Combined Proteomic and Phosphoproteomic Characterization of the Molecular Regulators and Functional Modules During Pancreatic Progenitor Cell Development.

Differentiated multipotent pancreatic progenitors have major advantages for both modeling pancreas development and preventing or treating diabetes. Despite significant advancements in inducing the differentiation of human pluripotent stem cells into insulin-producing cells, the complete mechanism governing proliferation and differentiation remains poorly understood. This study used large-scale mass spectrometry to characterize molecular processes at various stages of human embryonic stem cell (hESC) differentiation toward pancreatic progenitors. hESCs were induced into pancreatic progenitor cells in a five-stage differentiation protocol. A high-performance liquid chromatography-mass spectrometry platform was used to undertake comprehensive proteome and phosphoproteome profiling of cells at different stages. A series of bioinformatic explorations, including coregulated modules, gene regulatory networks, and phosphosite enrichment analysis, were then conducted. A total of 27,077 unique phosphorylated sites and 8122 proteins were detected, including several cyclin-dependent kinases at the initial stage of cell differentiation. Furthermore, we discovered that ERK1, a member of the MAPK cascade, contributed to proliferation at an early stage. Finally, Western blotting confirmed that the phosphosites from SIRT1 and CHEK1 could inhibit the corresponding substrate abundance in the late stage. Thus, this study extends our understanding of the molecular mechanism during pancreatic cell development.

Magnetic zirconium-based Prussian blue analog nanozyme: enhanced peroxidase-mimicking activity and colorimetric sensing of phosphate ion.

A magnetic zirconium hexacyanoferrate-based Prussian blue analog (MB@ZrHCF) nanozyme was synthesized using dopamine (DA) reduction-assisted method and employed for colorimetric PO43- sensing. The MB@ZrHCF exhibits enhanced peroxidase-mimicking activity and ultrafast catalytic rate via the color reaction of 3,3',5,5'-tetramethylbenzidine (TMB) oxidized by hydrogen peroxide (H2O2). The catalytic reaction mechanism of MB@ZrHCF catalyzing H2O2 to produce hydroxyl radical (∙OH) was studied. Then, MB@ZrHCF was successfully applied to the detection of H2O2. Additionally, the catalytic activity of the nanocomposite is inhibited due to the steric hindrance effect from the coordination of PO43- and Zr(IV) node. Based on this, the MB@ZrHCF nanozyme can be used to detect PO43- in two linear ranges (10-100 µM and 100-200 µM) with a limit of detection of 2.25 µM. The proposed colorimetric sensor possesses excellent selectivity and reliability for PO43- sensing, which can be successfully applied to detect PO43- in sea and tap water samples.

Improving Gene Annotation of the Peanut Genome by Integrated Proteogenomics Workflow.

Peanut (Arachis hypogaea L.) is a staple crop in semiarid tropical and subtropical regions. Although the genome of peanut has been fully sequenced, the current gene annotations are still incomplete. New technologies in genomics and proteomics have resulted in the emergence of proteogenomics, which can integrate genomic, transcriptomic, and proteomic data for improving gene annotation. In the present study, we collected RNA-seq and proteomic data from multiple tissues such as seed, shell, and gynophore of peanut and utilized a proteogenomic approach to improve the gene annotation of peanut based on these data. A total of 1 935 655 904 RNA-seq reads and 7 490 280 MS/MS spectra were collected. Ultimately, 13 767 annotated genes were found with evidence at the protein level, and seven novel protein-coding genes were found with both RNA-seq and proteomics evidence. In addition, 35 gene models were updated based on proteomics data. Proteogenomic approaches improved the gene annotation in certain aspects by integrating both RNA-seq and proteomic data. We expect that these approaches could help improve existing genome annotations of other species.

PPIP: Automated Software for Identification of Bioactive Endogenous Peptides.

Endogenous peptides play an important role in multiple biological processes in many species. Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) is an important technique for detecting these peptides on a large scale. We present PPIP, which is a dedicated peptidogenomics software for identifying endogenous peptides based on peptidomics and RNA-Seq data. This software automates the de novo transcript assembly based on RNA-Seq data, construction of a protein reference database based on the de novo assembled transcripts, peptide identification, function analysis, and HTML-based report generation. Different function components are integrated using Docker technology. The Docker image of PPIP is available at https://hub.docker.com/r/shawndp/ppip , and the source code under GPL-3 license is available at https://github.com/Shawn-Xu/PPIP . A user manual of PPIP is available at https://shawn-xu.github.io/PPIP .

