Nano-liquid Chromatography-tandem Mass Spectrometry Research Articles

Identification of umami peptides and mechanism of the interaction with umami receptors T1R1/T1R3 in pigeon meat.

Pigeon meat offer an ideal source for extracting fresh flavor peptides. These peptides not only enhance the taste of food but also have potential health benefits, including providing low-sugar, low-sodium, and low-calorie options for individuals with conditions like diabetes, hypertension, and obesity. Therefore, further research into the pigeon industry holds promise for addressing both economic and nutritional needs. To explore umami peptides and their molecular binding mechanisms with umami receptor type 1 member 1 in pigeon meat, an enzymatic hydrolysate product is isolated, analyzed, and subjected to sensory evaluation. Fifteen peptides with high freshness characteristics are separated and identified by ultrafiltration, gel separation, reverse performance liquid chromatography, and nano-liquid chromatography-tandem mass spectrometry (nano-LC-MS/MS). The molecular docking results show that the amino acid residue Glu128 is a common ligand binding site for all of the fresh-flavored peptides to taste T1R1/T1R3 receptors and it exerts freshness-presenting effects with 15 fresh-flavored peptides through hydrogen bonding, electrostatic interactions, salt bridges, and hydrophobic interactions. This study provides a theoretical basis and technical support for the subsequent development of flavor peptide products in pigeon meat.

Proteomic Profiling of Lymph Nodes Differentiates Classic Hodgkin Lymphoma With and Without Skeletal Involvement.

Classic Hodgkin lymphoma (CHL) is a highly curable disease, even in advanced stages. Controversy remains over whether bone involvement negatively affects overall and progression-free survival in patients treated with intensive chemotherapy regimens. Whether cases that present with bone lesions harbor specific tumor microenvironmental features is unknown. We investigated protein expression in diagnostic lymph node biopsies from CHL patients with and without skeletal involvement at diagnosis to identify potential markers of skeletal disease. Protein expression patterns in diagnostic formalin-fixed paraffin-embedded lymphoma lymph node samples from CHL patients were analyzed by nano-liquid chromatography-tandem mass spectrometry. Patients were grouped according to skeletal involvement, which was defined as the presence of one or more FDG-avid lesions on a diagnostic FDG-PET/CT scan. Protein profiles identified patients with skeletal disease at diagnosis and showed disrupted cellular pathways, including immune system processes, cell adhesion, and cell growth/survival. Immunohistochemical evaluation also demonstrated differential expressions of angiotensin-converting enzyme (ACE), intercellular adhesion molecule 3 (ICAM3), integrin alpha-X (ITGAX), and calreticulin (CALR). In conclusion, proteomics identified altered protein expression profiles in lymph nodes among CHL cases presenting with disease disseminated to the skeletal system, which implies altered disease pathogenesis for these patients.

Favourable HDL composition in endurance athletes is not associated with changes in HDL in vitro antioxidant and endothelial anti-inflammatory function.

Given the failure of high-density lipoprotein (HDL) raising therapies to reduce cardiovascular disease risk, attention has turned towards HDL composition and vascular protective functions. In individuals with insulin resistance, exercise interventions recover HDL function. However, the effect of exercise on HDL in otherwise healthy individuals is unknown. This cross-sectional study aimed to measure HDL composition and antioxidant / endothelial anti-inflammatory function in insulin sensitive endurance athlete and healthy control men. HDL was isolated using density gradient ultracentrifugation. HDL composition was measured using microplate assays for apolipoprotein A-I, total cholesterol content and apolipoprotein M. HDL protein composition was measured using nano-liquid chromatography tandem mass spectrometry. HDL subclass distribution was measured by native gel electrophoresis. HDL in vitro antioxidant function was measured by paraoxonase-1 activity assay and anti-inflammatory function assessed in endothelial cells. Compared to controls, endurance athlete HDL had higher apolipoprotein A-1 (1.65 ± 0.62 mg/mL vs 1.21 ± 0.34 mg/mL, p = 0.028) and higher total cholesterol content (1.54 ± 0.33 mmol/L vs 2.09 ± 0.44 mmol/L, p < 0.001). Proteomics revealed higher apolipoprotein A-II, A-IV and D and transthyretin in endurance athlete HDL versus controls. There was no difference observed in in vitro HDL antioxidant or anti-inflammatory functions between controls and endurance athletes. Despite a more favourable composition, endurance athlete HDL did not have higher in vitro antioxidant or anti-inflammatory function. It is possible that HDL has a ceiling of function, i.e. that healthy HDL function cannot be enhanced by endurance exercise.

