Protein Profiles Research Articles

In-depth analysis of lymph node metastasis-related sialylated protein profiling and their clinical and biological significance in colorectal cancer using mass spectrometry and multi-omics technologies

In-depth analysis of lymph node metastasis-related sialylated protein profiling and their clinical and biological significance in colorectal cancer using mass spectrometry and multi-omics technologies

Abstract C017: Exploring the composition of and interaction of the tumor microenvironment in Langerhans cell histiocytosis

Abstract Langerhans cell histiocytosis (LCH) is a rare inflammatory hematological neoplasm, characterized by the accumulation of LCH cells in various tissues and organs. These LCH cells originate from myeloid progenitors that carry a MAPK-pathway mutation. Once these mutated cells enter the tissue from the periphery, they differentiate into so-called LCH cells. In addition to LCH cells, the tumor microenvironment (TME) consists of a wide variety of immune cells. However, the interactions between these cells within the TME and their contribution to disease progression remain poorly understood. In this study, we focus on understanding the role of lymphocytes in LCH lesions. We used single-cell RNA sequencing to comprehensively profile the immune populations in LCH lesions on the transcriptomic level, which will be supplemented with spatial profiling. Our analysis identified a wide variety of infiltrating immune cells within LCH lesions with high cellular heterogeneity among patients. LCH- and T cells are the most prevalent cell types, suggesting a key role in lesion development and maintenance. Furthermore, the immune landscape includes several dendritic cell subsets – such as plasmacytoid DCs (pDCs), cDC1, and mature regulatory dendritic cells (mregDCs) – alongside tumor-associated macrophages (TAMs), monocytes, NK cells, B cells, and plasmablasts. Among T cells, regulatory T cells (Tregs) are particularly abundant and appear to be heavily influenced by LCH cells and other myeloid cells via the TNF-TNFR2 and CD86-CTLA4 pathways. Additionally, LCH cells express high levels of TGFβ – a known regulator of T cell activity – and MMP9 – which might hamper cytotoxic T cell infiltration. This suggests that LCH cells actively contribute to creating an immunosuppressive environment. Surprisingly, pDCs seem to be a major immune regulator within lesions and are likely to interact with T cells via TNFSF9 and Granzyme B. These insights into the immunosuppressive environment within LCH lesions highlight the critical role of Tregs as central regulators within the tumor microenvironment. The implications of these findings will be further explored and validated through in-vitro experiments and high-plex spatial protein profiling. Ultimately, our work may pave the way for developing targeted therapies aimed at modulating the TME, potentially enhancing anti-tumor immunity, and offering new strategies for treating LCH. Citation Format: Wouter van Midden, Raphaela Schwentner, Sebastian Eder, Philipp Ben Soussia-Weiss, Giulio Abagnale, Caroline Hutter. Exploring the composition of and interaction of the tumor microenvironment in Langerhans cell histiocytosis [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr C017.

In-Depth Proteome Profiling of the Hippocampus of LDLR Knockout Mice Reveals Alternation in Synaptic Signaling Pathway.

The low-density lipoprotein receptor (LDLR) is a major apolipoprotein receptor that regulates cholesterol homeostasis. LDLR deficiency is associated with cognitive impairment by the induction of synaptopathy in the hippocampus. Despite the close relationship between LDLR and neurodegenerative disorders, proteomics research for protein profiling in the LDLR knockout (KO) model remains insufficient. Therefore, understanding LDLR KO-mediated differential protein expression within the hippocampus is crucial for elucidating a role of LDLR in neurodegenerative disorders. In this study, we conducted first-time proteomic profiling of hippocampus tissue from LDLR KO mice using tandem mass tag (TMT)-based MS analysis. LDLR deficiency induces changes in proteins associated with the transport of diverse molecules, and activity of kinase and catalyst within the hippocampus. Additionally, significant alterations in the expression of components in the major synaptic pathways were found. Furthermore, these synaptic effects were verified using a data-independent acquisition (DIA)-based proteomic method. Our data will serve as a valuable resource for further studies to discover the molecular function of LDLR in neurodegenerative disorders.