Comparative Analysis of Proteomics and Transcriptomics during Fertility Transition in a Two-Line Hybrid Rice Line Wuxiang S.

The two-line hybrid rice is an important factor of a global crop, but its fertility transition mechanism is unclear. Here, a comparative proteomics and transcriptomics analysis was completed on the two-line hybrid rice line Wuxiang S (WXS) to explore its molecular mechanism and protein regulation during fertility transition. A total of 340 differentially abundant proteins (DAPs) were identified using iTRAQ between the pollen mother cell formation stage (P2) and the meiosis stage (P3). There were 3541 and 4247 differentially expressed genes (DEGs) in P2 and P3 between WXS (Sterile, S)-WXS(S) and WXS (Fertile, F)-WXS(F), respectively, of which 92 and 71 DEGs had corresponding DAPs. Among the DAPs and DEGs, 65 (SP2 vs. FP2) and 55 (SP3 vs. FP3) corresponding DEGs and DAPs (cor-DEGs-DAPs) showed the same expression trend, indicating the cor-DEGs-DAPs genes might play vital roles in WXS fertility transition. Further analysis indicated that cor-DEGs-DAPs proteins were related to energy metabolism-related proteins in anther development and were accompanied by the activation of the stress response pathway and modifications to the cell wall, which ultimately affected the fertility transition of the PTGMS rice line WXS.

Biomarker Discovery and Verification of Esophageal Squamous Cell Carcinoma Using Integration of SWATH/MRM.

We propose an efficient integration of SWATH with MRM for biomarker discovery and verification when the corresponding ion library is well established. We strictly controlled the false positive rate associated with SWATH MS signals and carefully selected the target peptides coupled with SWATH and MRM. We collected 10 samples of esophageal squamous cell carcinoma (ESCC) tissues paired with tumors and adjacent regions and quantified 1758 unique proteins with FDR 1% at protein level using SWATH, in which 467 proteins were abundance-dependent with ESCC. After carefully evaluating the SWATH MS signals of the up-regulated proteins, we selected 120 proteins for MRM verification. MRM analysis of the pooled and individual esophageal tissues resulted in 116 proteins that exhibited similar abundance response modes to ESCC that were acquired with SWATH. Because the ESCC-related proteins consisted of a high percentile of secreted proteins, we conducted the MRM assay on patient sera that were collected from pre- and postoperation. Of the 116 target proteins, 42 were identified in the ESCC sera, including 11 with lowered abundances postoperation. Coupling SWATH and MRM is thus feasible and efficient for the discovery and verification of cancer-related protein biomarkers.

Appraisal of the Missing Proteins Based on the mRNAs Bound to Ribosomes.

Considering the technical limitations of mass spectrometry in protein identification, the mRNAs bound to ribosomes (RNC-mRNA) are assumed to reflect the mRNAs participating in the translational process. The RNC-mRNA data are reasoned to be useful for appraising the missing proteins. A set of the multiomics data including free-mRNAs, RNC-mRNAs, and proteomes was acquired from three liver cancer cell lines. On the basis of the missing proteins in neXtProt (release 2014-09-19), the bioinformatics analysis was carried out in three phases: (1) finding how many neXtProt missing proteins have or do not have RNA-seq and/or MS/MS evidence, (2) analyzing specific physicochemical and biological properties of the missing proteins that lack both RNA-seq and MS/MS evidence, and (3) analyzing the combined properties of these missing proteins. Total of 1501 missing proteins were found by neither RNC-mRNA nor MS/MS in the three liver cancer cell lines. For these missing proteins, some are expected higher hydrophobicity, unsuitable detection, or sensory functions as properties at the protein level, while some are predicted to have nonexpressing chromatin structures on the corresponding gene level. With further integrated analysis, we could attribute 93% of them (1391/1501) to these causal factors, which result in the expression products scarcely detected by RNA-seq or MS/MS.

Systematic analysis of missing proteins provides clues to help define all of the protein-coding genes on human chromosome 1.

