. The key lipogenic enzymes fatty acid synthase along with ATP citrate lyase were quantified using an LC-MS/MS-based targeted proteomics approach in the samples along with the matched controls. The potential capacity of the proteins to distinguish between the tumor and controls was demonstrated using random forest-based class prediction analysis using the peptide intensities. Furthermore, in-depth proteomics of DNL inhibition in the CAC cell line revealed the significance of the pathway in proliferation and metastasis. DNL inhibition affected the major signaling pathways, including DNA repair, PI3K-AKT-mTOR pathway, membrane trafficking, proteasome, etc. The study revealed the upregulation of 26S proteasome machinery as a result of the treatment with subsequent induction of apoptosis. Again, in silico molecular docking-based drug repurposing was performed to find potential drug candidates. Furthermore, we have demonstrated that blocking DNL could be explored as a therapeutic option in CAC treatment.

Colon Adenocarcinoma Quantitative Proteomics Reveals Dysregulation in Key Cancer Signaling Pathways and a Candidate Protein Marker Panel

Colorectal cancer (CRC) is reportedly the second leading cause of cancer death worldwide. By the end of the decade, there will likely be more than one million fatalities worldwide from this cancer, with an estimated 2.2 million additional cases. We need new ways of thinking about cancer research. One approach is to deploy systems science using quantitative proteomics to obtain postgenomic and functional insights into cancer. The present study compares the tissue proteome of CRC (n = 10) with the matched peritumoral controls (n = 10) in samples obtained from the Indian subcontinent. When compared with the controls, a list of 22 substantially altered protein candidates was identified, which were associated with the growth, survival, and metastasis of the tumor. A list of the unique peptides from top significant proteins, including olfactomedin-4, alanyl aminopeptidase, and grancalcin was further validated using a parallel reaction monitoring-based targeted proteomics approach. In addition, biological pathway analysis showed perturbation in key biological processes, including dysregulation in purine metabolism, MYC targets in cancer, DNA repair, and replication, and leukocyte transendothelial migration, among others. The protein panel reported herein is also shown to be dysregulated in CRC and warrants further research toward understanding pathobiology, diagnostics, and therapeutics development in CRC.

Determination of porcine derived components in gelatin and gelatin-containing foods by high performance liquid chromatography-tandem mass spectrometry

Detection and quantitation of porcine derived components in bovine and donkey gelatins and gelatin-containing foods is required for religious practices and consumer economic interests. In this study, shot-gun proteomics approaches were implemented to identify species-specific peptide biomarkers of three main gelatin origins, including porcine, bovine and donkey. Tryptic gelatin peptides were separated and analyzed with ultra-performance liquid chromatography-tandem high-resolution mass spectrometry (UPLC-HRMS), and 11 peptide biomarkers were selected for porcine, bovine and donkey. The gelatin pretreatment methods in three different food matrixes were optimized, and a label-free quantitative method using the MRM method of the most sensitive biomarker peptides was established. Among them, two porcine gelatin quantitative peptides were confirmed to have good linearity. The method has been successfully applied to the adulteration detection of porcine-derived components in gelatin and the quantification of porcine gelatin in candy and dairy products, with LODs of 0.07, 0.08 and 0.12 mg/kg, respectively. • A strategy for identification of peptide biomarkers of common gelatin species was established and improved with untargeted and targeted proteomics approaches. • Eleven species-specific peptide biomarkers were selected for porcine, bovine and donkey gelatin. • The pretreatment method of gelatin in different food matrixes has been optimized, especially for gelatin in dairy products. • The label-free MRM method is used for quantitative determination of adulterated porcine gelatin in mixed gelatin and gelatin-containing food, with good linearity, specificity, sensitivity and repeatability.

First Identification of a Large Set of Serine Hydrolases by Activity-Based Protein Profiling in Dibutyl Phthalate-Exposed Zebrafish Larvae.