Dietary intervention of propolis and/or turmeric boosted growth, hematology, biochemical profile, and antioxidant-immune responses and their associated gene expression in Nile tilapia (Oreochromis niloticus) challenged with Edwardsiella tarda

AbstractImmunostimulant phytogenic feed additives are given great concern for improving fish health, growth, immune responses, and resistance to diseases. This research investigated the impact of dietary propolis (PRO), turmeric (TUR), and their combination on the growth, hematology, antioxidant-immune responses, and their regulating genes in Nile tilapia (Oreochromis niloticus) during Edwardsiella tarda challenge. For 8 weeks, a total number of 320 fish (20.70 ± 0.14 g) were allocated into four groups at random, each with eight replicates (10 fish each). The first group (1st) was given the basal diet (control) without any supplements. The 2nd, 3rd, and 4th groups were supplemented with 1% PRO, 1% TUR, and the mixture (1%PRO + 1%TUR), respectively. The experimental groups were challenged intraperitoneally with E. tarda at a dose of 0.1 mL (1 × 105 CFU) at the termination of the feeding trial, and the fish survival was estimated for an additional 7 days. The results demonstrated that fish-fed diets supplemented with PRO and/or TUR showed higher body weight, condition factor, specific growth rate, feed intake, and feed efficiency utilization than the control group (P < 0.05). The hematological, protein profile, and antioxidant-immune (total antioxidant capacity, lysozymes, and IgM) parameters were substantially improved in the challenged fish fed on PRO and/or TUR diets compared to the challenged non-fed fish. The lipid profile and malondialdehyde were substantially decreased in the challenged fish fed on PRO and/or TUR diets compared to the challenged non-fed group. Notably, a down-turning of nuclear factor kappa B (NFκB) and tumor necrosis factor-alpha (TNF-α) expression with up-turning of nuclear factor erythroid 2-related factor 2 (Nrf2) and transforming growth factor-beta (TGF-β) expression was noticed in the challenged fish fed on PRO and/or TUR diets compared to the challenged non-fed fish. Noteworthy, dietary PRO and/or TUR improved the fish survival during E. tarda challenge. The mixture of PRO and TUR can be added to Nile tilapia diets to enhance their growth, immune response, and resistance to E. tarda. These outcomes help in the sustainable development of the Nile tilapia culture industry. Graphical Abstract

Multi-omics analysis reveals the positive impact of differential chloroplast activity during in vitro regeneration of barley.

Existence of potent in vitro regeneration system is a prerequisite for efficient genetic transformation and functional genomics of crop plants. In this study, two contrasting cultivars differencing in their in vitro regeneration efficiency were identified. Tissue culture friendly cultivar Golden Promise (GP) and tissue culture resistant DWRB91(D91) were selected as contrasting cultivars to investigate the molecular basis of regeneration efficiency through multiomics analysis. Transcriptomics analysis revealed 1487 differentially expressed genes (DEGs), in which 795 DEGs were upregulated and 692 DEGs were downregulated in the GP-D91 transcriptome. Genes encoding proteins localized in chloroplast and involved in ROS generation were upregulated in the embryogenic calli of GP. Moreover, proteome analysis by LC-MS/MS revealed 3062 protein groups and 16,989 peptide groups, out of these 1586 protein groups were differentially expressed proteins (DEPs). Eventually, GC-MS based metabolomics analysis revealed the higher activity of plastids and alterations in key metabolic processes such as sugar metabolism, fatty acid biosynthesis, and secondary metabolism. TEM analysis also revealed differential plastid development. Higher accumulation of sugars, amino acids and metabolites corresponding to lignin biosynthesis were observed in GP as compared to D91. A comprehensive examination of gene expression, protein profiling and metabolite patterns unveiled a significant increase in the genes encompassing various functions, such as ion homeostasis, chlorophyll metabolic process, ROS regulation, and the secondary metabolic pathway.

Potentiality of Amaranthus viridis (L.) to accumulation of heavy metals and its relation to protein profile