Our first proteomic exploration of human chromosome 1 began in 2012 (CCPD 1.0), and the genome-wide characterization of the human proteome through public resources revealed that 32-39% of proteins on chromosome 1 remain unidentified. To characterize all of the missing proteins, we applied an OMICS-integrated analysis of three human liver cell lines (Hep3B, MHCC97H, and HCCLM3) using mRNA and ribosome nascent-chain complex-bound mRNA deep sequencing and proteome profiling, contributing mass spectrometric evidence of 60 additional chromosome 1 gene products. Integration of the annotation information from public databases revealed that 84.6% of genes on chromosome 1 had high-confidence protein evidence. Hierarchical analysis demonstrated that the remaining 320 missing genes were either experimentally or biologically explainable; 128 genes were found to be tissue-specific or rarely expressed in some tissues, whereas 91 proteins were uncharacterized mainly due to database annotation diversity, 89 were genes with low mRNA abundance or unsuitable protein properties, and 12 genes were identifiable theoretically because of a high abundance of mRNAs/RNC-mRNAs and the existence of proteotypic peptides. The relatively large contribution made by the identification of enriched transcription factors suggested specific enrichment of low-abundance protein classes, and SRM/MRM could capture high-priority missing proteins. Detailed analyses of the differentially expressed genes indicated that several gene families located on chromosome 1 may play critical roles in mediating hepatocellular carcinoma invasion and metastasis. All mass spectrometry proteomics data corresponding to our study were deposited in the ProteomeXchange under the identifiers PXD000529, PXD000533, and PXD000535.

Expansion of the ion library for mining SWATH-MS data through fractionation proteomics.

The strategy of sequential window acquisition of all theoretical fragment ion spectra (SWATH) is emerging in the field of label-free proteomics. A critical consideration for the processing of SWATH data is the quality of the ion library (or mass spectrometric reference map). As the availability of open spectral libraries that can be used to process SWATH data is limited, most users currently create their libraries in-house. Herein, we propose an approach to construct an expanded ion library using the data-dependent acquisition (DDA) data generated by fractionation proteomics. We identified three critical elements for achieving a satisfactory ion library during the iterative process of our ion library expansion, including a correction of the retention times (RTs) gained from fractionation proteomics, appropriate integrations of the fractionated proteomics into an ion library, and assessments of the impact of the expanded ion libraries to data mining in SWATH. Using a bacterial lysate as an evaluation material, we employed sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to fractionate the lysate proteins and constructed the expanded ion library using the fractionation proteomics data. Compared with the ion library built from the unfractionated proteomics, approximately 20% more peptides were extracted from the expanded ion library. The extracted peptides, moreover, were acceptable for further quantitative analysis.

Proteomic analysis reveals LRPAP1 as a key player in the micropapillary pattern metastasis of lung adenocarcinoma.

Objectives: Lung adenocarcinomas have different prognoses depending on their histological growth patterns. Micropapillary growth within lung adenocarcinoma, particularly metastasis, is related to dismal prognostic outcome. Metastasis accounts for a major factor leading to mortality among lung cancer patients. Understanding the mechanisms underlying early stage metastasis can help develop novel treatments for improving patient survival. Methods: Here, quantitative mass spectrometry was conducted for comparing protein expression profiles among various histological subtypes, including adenocarcinoma in situ, minimally invasive adenocarcinoma, and invasive adenocarcinoma (including acinar and micropapillary [MIP] types). To determine the mechanism of MIP-associated metastasis, we identified a protein that was highly expressed in MIP. The expression of the selected highly expressed MIP protein was verified via immunohistochemical (IHC) analysis and its function was validated by an in vitro migration assay. Results: Proteomic data revealed that low-density lipoprotein receptor-related protein-associated protein 1 (LRPAP1) was highly expressed in MIP group, which was confirmed by IHC. The co-expressed proteins in this study, PSMD1 and HSP90AB1, have been reported to be highly expressed in different cancers and play an essential role in metastasis. We observed that LRPAP1 promoted lung cancer progression, including metastasis, invasion and proliferation in vitro and in vivo. Conclusion: LRPAP1 is necessary for MIP-associated metastasis and is the candidate novel anti-metastasis therapeutic target.

Quantitative analysis of the human AKR family members in cancer cell lines using the mTRAQ/MRM approach.