Despite the involvement of several serine hydrolases (SHs) in the metabolism of xenobiotics such as dibutyl phthalate (DBP), no study has focused on mapping this enzyme class in zebrafish, a model organism frequently used in ecotoxicology. Here, we survey and identify active SHs in zebrafish larvae and search for biological markers of SH type after exposure to DBP. Zebrafish were exposed to 0, 5, and 100 µg/L DBP from 4 to 120 h post-fertilization. A significant decrease in vitellogenin expression level of about 2-fold compared to the control was found in larvae exposed to 100 µg/L DBP for 120 h. The first comprehensive profiling of active SHs in zebrafish proteome was achieved with an activity-based protein profiling (ABPP) approach. Among 49 SHs identified with high confidence, one was the carboxypeptidase ctsa overexpressed in larvae exposed to 100 µg/L DBP for 120 h. To the best of our knowledge, this is the first time that a carboxypeptidase has been identified as deregulated following exposure to DBP. The overall results indicate that targeted proteomics approaches, such as ABPP, can, therefore, be an asset for understanding the mechanism of action related to xenobiotics in ecotoxicology.

Maternal plasma proteome profiling of biomarkers and pathogenic mechanisms of early-onset and late-onset preeclampsia

Preeclampsia is still the leading cause of morbidity and mortality in pregnancy without a cure. There are two phenotypes of preeclampsia, early-onset (EOPE) and late-onset (LOPE) with poorly defined pathogenic differences. This study aimed to facilitate better understanding of the mechanisms of pathophysiology of EOPE and LOPE, and identify specific biomarkers or therapeutic targets. In this study, we conducted an untargeted, label-free quantitative proteomic analyses of plasma samples from pregnant women with EOPE (n = 17) and LOPE (n = 11), and age, BMI-matched normotensive controls (n = 18). Targeted proteomics approach was also employed to validate a subset of proteins (n = 17). In total, there were 26 and 20 differentially abundant proteins between EOPE or LOPE, and normotensive controls, respectively. A series of angiogenic and inflammatory proteins, including insulin-like growth factor-binding protein 4 (IGFBP4; EOPE: FDR = 0.0030 and LOPE: FDR = 0.00396) and inter-alpha-trypsin inhibitor heavy chain H2-4 (ITIH2-4), were significantly altered in abundance in both phenotypes. Through validation we confirmed that ITIH2 was perturbed only in LOPE (p = 0.005) whereas ITIH3 and ITIH4 were perturbed in both phenotypes (p < 0.05). Overall, lipid metabolism/transport proteins associated with atherosclerosis were highly abundant in LOPE, however, ECM proteins had a more pronounced role in EOPE. The complement cascade and binding and uptake of ligands by scavenger receptors, pathways, were associated with both EOPE and LOPE.

Subtype-specific plasma signatures of platelet-related protein releasate in acute pulmonary embolism

IntroductionThere is evidence that plasma protein profiles differ in the two subtypes of pulmonary embolism (PE), isolated PE (iPE) and deep vein thrombosis (DVT)-associated PE (DVT-PE), in the acute phase. The aim of this study was to determine specific plasma signatures for proteins related to platelets in acute iPE and DVT-PE compared to isolated DVT (iDVT). MethodsWithin the Genotyping and Molecular Phenotyping of Venous ThromboEmbolism (GMP-VTE) Project, a multicenter prospective cohort study of 693 confirmed VTE cases, a highly sensitive targeted proteomics approach based on dual-antibody proximity extension assay was applied. LASSO-regularized logistic regression analysis selected 33 and 30 of 135 platelet-related candidate proteins in iPE and DVT-PE vs. iDVT, respectively. ResultsAll regulated proteins were well associated with six prominently released platelet proteins and the majority showed specificity for iPE and DVT-PE compared to iDVT. While iPE-specific proteins were assigned to be predominantly released via shedding mechanisms and extracellular vesicles, granule secretion was identified as a major release mechanism assigned to DVT-associated PE-specific proteins. Network analysis demonstrated three interconnected clusters of specifically regulated proteins in iPE linked to immunoreceptor signaling, pathogen clearance and chemotaxis, whereas for DVT-associated PE one cluster linked to tissue remodeling and leukocyte trafficking. Machine learning-based analysis reveals specific plasma signatures and differential release mechanisms of proteins related to platelets in acute iPE and DVT-associated PE. ConclusionThese data suggest that the platelet protein releasate contributes to the differential regulation of plasma proteins in acute PE compared to iDVT, which may be associated with different platelet activation patterns.