The present study was undertaken to assess the levels of heavy metals (Cd, Fe, Ni and Zn) in soil and plant parts. The phytoremediation potential of Amaranthus viridis (amaranth) was determined by calculating the bioconcentration factor (BCF) and translocation factor (TF). Soil and plant samples were collected from eight different sites in their native habitats, distributed along Ismailia irrigation canal located in the east of the Nile Delta region, Egypt. Roots of amaranth had the higher concentrations of Cd, Fe and Ni means which were 2.75, 547.3 and 7.25 mg g-1 DW at sites SH8, SH6 and SH4 respectively, as compared to the other sampling sites. Whereas, shoots had the higher concentrations of Zn (283.6 mg g-1 DW) at site SH5. All sites were found with BCF more than 1 for all heavy metals. Except for Zn, TF values of Cd, Fe and Ni at all sites were lower than one. Depending on BCF and TF results, amaranth showed significant potential for phytostabilization of Cd, Fe and Ni and phytoextraction of Zn as well. Moreover, the protein profile showed different bands with varied molecular weights. Both relationships between studied accessions and similarity were elucidated, with high similarity (96% between SH2 and SH3 accessions), lowest similarity (72% between SH4 and SH8) and degree of polymorphism calculated as 62.5%. The variation in the similarity index may be due to the induction of a variety of proteins under heavy metal stress and also ecological features in various sites that cause protein polymorphism.

Abstract A029: Novel high-purity CTC isolation technology for downstream analysis: A “Cell- Circuit” magnetophoretic-microfluidic hybrid device

Abstract Circulating Tumor Cells are the only blood analytes that contain comprehensive multi-omic information regarding living tumor cells. As such, CTCs are an essential tool for the precise diagnosis of cancer. This has led to increased interest in both enumeration, and more significantly, the downstream analysis of bulk and single CTCs. One primary technical limitation that limits adoption of CTCs into routine clinical practice is the ability to effectively isolate CTCs from non-target cells. While several technologies have been developed and commercialized including initial clinical adoption, most technologies have fundamental limitations in sample purity which limits the quality of downstream analysis. Another limitation is the number of steps needed to complete enrichment, especially in high purity isolation where 2 techniques are usually required to be performed in sequence. The authors propose a novel, single-cell isolation technology called L:Cell™. This technology utilizes precise micro-magnetic patterns to allow immuno-magnetically labeled cells to be isolated individually and directed to user-defined locations. Such “Cell-Circuits” enable parallel, computer-circuit-like manipulation of a large array of single cells – combining both precision and throughput. Passive and active single-cell control is made possible by circuit components such as gates, single-cell capacitors, and transistors. The technology also enables the classification of cells based on size in addition to the presence of magnetic beads. A "passive component" performs its function under an external rotating magnetic field, manipulating cells without the need for an electrical current. The majority of circuitry on-chip are such passive components, which enable the device's key advantage of parallelized control. In contrast, an "active component" is designed in conjunction with passive components to create a local magnetic field at specific locations. This allows cells to be rerouted or manipulated on-demand. Using active components, users can selectively isolate individual cells (e.g., based on size, morphology, or fluorescence). While micro-magnetophoretic technology has been explored by various research teams, the authors have advanced this technology further by micro-magnetic simulation and integrating microfluidic technology. This integration improves the inlet and outlet structures for cell samples and the alignment of particles’ flow, thereby maximizing the commercialization potential of this sophisticated technology. In lab-scale modeling, the authors demonstrated a high separation purity level of 92%. By allowing manipulation at single-cell level using magnetic fields only, the technology minimizes any stress on the cells during isolation processes. The authors developed a user- friendly interface utilizing microfluidic channels for the extraction of CTCs from whole blood using this hybrid device. Additionally, the authors are conducting further adaptation studies to apply this technology to various downstream techniques, such as genomic analysis and protein profiling. Citation Format: Chanhee Lee, Hun Heo, Youngwoo Park, Donghee Ko. Novel high-purity CTC isolation technology for downstream analysis: A “Cell- Circuit” magnetophoretic-microfluidic hybrid device [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr A029.

Abstract B042: Molecular assessment of cell surface targets using integrated circulating tumor cell (CTC) RNAseq and single CTC phenotyping