Proteomic Profiling Identifies Predictive Signatures for Progression Risk in Patients with Advanced-Stage Follicular Lymphoma.

Background: Follicular lymphoma (FL) is characterized by an indolent nature and generally favorable prognosis, yet poses a particular clinical challenge, since disease progression is observed in a notable subset of patients. Currently, it is not possible to anticipate which patients will be at risk of progression, highlighting the need for reliable predictive biomarkers that can be detected early in the disease. Methods: We applied tandem-mass-tag labelled nano-liquid chromatography tandem mass spectrometry (nLC-MS/MS) on 48 diagnostic formalin-fixed, paraffin-embedded tumor samples from patients with advanced-stage FL. Of these, 17 experienced subsequent progression (subsequently-progressing, sp-FL) while 31 did not (non-progressing, np-FL). Results: We identified 99 proteins that were significantly differentially expressed between sp-FL samples and np-FL samples (p < 0.05; log2-fold changes between 0.2 and -1.3). Based on this subset of proteins, we classified patients into high-risk and low-risk subgroups using unsupervised machine learning techniques. Pathway analyses of the identified proteins revealed aberrancies within the immune system and cellular energy metabolism. In addition, two proteins were selected for immunohistochemical evaluation, namely stimulator of interferon genes 1 (STING1) and isocitrate dehydrogenase 2 (IDH2). Notably, IDH2 retained significantly lower expression levels in sp-FL samples compared with np-FL samples (p = 0.034). Low IDH2 expression correlated with shorter progression-free survival (PFS, p = 0.020). Conclusions: This study provides evidence for some of the biological mechanisms likely to be involved in FL progression and, importantly, identifies potential predictive biomarkers for improvement of risk stratification up-front at time of FL diagnosis.

Decoding of novel umami peptides from corn fermented powder and its mechanism via multisensory techniques, virtual screening, and molecular simulation approaches

This study aimed to identify umami peptides in corn fermented powder (CFP) and investigate their umami enhancing effect. Ultrafiltration and ethanol precipitation was used to separate the umami peptides in CFP. Dynamic sensory evaluations were used to identify the peptide fraction with the intense umami taste, and the peptides in the fraction were identified by nano-liquid chromatography-tandem mass spectrometry. Subsequently, ten umami-enhancing peptide candidates were screened using an integrated virtual screening strategy. Molecular docking revealed that Ser382, Ser104, Leu334, Glu338 and Glu148 of the T1R1 and T1R3 taste receptors are important amino acid residues for binding of the ten umami peptides. Three umami peptides (VDW, WGDDP, and WPAGE) exhibited the stronger binding affinity with the umami receptors. Moreover, molecular dynamics simulation revealed that the T1R1/T1R3 formed stable complexes with the three umami peptides during the simulation. Sensory evaluation indicated that the three peptides exhibited diverse taste characteristics (detection thresholds:0.0315–0.0625 mg/mL). The sigmoid curve analysis further confirmed peptides were identified as synergistically (VDW and WGDDP) or additively (WPAGE) enhancing the umami of 3 mg/mL MSG solution. This study uncovers the mechanism of umami-peptide-driven taste in fermented corn products.

Histidine-rich glycoprotein modulates neutrophils and thrombolysis-associated hemorrhagic transformation

Intravenous thrombolysis using recombinant tissue plasminogen activator (tPA) remains the primary treatment for patients with acute ischemic stroke (AIS). However, the mechanism of tPA-related hemorrhagic transformation (HT) remains poorly understood. Elevation of histidine-rich glycoprotein (HRG) expression was detected by nano-liquid chromatography tandem mass spectrometry at 1 h following tPA infusion as compared to baseline prior to tPA infusion (discovery cohort, n = 10), which was subsequently confirmed in a validation cohort (n = 157) by ELISA. Surprisingly, no elevation of HRG was detected in individuals who subsequently developed HT. During in vitro experiments, HRG reduced neutrophil NETosis, inflammatory cytokine production, and migration across the blood–brain barrier induced by tPA. In a photothrombotic murine AIS model, HRG administration ameliorated HT with delayed thrombolysis, by inhibiting neutrophil immune infiltration and downregulating pro-inflammatory signaling pathways. Neutrophil depletion or NETosis inhibition also alleviated HT, whereas HRG siRNA treatment exacerbated HT. In conclusion, fluctuations in HRG levels may reflect tPA therapy and its associated HT. The inhibitory effect of HRG on neutrophils may counteract tPA-induced immune abnormalities and HT in patients with AIS.