Members of human aldo-keto reductase (AKR) superfamily have been reported to be involved in cancer progression, whereas the final conclusion is not generally accepted. Herein, we propose a quantitative method to measure human AKR proteins in cells using mTRAQ-based multiple reaction monitoring (MRM). AKR peptides with multiple transitions were carefully selected upon tryptic digestion of the recombinant AKR proteins, while AKR proteins were identified by SDS-PAGE fractionation coupled with LC-MS/MS. Utilizing mTRAQ triplex labeling to produce the derivative peptides, calibration curves were generated using the mixed lysate as background, and no significantly different quantification of AKRs was elicited from the two sets of calibration curves under the mixed and single lysate as background. We employed this approach to quantitatively determine the 6 AKR proteins, AKR1A1, AKR1B1, AKR1B10, AKR1C1/C2, AKR1C3, and AKR1C4, in 7 different cancer cell lines and for the first time to obtain the absolute quantities of all the AKR proteins in each cell. The cluster plot revealed that AKR1A and AKR1B were widely distributed in most cancer cells with relatively stable abundances, whereas AKR1Cs were unevenly detected among these cells with diverse dynamic abundances. The AKR quantitative distribution in different cancer cells, therefore, may assist further exploration toward how the AKR proteins are involved in tumorigenesis.

A large-scale proteogenomic atlas of pear.

Pear is an important fruit tree that is widely distributed around the world. The first pear genome map was reported from our laboratory approximately 10 years ago. To further study global protein expression patterns in pear, we generated pear proteome data based on 24 major tissues. The tissue-resolved profiles provided evidence of the expression of 17 953 proteins. We identified 4294 new coding events and improved the pear genome annotation via the proteogenomic strategy based on 18 090 peptide spectra with peptide spectrum matches >1. Among the eight randomly selected new short coding open reading frames that were expressed in the style, four promoted and one inhibited the growth of pear pollen tubes. Based on gene coexpression module analysis, we explored the key genes associated with important agronomic traits, such as stone cell formation in fruits. The network regulating the synthesis of lignin, a major component of stone cells, was reconstructed, and receptor-like kinases were implicated as core factors in this regulatory network. Moreover, we constructed the online database PearEXP (http://www.peardb.org.cn) to enable access to the pear proteogenomic resources. This study provides a paradigm for in-depth proteogenomic studies of woody plants.

Citrus peel extract protects against diesel exhaust particle-induced chronic obstructive pulmonary disease-like lung lesions and oxidative stress.

Chronic obstructive pulmonary disease (COPD) is the third leading cause of death worldwide and characterized by emphysema, small airway remodeling and mucus hypersecretion. Citrus peels have been widely used as food spices and in traditional Chinese medicine for chronic lung disease. Given that citrus peels are known for containing antioxidants and anti-inflammatory compounds, we hypothesize that citrus peel intake can suppress oxidative stress and inflammatory response to air pollution exposure, thereby alleviating COPD-like pathologies. This study aimed to investigate the efficacy of citrus peel extract, namely Guang Chenpi (GC), in preventing the development of COPD induced by diesel exhaust particles (DEPs) and its potential mechanism. DEP-induced COPD-like lung pathologies, inflammatory responses and oxidative stress with or without GC treatment were examined in vivo and in vitro. Our in vivo study showed that GC was effective in decreasing inflammatory cell counts and inflammatory mediator (IL-17A and TNF-α) concentrations in bronchoalveolar lavage fluid (BALF). Pretreatment with GC extract also significantly decreased oxidative stress in the serum and lung tissue of DEP-induced COPD rats. Furthermore, GC pretreatment effectively reduced goblet cell hyperplasia (PAS positive cells) and fibrosis of the small airways, decreased macrophage infiltration as well as carbon loading in the peripheral lungs, and facilitated the resolution of emphysema and small airway remodeling in DEP-induced COPD rats. An in vitro free radical scavenging assay revealed robust antioxidant potential of GC in scavenging DPPH free radicals. Moreover, GC demonstrated potent capacities in reducing ROS production and enhancing SOD activity in BEAS-2B cells stimulated by DEPs. GC treatment significantly attenuated the increased level of IL-8 and MUC5AC from DEP-treated BEAS-2B cells. Mechanistically, GC treatment upregulated the protein level of Nrf-2 and could function via MAPK/NF-κB signaling pathways by suppressing the phosphorylation of p38, JNK and p65. Citrus peel extract is effective in decreasing oxidative stress and inflammatory responses of the peripheral lungs to DEP exposure. These protective effects further contributed to the resolution of COPD-like pathologies.

Dynamic Plasma Lipidomic Analysis Revealed Cholesterol Ester and Amides Associated with Sepsis Development in Critically Ill Patients after Cardiovascular Surgery with Cardiopulmonary Bypass.

