CETSA-MS-based target profiling of anti-aging natural compound quercetin.

BACKGROUND: Quercetin is widely distributed in nature and abundant in the human diet, which exhibits diverse biological activities and potential medical benefits. However, there remains a lack of comprehensive understanding about its cellular targets, impeding its in-depth mechanistic studies and clinical applications. PURPOSE: This study aimed to profile protein targets of quercetin at the proteome level. METHODS: A label-free CETSA-MS proteomics technique was employed for target enrichment and identification. The R package Inflect was used for melting curve fitting and target selection. D3Pocket and LiBiSco tools were used for binding pocket prediction and binding pocket analysis. Western blotting, molecular docking, site-directed mutagenesis and pull-down assays were used for target verification and validation. RESULTS: We curated a library of direct binding targets of quercetin in cells. This library comprises 37 proteins that show increased thermal stability upon quercetin binding and 33 proteins that display decreased thermal stability. Through Western blotting, molecular docking, site-directed mutagenesis and pull-down assays, we validated CBR1 and GSK3A from the stabilized protein group and MAPK1 from the destabilized group as direct binding targets of quercetin. Moreover, we characterized the shared chemical properties of the binding pockets of quercetin with targets. CONCLUSION: Our findings deepen our understanding of the proteins pivotal to the bioactivity of quercetin and lay the groundwork for further exploration into its mechanisms of action and potential clinical applications.

Precise diagnosis and typing of early-stage renal immunoglobulin-derived amyloidosis by label-free quantification of parallel reaction monitoring-based targeted proteomics.

BACKGROUND: Early diagnosis and typing are crucial for improving the prognosis of patients with renal amyloidosis. Currently, Untargeted proteomics based precise diagnosis and typing of amyloid deposits are crucial for guiding patient management. Although untargeted proteomics achieve ultra-high-throughput by selecting the most abundant eluting cationic peptide precursors in series for tandem MS events, it lacks in sensitivity and reproducibility, which may not be suitable for early-stage renal amyloidosis with minor damages. Here, we aimed to develop parallel reaction monitoring (PRM)-based targeted proteomics to achieve high sensitivity and specificity by determining absolute abundances and codetecting all transitions of highly repeatable peptides of preselected amyloid signature and typing proteins in identifying early-stage renal immunoglobulin-derived amyloidosis. METHODS AND RESULTS: In 10 discovery cohort cases, Congo red-stained FFPE slices were micro-dissected and analyzed by data-dependent acquisition-based untargeted proteomics for preselection of typing specific proteins and peptides. Further, a list of proteolytic peptides from amyloidogenic proteins and internal standard proteins were quantified by PRM-based targeted proteomics to validate performance for diagnosis and typing in 26 validation cohort cases. The diagnosis and typing effectiveness of PRM-based targeted proteomics in 10 early-stage renal amyloid cases was assessed via a comparison with untargeted proteomics. A peptide panel of amyloid signature proteins, immunoglobulin light chain and heave chain in PRM-based targeted proteomics showed significantly distinguishing ability and amyloid typing performance in patients. The diagnostic algorithm of targeted proteomics with a low amount of amyloid deposits in early-stage renal immunoglobulin-derived amyloidosis showed better performance than untargeted proteomics in amyloidosis typing. CONCLUSIONS: This study demonstrates that the utility of these prioritized peptides in PRM-based targeted proteomics ensure high sensitivity and reliability for identifying early-stage renal amyloidosis. Owing to the development and clinical application of this method, rapid acceleration of the early diagnosis, and typing of renal amyloidosis is expected.

Follicular free fatty acid metabolic signatures and their effects on oocyte competence in non-obese PCOS patients.