Jellyfish Venom Peptides Targeting Human Potassium Channels Identified through Ligand Screening: Morphometric and Molecular Identification of the Species and Antibiotic Potential.

The relative lack of marine venom could be attributed to the difficulty in dealing with venomous marine animals. Moreover, the venom of marine animals consists of various bioactive molecules, many of which are proteins with unique properties. In this study, we investigated the potential toxic proteins of jellyfish collected for ligand screening to understand the mechanism of the toxic effects of jellyfish. Since taxonomic identification is problematic due to the lack of proper keys, we conducted morphological and molecular mitochondrial DNA sequencing from COI and ITS regions. The venom extract from nematocysts found along the bell margins was used for protein characterization using SDS-gel electrophoresis and nano-liquid chromatography-tandem mass spectrometry. Ligand screening for the most potent toxin and antibacterial and cytotoxicity assays were carried out. The phylogenetic tree showed distinct clustering from other Catostylus sp. The proteomic analysis revealed venom with many bioactive proteins. Only 13 venom proteins were identified with molecular weights ranging from 4318 to 184,923 Da, exhibiting the venom's complexity. The overall toxin protein composition of Catostylus sp. venom was dominated by potassium channel toxin alpha-KTx. Molecular docking of toxin alpha-KTx 1.13 revealed high specificity towards the human voltage-gated potassium channel Kv3 with a high fitness score and a minimum energy barrier of -17.9 kcal/mol. Disc diffusion and cytotoxicity assays revealed potent antibacterial activity against Klebsiella pneumoniae with no cytotoxicity. Further studies on detailed characterization and therapeutic potentials are warranted.

Isolation, purification, characterization, and analysis of the antioxidant activity of antioxidant peptides from walnut milk fermented with Lactobacillus paracasei SMN-LBK

Microbial fermentation is an important method to obtain antioxidant peptides. In this study, Lactobacillus paracasei SMN-LBK was used to ferment biprotein (walnut milk) to enhance the antioxidant capacity of the active peptide. The optimal fermentation process of 5% inoculum, 1:7 plant protein ratio, 37 °C and 12 h resulted in 77.63% ± 0.36% DPPH scavenging, 93.31% ± 0.25% ABTS scavenging and 79.37% ± 0.02% metal ion chelating capacity. The hydrolysis products were classified into three molecular weight classes using an ultrafiltration membrane system. DPAP-3 with a molecular weight of less than 3 kDa had the highest antioxidant activity. DPAP-3 was further separated into three subclasses using gel filtration chromatography. These subclasses were characterized using nano-liquid chromatography-tandem mass spectrometry (NanoLC-MS/MS) and screened by searching https://biochemia.uwm.edu. Four antioxidant peptides were identified using a combination of hydrophobic amino acids, positive ion-charge interactions, and numerical values. Two of these peptides were from animals and one from plants. Their amino acid sequences are as follows PCRGVLLR, IHIPLPRWV, QSKVLPVPQK, and KVLPVPQKA, with molecular docking binding energies of −7.8, −8.3, −7.4, and −7.7 kcal/mol, respectively. The results suggest that fermentation of plant-animal proteins with Lactobacillus paracasei can produce antioxidant peptides that can be used as functional ingredients in food products. This has the potential to conserve animal protein resources.