Abstract Background: Androgen-targeted therapies are a mainstay of treatment for metastatic prostate cancer and have significantly improved patient outcomes. However, development of treatment resistance remains universal, occurring through androgen receptor (AR) alterations driving constitutive AR signaling, or lineage state transitions that bypass AR entirely and culminate in a small cell/neuroendocrine phenotype (NEPC). Cell surface targeted therapies, including antibody drug conjugates and targeted radioligand therapies, represent a new therapeutic class that has shown promise for treatment of androgen-resistant metastatic prostate cancer (mCRPC). Circulating tumor cells (CTCs) present an accessible source of tumor material to identify expression of known and novel targets for therapeutic development, as well as the unique potential for longitudinal profiling to understand the evolution of target expression and association with clinical resistance to cell surface targeted therapies. We have developed a CTC platform allowing for gene expression and protein profiling of cell surface targets on CTCs. Methods: Live CTCs were isolated with our automated microfluidic technology integrating negative and positive selection for CTC enrichment with a cohort of 273 samples. CTCs were captured immunomagnetically followed by RNA isolation on chip and RNA-seq, or protein staining on chip for single cell fluorescent quantification of prostate adenocarcinoma and NEPC cell surface targets including PSMA, TROP2, B7H3 and DLL3. Results: CTC RNA sequencing can be used to understand the expression of cell surface targets across different CTC phenotypes. In CTCs from patient with mCRPC, canonical prostate adenocarcinoma cell surface targets STEAP1, KLK2 and FOLH1 and epithelial cell surface target TACSTD2 had the highest expression, while prostate adenocarcinoma target STEAP2, pan-tumor target ERBB2, and tumor immune checkpoint cell surface protein CD276 (B7H3) had intermediate expression. Expression of DLL3 and SSTR2, targets associated with neuroendocrine differentiation, was lowest, though still detected in a subset of CRPC CTCs. Comparison of expression levels between CRPC and NEPC CTCs demonstrated lower expression of most adenocarcinoma cell surface targets in NEPC CTCs as expected, and a trend towards higher expression of DLL3 and SSTR2. Cell surface expression is being assessed for proteins including Trop-2, PSMA and DLL3. Evaluation of DLL3 at the protein level in a subset of patients including both CRPC (n=13) and NEPC (n=12) confirmed that DLL3 was detected in both adenocarcinoma and neuroendocrine CTCs, with a median of 45.8% DLL3-positive CTCs per sample. Conclusions: Biomarkers are needed to better predict response and resistance to targeted therapies. CTC RNAseq and single CTC phenotyping hold potential for predictive and on-treatment biomarkers of response to cell surface targeted therapies. Further study of mechanisms driving response and resistance can be evaluated with these biomarkers. Citation Format: Jamie M. Sperger, Marina N. Sharifi, Katherine R. Kaufmann, Matthew L. Bootsma, Shannon R. Reese, Viridiana Carreno, Alex H. Chang, Luke A. Nunamaker A. Nunamaker, Charlotte Linebarger, Amy K. Taylor, Katherine E Tippins, Kyle T. Helzer, Shuang G. Zhao, Joshua M Lang. Molecular assessment of cell surface targets using integrated circulating tumor cell (CTC) RNAseq and single CTC phenotyping [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr B042.

Proteomic Profiling of COVID-19 Patients Sera: Differential Expression with Varying Disease Stage and Potential Biomarkers

Background/Objectives: SARS-CoV-2 is one of the viruses that caused worldwide health issues. This effect is mainly due to the wide range of disease prognoses it can cause. The aim of this study is to determine protein profiles that can be used as potential biomarkers for patients’ stratification, as well as potential targets for drug development. Methods: Eighty peripheral blood samples were collected from heathy as well as SARS-CoV-2 patients admitted at a major tertiary care center in Riyadh, Saudi Arabia. A label-free quantitative mass spectrometry-based proteomic analysis was conducted on the extracted sera. Protein–protein interactions and functional annotations of identified proteins were performed using the STRING. Results: In total, two-hundred-eighty-eight proteins were dysregulated among all four categories. Dysregulated proteins were mainly involved in the network map of SARS-CoV-2, immune responses, complement activation, and lipid transport. Compared to healthy subjects, the most common upregulated protein in all three categories were CRP, LGALS3BP, SAA2, as well as others involved in SARS-CoV-2 pathways such as ZAP70 and IGLL1. Notably, we found fifteen proteins that significantly discriminate between healthy/recovered subjects and moderate/under medication patients, among which are the SERPINA7, HSPD1 and TTC41P proteins. These proteins were also significantly downregulated in under medication versus moderate patients. Conclusions: Our results emphasize the possible association of specific proteins with the SARS-CoV-2 pathogenesis and their potential use as disease biomarkers and drug targets. Our study also gave insights about specific proteins that are likely increased upon infection but are likely restored post recovery.