In Brief: Polycystic ovary syndrome (PCOS) is a common cause of anovulatory infertility in women. This study identified changes in free fatty acids profiles in the follicular fluid that may lead to better diagnosis and management of infertility in PCOS women. Abstract: Polycystic ovary syndrome (PCOS) is a heterogeneous disease characterized by various endocrine/metabolic disorders and impaired reproductive potential. Alterations in oocyte competence are considered potentially causative factors for infertility in PCOS women and analyzing the composition of follicular fluid in these patients may help to identify which changes have the potential to alter oocyte quality. In this study, free fatty acid metabolic signatures in follicular fluid were performed to identify changes that may impact oocyte competence in non-obese PCOS women. Sixty-four non-obese women (32 with PCOS and 32 age- and BMI-matched controls) undergoing in vitro fertilization were recruited. Embryo quality was morphologically assessed. Free fatty acid metabolic profiling in follicular fluid was performed using gas/liquid chromatography-mass spectrometry. Principal component analysis and orthogonal partial least squares-discriminant analysis models were further constructed. Nine free fatty acids and 24 eicosanoids were identified and several eicosanoids synthesized by the cyclooxygenase pathway were significantly elevated in PCOS patients compared to controls. The combination of PGE2, PGF2α, PGJ2, and TXB2 had an area under the curve of 0.867 (0.775-0.960) for PCOS discrimination. Furthermore, follicular fluid levels of PGE2 and PGJ2 were negatively correlated with high-quality embryo rate in PCOS patients (P < 0.05). Metabolomic analysis revealed that follicular fluid lipidomic profiles undergo changes in non-obese PCOS women, which suggests that identifying changes in important metabolic signatures may give us a better understanding of the pathogenesis of PCOS. Furthermore, elevated PGE2 and PGJ2 concentrations may contribute to impaired oocyte competence in non-obese PCOS patients.

Open Search-Based Proteomics Reveals Widespread Tryptophan Modifications Associated with Hypoxia in Lung Cancer.

The tryptophan residue has a large hydrophobic surface that plays a unique role in the folded protein conformation and functions. Tryptophan modifications are presumably to be readily detected in proteins due to the vulnerability of the indole structure to electrophilic attacks. In this study, we report a systematic identification of sequence variations at tryptophan, termed tryptophan variants, from the proteome of patients with nonsmall cell lung cancer (NSCLC). Using shotgun proteomics and a modified open search algorithm, 25 tryptophan variants on 2481 sites in over 858 proteins were identified. Among these, 6 tryptophan variants are previously identified, 15 are newly annotated, and 4 are still unknown, most of which are involved in the cascade of oxidation in the blood microparticle. Remarkably, Trp313 of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was up-oxidized whereas Trp16 and Trp38 of hemoglobin (HBB) were down-oxidized in NCSLC tissues. The results were further supported by an independent cohort of 103 lung adenocarcinoma samples, reflecting a negative feedback and potential detoxification mechanism against tumor glycolysis and hypoxia. Overall, the study reports a quick approach to explore tryptophan variants at the proteomic scale. Our findings highlight the predominant role of tryptophan oxidation in regulating the redox balance of cancer cells and its potential role as prognostic biomarker for patients with NSCLC.

Ceramide subclasses identified as novel lipid biomarker elevated in women with polycystic ovary syndrome: a pilot study employing shotgun lipidomics.

This study aimed to identify potential lipid biomarkers in women with polycystic ovary syndrome (PCOS) and determine their predictive value for PCOS. Eighteen women with PCOS and 17 healthy controls were enrolled. A multi-dimensional mass spectrometry-based shotgun lipidomics approach was employed to analyze serum lipid profiles. Shotgun lipidomics revealed that the concentrations of ceramide (Cer) and phosphatidylcholine (PC) were higher (PC: 831.6 ± 217.4 vs. 605.2 ± 164.2 µmol/l; Cer: 3,387.6 ± 829.9 vs. 2,552.2 ± 679.4 nmol/l, respectively), whereas that of lysophosphatidylcholine was lower, in PCOS women than in healthy controls (82.02 ± 39.49 vs. 133.62 ± 65.36 µmol/l, respectively). Receiver operating characteristic analysis showed that the combination of Cer (OH_N16:0/N18:0) and Cer (N22:0) had the greatest discriminatory power to differentiate between women with and without PCOS (area under the curve: 0.889, 95% confidence interval: 0.784-0.994). These results indicate that the combination of Cer (OH_N16:0/N18:0) and Cer (N22:0) may represent a novel lipid predictor of PCOS.

Endogenous cholesterol ester hydroperoxides modulate cholesterol levels and inhibit cholesterol uptake in hepatocytes and macrophages.

Dysregulation of cholesterol metabolism represents one of the major risk factors for atherosclerotic cardiovascular disease (CVD). Oxidized cholesterol esters (oxCE) in low-density lipoprotein (LDL) have been implicated in CVD but the underlying mechanisms remain poorly defined. We use a targeted lipidomic approach to demonstrate that levels of oxCEs in human plasma are associated with different types of CVD and significantly elevated in patients with myocardial infarction. We synthesized a major endogenous cholesterol ester hydroperoxide (CEOOH), cholesteryl-13(cis, trans)-hydroperoxy-octadecadienoate (ch-13(c,t)-HpODE) and show that this endogenous compound significantly increases plasma cholesterol level in mice while decrease cholesterol levels in mouse liver and peritoneal macrophages, which is primarily due to the inhibition of cholesterol uptake in macrophages and liver. Further studies indicate that inhibition of cholesterol uptake by ch-13(c,t)-HpODE in macrophages is dependent on LXRα-IDOL-LDLR pathway, whereas inhibition on cholesterol levels in hepatocytes is dependent on LXRα and LDLR. Consistently, these effects on cholesterol levels by ch-13(c,t)-HpODE are diminished in LDLR or LXRα knockout mice. Together, our study provides evidence that elevated plasma cholesterol levels by CEOOHs are primarily due to the inhibition of cholesterol uptake in the liver and macrophages, which may play an important role in the pathogenesis of CVD.