Quantitative proteomics reveals CLR interactome in primary human cells

The G protein-coupled receptor (GPCR) calcitonin receptor-like receptor (CLR) mediates essential functions in several cell types and is implicated in cardiovascular pathologies, skin diseases, migraine and cancer. To date, the network of proteins interacting with CLR (“CLR interactome”) in primary cells, where this GPCR is expressed at endogenous (physiologically relevant) levels, remains unknown. To address this knowledge gap, we established a novel integrative methodological workflow/approach for conducting a comprehensive/proteome-wide analysis of Homo sapiens CLR interactome. We used primary human dermal lymphatic endothelial cells and combined immunoprecipitation (IP) utilising anti-human CLR antibody with label-free quantitative nano-liquid chromatography-tandem mass spectrometry (nano LC-MS/MS) and quantitative in situ proximity ligation assay (PLA). By using this workflow, we identified 37 proteins interacting with endogenously expressed CLR amongst 4,902 detected members of the cellular proteome (by quantitative nano LC-MS/MS) and revealed direct interactions of two kinases and two transporters with this GPCR (by in situ PLA). All identified interactors have not been previously reported as members of CLR interactome. Our approach and findings uncover the hitherto unrecognized compositional complexity of the interactome of endogenously expressed CLR and contribute to fundamental understanding of the biology of this GPCR. Collectively, our study provides a first-of-its-kind integrative methodological approach and datasets as valuable resources and robust platform/springboard for advancing the discovery and comprehensive characterization of physiologically relevant CLR interactome at a proteome-wide level in a range of cell types and diseases in future studies.

(Sialyl)Lewis Antigen Expression on Glycosphingolipids, N-, and O-Glycans in Colorectal Cancer Cell Lines is Linked to a Colon-Like Differentiation Program

Alterations in the glycomic profile are a hallmark of cancer, including colorectal cancer (CRC). While, the glycosylation of glycoproteins and glycolipids has been widely studied for CRC cell lines and tissues, a comprehensive overview of CRC glycomics is still lacking due to the usage of different samples and analytical methods. In this study, we compared glycosylation features of N-, O-glycans, and glycosphingolipid glycans for a set of 22 CRC cell lines, all measured by porous graphitized carbon nano-liquid chromatography-tandem mass spectrometry. An overall, high abundance of (sialyl)Lewis antigens for colon-like cell lines was found, while undifferentiated cell lines showed high expression of H blood group antigens and α2-3/6 sialylation. Moreover, significant associations of glycosylation features were found between the three classes of glycans, such as (sialyl)Lewis and H blood group antigens. Integration of the datasets with transcriptomics data revealed positive correlations between (sialyl)Lewis antigens, the corresponding glycosyltransferase FUT3 and transcription factors CDX1, ETS, HNF1/4A, MECOM, and MYB. This indicates a possible role of these transcription factors in the upregulation of (sialyl)Lewis antigens, particularly on glycosphingolipid glycans, via FUT3/4 expression in colon-like cell lines. In conclusion, our study provides insights into the possible regulation of glycans in CRC and can serve as a guide for the development of diagnostic and therapeutic biomarkers.

Simultaneous quantification of six proteins related to liver injury using nano liquid chromatography-tandem mass spectrometry.

In clinical diagnosis of liver injury, which is an important health concern, serum aminotransferase assays have been the go-to method used worldwide. However, the measurement of serum enzyme activity has limitations, including inadequate disease specificity and enzyme specificity. With the high selectivity and specificity provided by nano liquid chromatography-tandem mass spectrometry (LC/MS/MS), this work describes a method for the simultaneous determination of six proteins in liver that can be potentially used as biomarkers for liver injury: glutamic-pyruvic transaminase 1 (GPT1), glutamic oxaloacetic transaminase 1 (GOT1), methionine adenosyl transferase 1A (MAT1A), glutathione peroxidase 1 (GPX1), cytokeratin 18 (KRT18) and apolipoprotein E (APOE). In validation, the method was shown to have good selectivity and sensitivity (limits of detection at pg/mL level). The analytical method revealed that, compared with normal mice, in carbon tetrachloride-induced acute liver injury mice, liver MAT1A and GPX1 were significantly lower (p < 0.01 and p < 0.05, respectively), KRT18 was significantly higher (p < 0.05) and APOE and GPT1 were marginally significantly lower (p between 0.05 and 0.1). This is the first work reporting the absolute contents of GPT1, GOT1, MAT1A, GPX1 and KRT18 proteins based on LC/MS. The proposed method provides a basis for establishing more specific diagnostic indicators of liver injury.

Wearable sampling of proteins from human exhaled aerosols for nano-liquid chromatography-tandem mass spectrometry analysis: A pilot study.