Abstract 4119195: Proteomic Analysis of Primary Angioplasty in Acute Myocardial Infarction: Implications for Left Ventricular Remodelling and Recovery of Function

Introduction: Acute myocardial infarction (AMI) is a leading global cause of mortality. Following primary percutaneous coronary intervention (PCI), 25% require hospitalisation for symptomatic heart failure. Further understanding of the molecular pathogenesis and the temporal relationship of the protein profile associated with ischaemic insult and subsequent cardiac remodelling is required to improve risk stratification following AMI. Methods: 20 patients with AMI presenting to Royal Free Hospital in London were recruited for proteomic analysis (O link Proximity Extension Immunoassay) at presentation and at 24 and 72 hours following primary PCI. All underwent initial CMR on admission and at 6 month follow up to assess for myocardial oedema, left ventricular volumes and function and assessment of scar. Results: Proteomic biomarkers that increased on discharge following AMI were: IGFBP2 (8% increase, p=0.001), MMP-10 (5% increase, p=0.001), TIMP4 (14.4% increase, p<0.001), CSF-1 (3.4% increase, p<0.001), CHI3L1 (15% increase, p<0.001), ST2 (19% increase, p=0.001), TGF-alpha (17% increase, p=0.001), IL6 (38% increase, p<0.001), and NT-proBNP (93% increase, p<0.001). The only biomarker that significantly decreased was IGFBP-1 (24% decrease, p=0.003). Notably, increased post-PCI biomarkers correlated with reduced infarct size at follow-up, including CCL23 (R=-0.6, p=0.016), IL6 (R=-0.051, p=0.04), NT-proBNP (R=-0.72, p=0.002). High biomarkers at initial presentation associated with reduced left ventricular end diastolic volume (LVEDV) were CSF-1 (R=-0.64, p=0.03) and CCL23 (R=-0.76, p=0.006). Discussion: Following AMI, we observed increase in proteins implicated in inflammation, angiogenesis and tissue repair. IGFBP1 as a stress hormone significantly decreased after PCI. Markers that showed linear association with reduced cardiac preload measured by LVEDV were linked with angiogenesis promotion and resolution of inflammation.

Abstract 4136813: Redox Serum Proteomics Of Left Ventricular Assist Device Unloaded Human Heart

Heart failure (HF) is a global epidemic claiming millions of lives worldwide. Unloading the heart of end-stage HF patients via left ventricular assist devices (LVADs) is used not only as a bridge to transplantation but also as a bridge to recovery in certain selected patients. To date, assessment of patients’ recovery from HF is based on clinical and symptomatic evaluation. The use of non-invasive biomarkers that monitor the remodeling/reverse remodeling associated with HF progression/recovery is still lacking. In this study we applied mass spectrometry-based quantitative redox proteomics to identify serum biomarkers to monitor both the progression of HF and its recovery after LVAD treatment. A fingerprint of reversed behavior at the protein and at the oxidized Cys peptide level has been discovered when comparing HF and LVAD patients. HDL proteins related to lipid metabolism were decreased in HF samples and increased in LVAD patients. In addition, a number of inflammatory proteins showed a decrease in LVAD samples and an increase in HF samples. In particular APOA4, C7, F2 or SAA2 discriminated between HF and LVAD patients. A pool of peptides containing specific oxidized Cys residues showed an inverse relationship in HF as opposed to LVAD samples. Oxidized Cys-containing peptides derived from Albumin, IGK and IGG1 discriminated HF from LVAD samples. Discriminatory sensitivity and specificity were enhanced by using a combination of the proteins APOA4, C7, F2 and the above-mentioned peptide derived from Albumin. Responders and non-responders could also be separated by proteins such as APOA4, and Cys-oxidized peptides, such as the ones derived from Albumin or IGK mentioned above. This is the first study to apply redox proteomics to explore the protein profile of the LVAD unloaded human heart. These data suggest that HDL plays an important role in LVAD recovery and that a specific group of peptides is associated with the recovery process; the mechanisms involved need further study. Our data could have important implications to the management of patients in the LVAD recovery program and could serve to identify new therapeutic targets.

Protein profile changes during priming explants to embryogenic response in Coffea canephora: identification of the RPN12 proteasome subunit involved in the protein degradation.