Background: Sepsis in patients after cardiovascular surgery with cardiopulmonary bypass (CPB) has a high rate of mortality. We sought to determine whether changes in lipidomics can predict sepsis after cardiac surgery. Methods: We used high-performance liquid chromatography coupled to tandem mass spectrometry to explore global lipidome changes in samples from a prospective case-control cohort (30 sepsis vs. 30 nonsepsis) hospitalized with cardiovascular surgery. All patients were sampled before and within 48−72 h after surgery. A bioinformatic pipeline was applied to acquire reliable features and MS/MS-driven identifications. Furthermore, a multiple-step machine learning framework was performed for signature discovery and performance evaluation. Results: Compared with preoperative samples, 94 features were upregulated and 282 features were downregulated in the postoperative samples of the sepsis group, and 73 features were upregulated and 265 features were downregulated in the postoperative samples of the nonsepsis group. "Autophagy", "pathogenic Escherichia coli infection" and "glycosylphosphatidylinositol-anchor biosynthesis" pathways were significantly enriched in the pathway enrichment analysis. A multistep machine learning framework further confirmed that two cholesterol esters, CE (18:0) and CE (16:0), were significantly decreased in the sepsis group (p < 0.05). In addition, oleamide and stearamide were increased significantly in the postoperative sepsis group (p < 0.001). Conclusions: This study revealed characteristic lipidomic changes in the plasma of septic patients before and after cardiac surgery with CPB. We discovered two cholesterol esters and two amides from peripheral blood that could be promising signatures for sepsis within a dynamic detection between the preoperative and postoperative groups.

Lysophosphatidylcholines and phosphatidylcholines as biomarkers for stroke recovery.

Stroke is a serious global public health issue, associated with severe disability and high mortality rates. Its early detection is challenging, and no effective biomarkers are available. To obtain a better understanding of stroke prevention, management, and recovery, we conducted lipidomic analyses to characterize plasma metabolic features. Lipid species were measured using an untargeted lipidomic analysis with liquid chromatography-tandem mass spectrometry. Sixty participants were recruited in this cohort study, including 20 healthy individuals and 40 patients with stroke. To investigate the association between lipids related to long-term functional recovery in stroke patients. The primary independent variable was activities of daily living (ADL) dependency upon admission to the stroke unit and at the 3-month follow-up appointment. ADL dependency was assessed using the Barthel Index. Eleven significantly altered lipid species between the stroke and healthy groups were detected and displayed in a hierarchically clustered heatmap. Acyl carnitine, triacylglycerol, and ceramides were detected as potential lipid markers. Regarding the association between lipid profiles and functional status of patients with stroke the results indicated, lysophosphatidylcholines (LPC) and phosphatidylcholines were closely associated with stroke recovery. LPC may contribute positively role in patient's rehabilitation process via an anti-inflammatory mechanism. Appropriate management or intervention for lipid levels is expected to lead to better clinical outcomes.

Clinical Multi-Omics Study on the Gut Microbiota in Critically Ill Patients After Cardiovascular Surgery Combined With Cardiopulmonary Bypass With or Without Sepsis (MUL-GM-CSCPB Study): A Prospective Study Protocol.

Introduction: Fever of unknown origin (FUO) and hemodynamic instability are complications that develop after cardiac surgery combined with cardiopulmonary bypass (CPB) for heart disease. Patients who develop fever with hemodynamic instability after cardiac surgery may have systemic inflammatory response syndrome or sepsis. Cardiopulmonary bypass (CPB) is a technique that temporarily takes over the function of the heart and lungs during cardiac surgery. Recent reports suggest that early bloodstream infections of patients undergoing CPB are due to gram-negative bacteria that are present in the intestinal flora. The theory of intestinal flora translocation has growing evidence. Intestinal ischemia-reperfusion that occurs during cardiac surgery with CPB will induce a systemic inflammatory reaction and may cause intestinal flora translocation. Does this systemic reaction cause sepsis? We therefore propose this protocol to determine whether the changes in the intestinal flora in patients after cardiac surgery with CPB are related to sepsis. Methods and Analysis: This study is a prospective observational case-control study to analyze the variation in the intestinal microflora and metabolites in patients undergoing cardiac surgery with CPB and to observe the outcomes of patients with routine clinical interventions. The control group will include healthy people without intestinal illness. Feces and blood samples will be acquired 1 day before cardiac surgery and within 24-72 h after cardiac surgery, and will be used for genomics and metabolomics analyses. Demographic data describing age, sex, main diagnosis, and past medical history and data related to the CPB time and application of antibiotics are available. Sequential (sepsis-related) organ failure assessment, infection-related laboratory items, infection site, and pathogenic microorganisms, and nutrition, and gastrointestinal function assessment are additionally recorded. Group analysis of data will be conducted according to the outcomes (sepsis vs. non-sepsis and survivors vs. non-survivors). Ethics and Dissemination: This protocol has been ethically approved by the Ethics Committee of Peking Union Medical College (ID: ZS-1612). Informed consent will be obtained before subject enrolment, and data will be stored in a secured database. The results will be submitted to international peer-reviewed journals and presented at international conferences. Trial Registration Number: NCT04032938.