Human exhaled breath usually contains unique proteins that may provide clues to characterize individual physiological activities and many diseases. However, the concentration of exhaled proteins in exhaled breath is extremely low and usually does not reach the detection limits of all online breath mass spectrometry instruments. Therefore, developing a new breath sampler for collecting and characterizing exhaled proteins is important. In this study, a new mask-based wearable sampler was developed by fixing metal materials into the inner surface of the KN95 mask. Human exhaled proteins could be directly adsorbed onto the metal material while wearing the mask. After sampling, the collected proteins were eluted, digested, and identified using nano-liquid chromatography-tandem mass spectrometry (nano-LC-MS/MS). The adsorption of exhaled proteins was evaluated, showing that modified gold foil is an effective material for collecting exhaled proteins. Various endogenous proteins were successfully identified from exhaled breath, many of which can be potential biomarkers for disease diagnosis. By coupling the newly developed mask sampler with nano-LC-MS/MS, human exhaled proteins were successfully collected and identified. Our results show that the mask sampler is wearable, simple, and convenient, and the method is noninvasive for investigating disease diagnosis and human health.

Site-Specific Glycosylation Analysis of Murine and Human Fcγ Receptors Reveals High Heterogeneity at Conserved N-Glycosylation Site.

Fc γ-receptors (FcγRs) on leukocytes bind immunoglobulin G (IgG) immune complexes to mediate effector functions. Dysregulation of FcγR-mediated processes contributes to multiple inflammatory diseases, including rheumatoid arthritis, lupus, and immune thrombocytopenia. Critically, immunoregulatory N-glycan modifications on both FcγRs and IgGs alter FcγR-IgG binding affinity. Rapid methods for the characterization of N-glycans across multiple Fcγ receptors are needed to propel investigations into disease-specific contributions of FcγR N-glycans. Here, we utilize nanoliquid chromatography tandem mass spectrometry (nLC-MS/MS) to characterize FcγR glycosylation and report quantitative and site-specific N-glycan characterization of recombinant human FcγRI, FcγRIIIA V158, and FcγRIIIA F158 from CHO cells and murine FcγRI, FcγRIII, and FcγRIV from NS0 cells. Data are available via ProteomeXchange with identifier PXD043966. Broad glycoform distribution (≥30) was observed at mouse FcγRIV site N159 and human FcγRIIIA site N162, an evolutionarily conserved site. Further, mouse FcγRIII N-glycopeptides spanning all four predicted N-glycosylation sequons were detected. Glycoform relative abundances for hFcγRIIIA V/F158 polymorphic variants are reported, demonstrating the clinical potential of this workflow to measure differences in glycosylation between common human FcγRIIIA allelic variants with disease-associated outcomes. The multi-Fcγ receptor glycoproteomic workflow reported here will empower studies focused on the role of FcγR N-glycosylation in autoimmune diseases.

Comparison of sialylated and fucosylated N-glycans attached to Asn 6 and Asn 41 with different roles in hyaluronan and proteoglycan link protein 1 (HAPLN1)

Hyaluronan and proteoglycan link protein 1 (HAPLN1) is an extracellular matrix protein stabilizing interactions between hyaluronan and proteoglycan. Although HAPLN1 is being investigated for various biological roles, its N-glycosylation is poorly understood. In this study, the structure of N-glycopeptides of trypsin-treated recombinant human HAPLN1 (rhHAPLN1) expressed from CHO cells were identified by nano-liquid chromatography-tandem mass spectrometry. A total of 66 N-glycopeptides were obtained, including 16 and 12 N-glycans at sites Asn 6 (located in the N-terminal region) and Asn 41 (located in the Ig-like domain, which interacts with proteoglycan), respectively. The quantities (%) of each N-glycan relative to the totals (100 %) at each site were calculated. Tri- and tetra-sialylation (to resist proteolysis and extend half-life) were more abundant at Asn 6, and di- (core- and terminal-) fucosylation (to increase binding affinity and stability) and sialyl-Lewis X/a epitope (a major ligand for E-selectin) were more abundant at Asn 41. These results indicate that N-glycans attached to Asn 6 (protecting HAPLN1) and Asn 41 (supporting molecular interactions) play different roles in HAPLN1. This is the first study of site-specific N-glycosylation in rhHAPLN1, which will be useful for understanding its molecular interactions in the extracellular matrix.