Plant somatic embryogenesis encompasses somatic cells switch into embryogenic cells that can later produce somatic embryos with the ability to produce plantlets. Previously, we defined in vitro culture settings for the somatic embryogenesis process of Coffea canephora that comprise adequate plantlets with auxin plus cytokinin followed by cut-leaf explant cultivation with cytokinin, producing embryos with the ability to regenerate plantlets. Here, we confirmed that cultivating cut-leaf explants with cytokinin is sufficient to promote somatic embryos proliferation and the high yield of somatic embryos in the protocol requires adequate plantlets with auxin plus cytokinin. Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels reveal auxin-plus cytokinin-dependent regulated proteins in plantlets with up and down abundance. Chitinase A class III, proteins involved in the metabolism and folding of proteins, photosynthesis, antioxidant activity, and chromatin organization were identified. The RPN12 protein, which is a subunit of the proteasome 26S, has an abundance that is not associated with transcript changes, suggesting post-translational regulation.

TMIC-37. GEMISTOCYTIC TUMOR CELLS AND IMMUNE REACTIVE MICROGLIA PROGRAMMED FOR GLIAL SCARRING CHARACTERIZE T CELL CONTAINMENT IN IDH-MUTANT ASTROCYTOMA

Abstract Isocitrate dehydrogenase 1/2 mutant (IDHmt) astrocytoma is considered a T cell-deprived tumor, yet little is known regarding the phenotype that relates to T cell exclusion. To aid better understanding of this mechanism, we systematically characterized the IDHmt astrocytoma immune microenvironment in a large cohort by multiplex immunofluorescence (IF) (n=75), bulk RNA sequencing (n=158), single nucleus RNA sequencing (n=7) and spatial RNA and protein profiling (n=12). We integrated and analyzed all datasets to acquire a robust characterization of the microenvironment organization and validated our results using IF stainings. We demonstrated that in IDHmt astrocytomas, T cells were contained in the perivascular space (i.e., T cell cuffs). In the tumor stroma surrounding T cell cuffs, we found a dense accumulation of gemistocytic tumor cells (GTCs), a histologically distinct tumor cell subtype. GTC-high tumors demonstrated enhanced expression of immune cell migration and activation pathways that were reliably expressed by TAMs and T cells. Notably, we found that GTCs formed a unique transcriptional cell sub-cluster that expressed markers including CD44, TNC and CRYAB, which are specific for glial scarring, a central nervous system specific mechanism for immune exclusion. Surrounding T cell cuffs, GTCs co-localized with immune-reactive microglia that expressed the chemoattractants CCL3 and CCL4. Together, co-localizing GTCs and reactive microglia expressed receptor-ligand pairs that take part in reactive astrogliosis and glial scarring, including Osteopontin – CD44 and IL-1β – IL1R1. Validation whole-slide multiplex IF stainings for this spatial relationship showed specific local accumulation of CD44 positive GTCs and TAMs around T cell cuffs in GTC-high tumors, which was absent in GTC-low tumors. Together, we show that IDHmt astrocytomas organize a network of tumor cells and leukocytes that mimics glial scarring and can aid T cell exclusion. Elucidating such specific immune exclusion mechanisms is vital for more appropriate target selection in novel immune therapies.

Breeding and biotechnology approaches to enhance the nutritional quality of rapeseed byproducts for sustainable alternative protein sources- a critical review

Global protein consumption is increasing exponentially, which requires efficient identification of potential, healthy, and simple protein sources to fulfil the demands. The existing sources of animal proteins are high in fat and low in fiber composition, which might cause serious health risks when consumed regularly. Moreover, protein production from animal sources can negatively affect the environment, as it often requires more energy and natural resources and contributes to greenhouse gas emissions. Thus, finding alternative plant-based protein sources becomes indispensable. Rapeseed is an important oilseed crop and the world’s third leading oil source. Rapeseed byproducts, such as seed cakes or meals, are considered the best alternative protein source after soybean owing to their promising protein profile (30%–60% crude protein) to supplement dietary requirements. After oil extraction, these rapeseed byproducts can be utilized as food for human consumption and animal feed. However, anti-nutritional factors (ANFs) like glucosinolates, phytic acid, tannins, and sinapines make them unsuitable for direct consumption. Techniques like microbial fermentation, advanced breeding, and genome editing can improve protein quality, reduce ANFs in rapeseed byproducts, and facilitate their usage in the food and feed industry. This review summarizes these approaches and offers the best bio-nutrition breakthroughs to develop nutrient-rich rapeseed byproducts as plant-based protein sources.