Protein Carbonylation as a Reliable Read-Out of Urban Pollution Damage/Protection of Hair Fibers

(1) Background: Environmental factors, such as airborne pollutants and solar UV, induce oxidative damage to proteins and lipids on hair fibers, leading to decreased hair strength and shine, increased fiber porosity, brittleness, dryness, and stiffness. Traditional methods used for hair damage/protection/reparation assessment show limitations in sensitivity or specificity for evidencing the benefits to be gained from the protection/reparation of hair fibers against environmental stressors. (2) Methods: Ex vivo experimental models of hair fibers exposed to urban pollutants and UV irradiation were developed. Targeted proteomics approaches for the quantification of oxidatively damaged (carbonylated) proteins on hair fibers were optimized. (3) Results: A significant dose-dependent increase in carbonylation both in the cuticle and cortex proteins was observed upon exposure of hair fibers to particulate matter and UV-A radiation, at daily stress equivalent doses. Increased protein carbonylation on keratins and keratin-associated proteins led to loss of hair fiber structural integrity. The oxidative modification of proteins induced by urban pollution exposure led to hair cuticle structural damage revealed by an increased permeability. However, protein carbonylation was prevented in the presence of antioxidant compounds. (4) Conclusions: Protein carbonylation is an early event in hair fiber damage which can be used as a reliable biomarker for the efficacy of hair care interventions against environmental stressors.

Targeted proteomics identifies potential biomarkers of dysglycaemia, beta cell function and insulin sensitivity in Black African men and women.

Using a targeted proteomics approach, we aimed to identify and validate circulating proteins associated with impaired glucose metabolism (IGM) and type 2 diabetes in a Black South African cohort. In addition, we assessed sex-specific associations between the validated proteins and pathophysiological pathways of type 2 diabetes. This cross-sectional study included Black South African men (n=380) and women (n=375) who were part of the Middle-Aged Soweto Cohort (MASC). Dual-energy x-ray absorptiometry was used to determine fat mass and visceral adipose tissue, and fasting venous blood samples were collected for analysis of glucose, insulin and C-peptide and for targeted proteomics, measuring a total of 184 pre-selected protein biomarkers. An OGTT was performed on participants without diabetes, and peripheral insulin sensitivity (Matsuda index), HOMA-IR, basal insulin clearance, insulin secretion (C-peptide index) and beta cell function (disposition index) were estimated. Participants were classified as having normal glucose tolerance (NGT; n=546), IGM (n=116) or type 2 diabetes (n=93). Proteins associated with dysglycaemia (IGM or type 2 diabetes) in the MASC were validated in the Swedish EpiHealth cohort (NGT, n=1706; impaired fasting glucose, n=550; type 2 diabetes, n=210). We identified 73 proteins associated with dysglycaemia in the MASC, of which 34 were validated in the EpiHealth cohort. Among these validated proteins, 11 were associated with various measures of insulin dynamics, with the largest number of proteins being associated with HOMA-IR. In sex-specific analyses, IGF-binding protein 2 (IGFBP2) was associated with lower HOMA-IR in women (coefficient -0.35; 95% CI -0.44, -0.25) and men (coefficient -0.09; 95% CI -0.15, -0.03). Metalloproteinase inhibitor 4 (TIMP4) was associated with higher insulin secretion (coefficient 0.05; 95% CI 0.001, 0.11; p for interaction=0.025) and beta cell function (coefficient 0.06; 95% CI 0.02, 0.09; p for interaction=0.013) in women only. In contrast, a stronger positive association between IGFBP2 and insulin sensitivity determined using an OGTT (coefficient 0.38; 95% CI 0.27, 0.49) was observed in men (p for interaction=0.004). A posteriori analysis showed that the associations between TIMP4 and insulin dynamics were not mediated by adiposity. In contrast, most of the associations between IGFBP2 and insulin dynamics, except for insulin secretion, were mediated by either fat mass index or visceral adipose tissue in men and women. Fat mass index was the strongest mediator between IGFBP2 and insulin sensitivity (total effect mediated 40.7%; 95% CI 37.0, 43.6) and IGFBP2 and HOMA-IR (total effect mediated 39.1%; 95% CI 31.1, 43.5) in men. We validated 34 proteins that were associated with type 2 diabetes, of which 11 were associated with measures of type 2 diabetes pathophysiology such as peripheral insulin sensitivity and beta cell function. This study highlights biomarkers that are similar between cohorts of different ancestry, with different lifestyles and sociodemographic profiles. The African-specific biomarkers identified require validation in African cohorts to identify risk markers and increase our understanding of the pathophysiology of type 2 diabetes in African populations.