Integrated Analysis of Comparative Lipidomics and Proteomics Reveals the Dynamic Changes of Lipid Molecular Species in High-Oleic Acid Peanut Seed.

Modern peanut contains fatty acid desaturase 2 (FAD2) mutation, which is capable of producing high oleic acid for human health. However, the dynamic changes of the lipidome regarding fad2 remain elusive in peanut seed. In the present study, 547 lipid features were identified in high- and normal-oleic peanut seeds by utilizing the mass spectrometric approach. The fad2-induced differently expressed lipids (DELs) were polarly distributed at early and maturation stages during high-oleic acid (OA) seed development. Subsequently, integration of previously published proteomic data and lipidomic data revealed that 21 proteins and 149 DELs were annotated into the triacylglycerol assembly map, of which nine enzymes and 31 lipid species shared similar variation tendencies. Additionally, the variation tendencies of 17 acyl fatty acids were described in a hypothetical biosynthetic pathway. Collectively, the understanding of the lipid composition correlated with fad2 established a foundation for future high-OA peanut breeding based on lipidomic data.

A proteomic analysis of peanut seed at different stages of underground development to understand the changes of seed proteins.

In order to obtain more valuable insights into the protein dynamics and accumulation of allergens in seeds during underground development, we performed a proteomic study on developing peanut seeds at seven different stages. A total of 264 proteins with altered abundance and contained at least one unique peptide was detected by matrix-assisted laser desorption ionization time-of-flight/time-of-flight mass spectrometry (MALDI-TOF/TOF MS). All identified proteins were classified into five functional categories as level 1 and 20 secondary functional categories as level 2. Among them, 88 identified proteins (IPs) were related to carbohydrate/ amino acid/ lipid transport and metabolism, indicating that carbohydrate/amino acid/ lipid metabolism played a key role in the underground development of peanut seeds. Hierarchical cluster analysis showed that all IPs could be classified into eight cluster groups according to the abundance profiles, suggesting that the modulatory patterns of these identified proteins were complicated during seed development. The largest group contained 41 IPs, the expression of which decreased at R 2 and reached a maximum at R3 but gradually decreased from R4. A total of 14 IPs were identified as allergen-like proteins by BLAST with A genome (Arachis duranensis) or B genome (Arachis ipaensis) translated allergen sequences. Abundance profile analysis of 14 identified allergens showed that the expression of all allergen proteins was low or undetectable by 2-DE at the early stages (R1 to R4), and began to accumulate from the R5 stage and gradually increased. Network analysis showed that most of the significant proteins were involved in active metabolic pathways in early development. Real time RT-PCR analysis revealed that transcriptional regulation was approximately consistent with expression at the protein level for 8 selected identified proteins. In addition, some amino acid sequences that may be associated with new allergens were also discussed.

Comparative Analysis of the Metabolic Profiles of Yellow- versus Black-Seeded Rapeseed Using UPLC-HESI-MS/MS and Transcriptome Analysis.

The high levels of secondary metabolites in rapeseed play important roles in determining the oil quality and feeding value. Here, we characterized the metabolic profiles in seeds of various yellow- and black-seeded rapeseed accessions. Two hundred and forty-eight features were characterized, including 31 phenolic acids, 54 flavonoids, 24 glucosinolates, 65 lipid compounds, and 74 other polar compounds. The most abundant phenolic acids and various flavonoids (epicatechin, isorhamnetin, kaempferol, quercetin, and their derivatives) were widely detected and showed significant differences in distribution between the yellow- and black-seeded rapeseed. Furthermore, the related genes (e.g., BnTT3, BnTT18, BnTT10, BnTT12, and BnBAN) involved in the proanthocyanidin pathway had lower expression levels in yellow-seeded rapeseed, strongly suggesting that the seed coat color could be mainly determined by the levels of epicatechin and their derivatives. These results improve our understanding of the primary constituents of rapeseed and lay the foundation for breeding novel varieties with a high nutritional value.