Alterations of polyunsaturated fatty acid metabolism in ovarian tissues of polycystic ovary syndrome rats.

The metabolism of polyunsaturated fatty acids (PUFAs) remains poorly characterized in ovarian tissues of patients with polycystic ovary syndrome (PCOS). This study aimed to explore alterations in the levels of PUFAs and their metabolites in serum and ovarian tissues in a PCOS rat model treated with a high-fat diet and andronate. Levels of PUFAs and their metabolites were measured using gas/liquid chromatography-mass spectrometry after the establishment of a PCOS rat model. Only 3 kinds of PUFAs [linoleic acid, arachidonic acid (AA) and docosahexaenoic acid] were detected in both the circulation and ovarian tissues of the rats, and their concentrations were lower in ovarian tissues than in serum. Moreover, significant differences in the ovarian levels of AA were observed between control, high-fat diet-fed and PCOS rats. The levels of prostaglandins, AA metabolites via the cyclooxygenase (COX) pathway, in ovarian tissues of the PCOS group were significantly increased compared to those in the controls. Further studies on the mechanism underlying this phenomenon showed a correlation between decreased expression of phosphorylated cytosolic phospholipase A2 (p-cPLA2) and increased mRNA and protein expression of COX2, potentially leading to a deeper understanding of altered AA and prostaglandin levels in ovarian tissues of PCOS rats.

TiO2 nanoparticles cause mitochondrial dysfunction, activate inflammatory responses, and attenuate phagocytosis in macrophages: A proteomic and metabolomic insight.

Titanium dioxide nanoparticles (TiO2 NPs) are widely used in food and cosmetics but the health impact of human exposure remains poorly defined. Emerging evidence suggests that TiO2 NPs may elicit immune responses by acting on macrophages. Our proteomic study showed that treatment of macrophages with TiO2 NPs led to significant re-organization of cell membrane and activation of inflammation. These observations were further corroborated with transmission electron microscopy (TEM) experiments, which demonstrated that TiO2 NPs were trapped inside of multi-vesicular bodies (MVB) through endocytotic pathways. TiO2 NP caused significant mitochondrial dysfunction by increasing levels of mitochondrial reactive oxygen species (ROS), decreasing ATP generation, and decreasing metabolic flux in tricarboxylic acid (TCA) cycle from 13C-labelled glutamine using GC-MS-based metabolic flux analysis. Further lipidomic analysis showed that TiO2 NPs significantly decreased levels of cardiolipins, an important class of mitochondrial phospholipids for maintaining proper function of electron transport chains. Furthermore, TiO2 NP exposure activates inflammatory responses by increasing mRNA levels of TNF-α, iNOS, and COX-2. Consistently, our targeted metabolomic analysis showed significantly increased production of COX-2 metabolites including PGD2, PGE2, and 15d-PGJ2. In addition, TiO2 NP also caused significant attenuation of phagocytotic function of macrophages. In summary, our studies utilizing multiple powerful omic techniques suggest that human exposure of TiO2 NPs may have profound impact on macrophage function through activating inflammatory responses and causing mitochondrial dysfunction without physical presence in mitochondria.

Comprehensive metabolomics identified lipid peroxidation as a prominent feature in human plasma of patients with coronary heart diseases.