Discovery and identification of interspecies peptide biomarkers in the seahorse species using liquid chromatography tandem mass spectrometry and chemometrics

Seahorses have important edible and medicinal values including strengthening the body, tonifying the liver and kidneys, and reducing swelling. And there are abundant seahorse species on Earth. Many seahorses have large price differences due to the scarcity of resources, and some seahorses with similar appearances appear to be confused for use. While in market trading, Hippocampus is susceptible to loss of specialized morphology characteristics, making it difficult to distinguish between specific species. Here we report an effective method based on peptide biomarkers for the identification of seahorse species. Peptide biomarkers for each species were predicted using nano-liquid chromatography-tandem mass spectrometry (Nano-LC-MS/MS) combined with chemometrics software. One unique biomarker peptide for each species was synthesized and verified, and finally developed a liquid chromatography tandem mass spectrometry (LC-MS/MS) multiple reaction monitoring method. The results indicate that the method has great potential for species-specific identification of seahorses and their preparations, among others.

In-Gel Cyanoethylation for Pseudouridines Mass Spectrometry Detection of Bacterial Regulatory RNA.

Regulatory RNAs, as well as many RNA families, contain chemically modified nucleotides, including pseudouridines (ψ). To map nucleotide modifications, approaches based onenzymatic digestion of RNA followed by nano liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS) analysis were implemented several years ago. However, detection of ψ by mass spectrometry (MS) is challenging as ψ exhibits the same mass as uridine. Thus, a chemical labeling strategy using acrylonitrile was developed to detect this mass-silent modification. Acrylonitrile reacts specifically to ψ to form 1-cyanoethylpseudouridine (Ceψ), resulting in a mass shift of ψ detectable by MS. Here, a protocol detailing the steps from the purification of RNA by polyacrylamide gel electrophoresis, including in-gel labeling of ψ, to MS data interpretation to map ψ and other modifications is proposed. To demonstrate its efficiency,theprotocol was applied to bacterial regulatory RNAs from E. coli: 6S RNA and transfer-messenger RNA (tmRNA, also known as 10Sa RNA). Moreover, ribonuclease P (RNase P)was also mapped using this approach. This method enabled the detection of several ψ at single nucleotide resolution.

One-step functionalization of paper and simplified antibody immobilization for on-the-spot immunocapture from dried serum in liquid chromatography-tandem mass spectrometry based targeted protein determination.

This work aimed to simplify and improve the process of binding monoclonal antibodies (mAbs) covalently to filter paper for use in dried blood spot sampling, enabling instant capture of protein biomarkers for targeted protein determination. Incorporating the necessary immunocapture sample preparation step in the initial sampling stage saves time and reduces the workload. The biomarker human chorionic gonadotropin (hCG) was used as the model analyte. The antibody-based paper samplers were prepared by functionalizing paper discs (6mm) through a simple reaction using divinyl sulfone (DVS). After DVS activation, the paper discs were incubated with E27 hCG mAbs, followed by 0.05% tween/phosphate buffer saline to block the surface. After sample application and drying, the discs only needed to be washed before tryptic digestion and finally analysed on a nanoliquid chromatography-tandem mass spectrometry system. The finished DVS-mAbs samplers could selectively capture hCG (100 ng/mL) from human serum, with a recovery of 50%. Sample clean-up reduced the number of identified proteins from 132 to 82 before and after wash, respectively, with a 70% reduction in serum albumin signal while still retaining hCG on the sampler during the washing protocol. An evaluation of the samplers revealed excellent linearity (R2 = 0.9995) for hCG in serum with relative standard deviations below 15%. This work has presented the first ever reported paper samplers immobilized with antibodies utilizing DVS chemistry, showing promise in the future of paper-based sampling.

The Globodera rostochiensis Gr29D09 Effector with a Role in Defense Suppression Targets the Potato Hexokinase 1 Protein.