Profiling the Misfolded Proteome in Human Disease

AbstractChanges in protein homeostasis are broadly implicated in many disease states, including amyloidoses, neurodegenerative diseases, cancer, and normal aging. Although this relationship has been fruitful for identifying and developing therapeutic strategies, it is challenging to identify which proteins are misfolding. New technologies have recently emerged that enable proteome‐wide interrogation of protein conformation and stability. In this review, we describe these technologies, and how they have been used to identify proteins whose folding changes between disease states. We discuss some of the challenges in this emerging field, and the potential for misfolded protein profiling to provide insight into human disease.

Myocardial Posttranscriptional Landscape in Peripartum Cardiomyopathy.

Pregnancy imposes significant cardiovascular adaptations, including progressive increases in plasma volume and cardiac output. For most women, this physiological adaptation resolves at the end of pregnancy, but some women develop pathological dilatation and ultimately heart failure late in pregnancy or in the postpartum period, manifesting as peripartum cardiomyopathy (PPCM). Despite the mortality risk of this form of heart failure, the molecular mechanisms underlying PPCM have not been extensively examined in human hearts. Protein and metabolite profiles from left ventricular tissue of end-stage PPCM patients (N=6-7) were compared with dilated cardiomyopathy (DCM; N=5-6) and nonfailing donors (N=7-18) using unbiased quantitative mass spectrometry. All samples were derived from the Sydney Heart Bank. Data are available via ProteomeXchange with identifier PXD055986. Differential protein expression and metabolite abundance and Kyoto Encyclopedia of Genes and Genomes pathway analyses were performed. Proteomic analysis identified 2 proteins, SBSPON (somatomedin B and thrombospondin type 1 domain-containing protein precursor) and TNS3 (tensin 3), that were uniquely downregulated in PPCM. SBSPON, an extracellular matrix protein, and TNS3, involved in actin remodeling and cell signaling, may contribute to impaired tissue remodeling and fibrosis in PPCM. Metabolomic analysis revealed elevated levels of homogentisate and deoxycholate and reduced levels of lactate and alanine in PPCM, indicating disrupted metabolic pathways and glucose utilization. Both PPCM and DCM shared pathways related to inflammation, immune responses, and signal transduction. However, thyroid hormone signaling was notably reduced in PPCM, affecting contractility and calcium handling through altered expression of PLN (phospholamban) and Sarcoendoplasmic Reticulum Calcium ATPase (SERCA). Enhanced endoplasmic reticulum stress and altered endocytosis pathways in PPCM suggested additional mechanisms of energy metabolism disruption. The present study reveals unique posttranslational molecular features of the PPCM myocardium, which mediates cellular and metabolic remodeling, and holds promise as potential targets for therapeutic intervention.

Tunable Activated Esters Enable Lysine-Selective Protein Labeling and Profiling.

Lysine residues on protein surfaces are abundant and often found in enzyme active sites, making them critical targets for studying undruggable proteins. However, the varied microenvironment surrounding lysine residues results in a wide range of pKa values, complicating site-specific covalent binding. In this study, we address the challenges posed by the diverse reactivity of amino side chains by modulating the amide reaction activity of heteroaromatic activated esters. By fine-tuning the type, position, and number of heteroatoms, we successfully rationalized the regulation of their amide reaction activity, leading to the design of probes for selective lysine labeling within the proteome for profiling purposes. Systematic optimization of these esters' reactivity and selectivity has yielded a series of effective probes suitable for both in vitro and cellular applications. These findings significantly enhance our understanding of protein functions and mechanisms, facilitated by the precise identification and analysis of protein labeling and profiling.

Insights into larval development and protein biochemical alterations of Agrotis ipsilon (Hufnagel) (Lepidoptera: Noctuidae) following Beauveria bassiana and Solanum lycopersicum treatments