A Targeted Proteomics Approach for Screening Serum Biomarkers Observed in the Early Stage of Type I Endometrial Cancer.

Endometrial cancer (EC) is the most common gynecologic malignancy, and it arises in the inner part of the uterus. Identification of serum biomarkers is essential for diagnosing the disease at an early stage. In this study, we selected 44 healthy controls and 44 type I EC at tumor stage 1, and we used the Immuno-oncology panel and the Target 96 Oncology III panel to simultaneously detect the levels of 92 cancer-related proteins in serum, using a proximity extension assay. By applying this methodology, we identified 20 proteins, associated with the outcome at binary logistic regression, with a p-value below 0.01 for the first panel and 24 proteins with a p-value below 0.02 for the second one. The final multivariate logistic regression model, combining proteins from the two panels, generated a model with a sensitivity of 97.67% and a specificity of 83.72%. These results support the use of the proposed algorithm after a validation phase.

Data independent acquisition mass spectrometry in relation to potential diagnostic and prognostic biomarkers for high-grade serous ovarian cancer.

e17564 Background: Ovarian cancer (OC) is one of the most common causes of cancer-related death among women worldwide. High-grade serous ovarian cancer (HGSOC) represents the major OC histological type, and challenges associated with its early detection and prediction of clinical outcomes significantly contribute to the high prevalence and poor prognosis of OC. Lack of reliable biomarkers further exacerbates the low survival rate of patients with HGSOC. Methods: In this study, we performed liquid chromatography data independent acquisition tandem-mass spectrometry (LC-DIA-MS/MS) experiments on depleted serum samples [26 HGSOC cases, 24 healthy controls (HCs)] to identify potential diagnostic and prognostic biomarkers for HGSOC. Results: A total of 1,148 proteins were identified in all samples combined, representing one of the largest quantifications in the serum proteome of OC to date. Among them, 122 proteins showed differential expressions between the serum proteome of HGSOC patients and HCs, including 109 up- and 14 down-regulated proteins in HGSOC. These potential diagnostic biomarkers were involved in the pathways related to immune response, TGF- β and IGF signaling, and lipid metabolism. Moreover, we performed Kaplan-Meier survival analysis on the HGSOC patients with and without recurrence, and identified several potential prognostic biomarkers highly associated with progression free survival (PFS). Using independent cohort samples, we validated the expression levels of HGSOC serum biomarker candidates using targeted proteomics approaches (MRM and PRM). In vitro functional assays revealed that some of these biomarkers may play an essential role in cancer cell migration and proliferation in HGSOC. Conclusions: In summary, our LC-DIA-MS/MS analysis of the HGSOC serum proteome successfully identified novel diagnostic and prognostic biomarkers for HGSOC, some of which were experimentally shown to have functional relevance to the pathophysiology of OC.