The potato cyst nematode (Globodera rostochiensis) is an obligate root pathogen of potatoes. G. rostochiensis encodes several highly expanded effector gene families, including the Gr4D06 family; however, little is known about the function of this effector family. We cloned four 29D09 genes from G. rostochiensis (named Gr29D09v1/v2/v3/v4) that share high sequence similarity and are homologous to the Hg29D09 and Hg4D06 effector genes from the soybean cyst nematode (Heterodera glycines). Phylogenetic analysis revealed that Gr29D09 genes belong to a subgroup of the Gr4D06 family. We showed that Gr29D09 genes are expressed exclusively within the nematode's dorsal gland cell and are dramatically upregulated in parasitic stages, indicating involvement of Gr29D09 effectors in nematode parasitism. Transgenic potato lines overexpressing Gr29D09 variants showed increased susceptibility to G. rostochiensis. Transient expression assays in Nicotiana benthamiana demonstrated that Gr29D09v3 could suppress reactive oxygen species (ROS) production and defense gene expression induced by flg22 and cell death mediated by immune receptors. These results suggest a critical role of Gr29D09 effectors in defense suppression. The use of affinity purification coupled with nanoliquid chromatography-tandem mass spectrometry identified potato hexokinase 1 (StHXK1) as a candidate target of Gr29D09. The Gr29D09-StHXK1 interaction was further confirmed using in planta protein-protein interaction assays. Plant HXKs have been implicated in defense regulation against pathogen infection. Interestingly, we found that StHXK1 could enhance flg22-induced ROS production, consistent with a positive role of plant HXKs in defense. Altogether, our results suggest that targeting StHXK1 by Gr29D09 effectors may impair the positive function of StHXK1 in plant immunity, thereby aiding nematode parasitism. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Comparative Serum Proteome Profiling of Canine Benign Prostatic Hyperplasia before and after Castration.

BPH is the most prevalent prostatic condition in aging dogs. Nevertheless, clinical diagnosis and management remain inconsistent. This study employed in-solution digestion coupled with nano-liquid chromatography tandem mass spectrometry to assess serum proteome profiling of dogs with BPH and those dogs after castration. Male dogs were divided into two groups; control and BPH groups. In the BPH group, each dog was evaluated at two time points: Day 0 (BF subgroup) and Day 30 after castration (AT subgroup). In the BF subgroup, three proteins were significantly upregulated and associated with dihydrotestosterone: solute carrier family 5 member 5, tyrosine-protein kinase, and FRAT regulator of WNT signaling pathway 1. Additionally, the overexpression of polymeric immunoglobulin receptors in the BF subgroup hints at its potential as a novel protein linked to the BPH development process. Conversely, alpha-1-B glycoprotein (A1BG) displayed significant downregulation in the BF subgroup, suggesting A1BG's potential as a predictive protein for canine BPH. Finasteride was associated with increased proteins in the AT subgroup, including apolipoprotein C-I, apolipoprotein E, apolipoprotein A-II, TAO kinase 1, DnaJ homolog subfamily C member 16, PH domain and leucine-rich repeat protein phosphatase 1, neuregulin 1, and pseudopodium enriched atypical kinase 1. In conclusion, this pilot study highlighted alterations in various serum proteins in canine BPH, reflecting different pathological changes occurring in this condition. These proteins could be a source of potential non-invasive biomarkers for diagnosing this disease.

A clinical proteomics study of exhaled breath condensate and biomarkers for pulmonary embolism

Pulmonary embolism (PE) can be a diagnostic challenge. Current diagnostic markers for PE are unspecific and new diagnostic tools are needed. The air we exhale is a possible new source for biomarkers which can be tapped into by analysing the exhaled breath condensate (EBC). We analysed the EBC from patients with PE and controls to investigate if the EBC is a useful source for new diagnostic biomarkers of PE. We collected and analysed EBC samples from patients with suspected PE and controls matched on age and sex. Patients in whom PE was ruled out after diagnostic work-up were included in the control group to increase the sensitivity and generalizability of the identified markers. EBC samples were collected using an RTube™. The protein composition of the EBCs were analysed using data dependent label-free quantitative nano liquid chromatography–tandem mass spectrometry. EBC samples from 28 patients with confirmed PE, and 49 controls were analysed. A total of 928 EBC proteins were identified in the 77 EBC samples. As expected, a low protein concentration was determined which resulted in many proteins with unmeasurable levels in several samples. The levels of HSPA5, PEBP1 and SFTPA2 were higher and levels of POF1B, EPPK1, PSMA4, ALDOA, and CFL1 were lower in PE compared with controls. In conclusion, the human EBC contained a variety of endogenous proteins and may be a source for new diagnostic markers of PE and other diseases.