BackgroundThe polyphagous notorious pest, black cutworm, Agrotis ipsilon (Hufnagel) (Lepidoptera: Noctuidae), cause significant production losses due to its distinctive feeding and hiding behavior, making it particularly challenging to control it with conventional methods. Therefore, sustainable agriculture demands more effective and environmentally safe pest control solutions. This study aimed to investigate the toxicity of two insecticide alternatives, the entomopathogenic fungus (EPF) Beauveria bassiana and Solanum lycopersicum extract (Tomato plant crude extract, TPCE), using two bioassay methods: the poisoned bait method and the leaf dipping method. In addition, the impact of these biological tools on larval development and protein profiles was evaluated.ResultsThe bait application of both tested materials exhibited higher toxicity than the leaf dipping method, as indicated by the toxicity index. The LC50 values for B. bassiana were 1.6 × 10⁸ and 1.8 × 10⁶ conidia ml−1 using the leaf dipping method and poisoned baits method, respectively. For TPCE, the LC50 values were 4.35 and 1.51 mg ml−1 for the same methods, respectively. In addition, sublethal concentrations of both materials altered the larval and pupal durations. B. bassiana significantly reduced the concentration of larval hemolymph protein. A maximum of 12 protein bands in the control sample, with molecular weights (Mw) ranging between 35 and 120 kDa, were detected by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). In B. bassiana-treated larvae, ten bands were detected with Mw ranging from 35 to 120 kDa. At least seven bands were detected in TPCE-treated larvae, with Mw ranging from 35 to 97 kDa.ConclusionsThe findings of this study can be integrated into management programs for A. ipsilon. In addition, the availability of B. bassiana and TPCE in Egypt and their cost-effectiveness as insecticide alternatives support their use in the management programs of this critical pest. These methods are particularly effective when applied in bait form.Graphical

Proteome integral solubility alteration via label-free DIA approach (PISA-DIA), game changer in drug target deconvolution.

Drug protein-target identification in past decades required screening compound libraries against known proteins to determine drugs binding to specific protein. Protein targets used in drug-target screening were selected predominantly used laborious genetic manipulation assays. In 2013, a team led by Pär Nordlund from Karolinska Institutet(Stockholm, Sweden) developed Cellular Thermal Shift Assay (CETSA), a method which, for the first time, enabled the possibility of drug protein-target identification in the complex cellular proteome. High throughput, quantitative mass spectrometry (MS) proteomics appeared as a compatible analytical method of choice to complement CETSA, aka Thermal Protein Profiling assay (TPP). Since the seminal CETSA-MS/ TPP-MS publications, different protein-target deconvolution strategies emerged including Proteome Integral Solubility Alteration (PISA). The work of Emery-Corbin etal.(Proteomics2024,2300644), titled Proteome Integral Solubility Alteration via label-free DIA approach (PISA-DIA), introduces Data-Independent Acquisition (DIA) as a quantification method, opening new avenues in drug target-deconvolution field. Application of DIA for target deconvolution offers attractive alternative to widely used data dependent methodology.

Comparison of three methods for generating the coccoid form of Helicobacter pylori and proteomic analysis

BackgroundHelicobacter pylori changes from spiral to coccoid depending on the host state, environmental factors, and surrounding microbial communities. The coccoid form of H. pylori still maintains its complete cellular structure, retains virulence genes, and thus plays a role in pathogenicity. To understand the coccoid form, it is crucial to establish the in vitro generation of the coccoid H. pylori. Although some conditions have been studied for the generation of the coccoid form, few studies have compared these conditions for coccoid generation. Here, we generated coccoid forms via three methods and compared the differences in morphology, viability, culturability, and protein expression.ResultsThe coccoid H. pylori was generated in vitro via three methods: a starvation method, a method using amoxicillin, and a method using the culture supernatant of Streptococcus mitis. The morphology and viability of the cells were examined by fluorescence microscopy after staining with SYTO9 and propidium iodide. The culturability of H. pylori was examined by counting colony-forming units on chocolate agar plates. In the starvation group, no colonies formed after 7 days, but viable coccoids were continuously observed. In the amoxicillin-treated group, the culturability decreased rapidly after 12 h, and showed a viable but non culturable (VBNC) state after the third day. Most cells treated with S. mitis supernatant changed to coccoid forms after 7 days, but colonies were continuously formed, probably due to living spiral forms. We performed proteomics to analyse the differences in protein profiles between the spiral and coccoid forms and protein profiles among the coccoid forms generated by the three methods.ConclusionAmoxicillin treatment changed H. pylori to VBNC cells faster than starvation. Treatment with the S. mitis supernatant prolonged the culturability of H. pylori, suggesting that the S. mitis supernatant may contain substances that support spiral form maintenance. Proteomic analysis revealed that the expression of proteins differed between the spiral form and coccoid form of H. pylori, and this variation was observed among the coccoid forms produced via three different methods. The proteins in the coccoid forms produced by the three methods differed from each other, but common proteins were also observed among them.