Coronary heart disease (CHD) is a complex human disease associated with inflammation and oxidative stress. The underlying mechanisms and diagnostic biomarkers for the different types of CHD remain poorly defined. Metabolomics has been increasingly recognized as an enabling technique with the potential to identify key metabolomic features in an attempt to understand the pathophysiology and differentiate different stages of CHD. We performed comprehensive metabolomic analysis in human plasma from 28 human subjects with stable angina (SA), myocardial infarction (MI), and healthy control (HC). Subsequent analysis demonstrated a uniquely altered metabolic profile in these CHD: a total of 18, 37 and 36 differential metabolites were identified to distinguish SA from HC, MI from SA, and MI from HC groups respectively. Among these metabolites, glycerophospholipid (GPL) metabolism emerged as the most significantly disturbed pathway. Next, we used a targeted metabolomic approach to systematically analyze GPL, oxidized phospholipid (oxPL), and downstream metabolites derived from polyunsaturated fatty acids (PUFAs), such as arachidonic acid and linoleic acid. Surprisingly, lipids associated with lipid peroxidation (LPO) pathways including oxidized PL and isoprostanes, isomers of prostaglandins, were significantly elevated in plasma of MI patients comparing to HC and SA, consistent with the notion that oxidative stress-induced LPO is a prominent feature in CHD. Our studies using the state-of-the-art metabolomics help to understand the underlying biological mechanisms involved in the pathogenesis of CHD; LPO metabolites may serve as potential biomarkers to differentiation MI from SA and HC.

Identification of a novel series of anti-inflammatory and anti-oxidative phospholipid oxidation products containing the cyclopentenone moiety in vitro and in vivo: Implication in atherosclerosis.

Oxidative stress and inflammation are two major contributing factors to atherosclerosis, a leading cause of cardiovascular disease. Oxidation of phospholipids on the surface of low density lipoprotein (LDL) particles generated under oxidative stress has been associated with the progression of atherosclerosis, but the underlying molecular mechanisms remain poorly defined. We identified a novel series of oxidation products containing the cyclopentenone moiety, termed deoxy-A2/J2-isoprostanes-phosphocholine, from 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine in vivo using mass spectrometry and by comparison to a chemically synthesized standard. Transcriptomic analysis (RNA-seq) demonstrated that these compounds affected >200 genes in bone marrow-derived macrophages, and genes associated with inflammatory and anti-oxidative responses are among the top 5 differentially expressed. To further investigate the biological relevance of these novel oxidized phospholipids in atherosclerosis, we chemically synthesized a representative compound 1-palmitoyl-2-15-deoxy-δ-12,14-prostaglandin J2-sn-glycero-3-phosphocholine (15d-PGJ2-PC) and found that it induced anti-inflammatory and anti-oxidant responses in macrophages through modulation of NF-κB, peroxisome proliferator-activated receptor γ (PPARγ), and Nrf2 pathways; this compound also showed potent anti-inflammatory properties in a mice model of LPS-induced systematic inflammatory response syndrome. Additionally, 15d-PGJ2-PC inhibited macrophage foam cell formation, suggesting a beneficial role against atherosclerosis. These properties were consistent with decreased levels of these compounds in the plasma of patients with coronary heart disease compared with control subjects. Our findings uncovered a novel molecular mechanism for the negative regulation of inflammation and positive enhancement of anti-oxidative responses in macrophages by these oxidized phospholipids in LDL in the context of atherosclerosis.

Discovery of Novel Lipid Profiles in PCOS: Do Insulin and Androgen Oppositely Regulate Bioactive Lipid Production?

Context: Polycystic ovary syndrome (PCOS) is a complex syndrome showing clinical features of an endocrine/metabolic disorder, including hyperinsulinemia and hyperandrogenism. Polyunsaturated fatty acids (PUFAs) and their derivatives, both tightly linked to PCOS and obesity, play important roles in inflammation and reproduction. Objective: This study aimed to investigate serum lipid profiles in newly diagnosed patients with PCOS using lipidomics and correlate these features with the hyperinsulinemia and hyperandrogenism associated with PCOS and obesity. Design and Setting: Thirty-two newly diagnosed women with PCOS and 34 controls were divided into obese and lean subgroups. A PCOS rat model was used to validate results of the human studies. Main Outcome Measures: Serum lipid profiles, including phospholipids, free fatty acids (FFAs), and bioactive lipids, were analyzed using gas chromatography-mass spectrometry (MS) and liquid chromatography-MS. Results: Elevation in phosphatidylcholine and a concomitant decrease in lysophospholipid were found in obese patients with PCOS vs lean controls. Obese patients with PCOS had decreased PUFA levels and increased levels of long-chain saturated fatty acids vs lean controls. Serum bioactive lipids downstream of arachidonic acid were increased in obese controls, but reduced in both obese and lean patients with PCOS vs their respective controls. Conclusions: Patients with PCOS showed abnormal levels of phosphatidylcholine, FFAs, and PUFA metabolites. Circulating insulin and androgens may have opposing effects on lipid profiles in patients with PCOS, particularly on the bioactive lipid metabolites derived from PUFAs. These clinical observations warrant further studies of the molecular mechanisms and clinical implications of PCOS and obesity.