Nano-liquid Chromatography-tandem Mass Research Articles

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.

Combination of virtual screening, machine learning, molecular dynamics simulations and sensory evaluation to discover novel umami peptides from fermented Atlantic cod

The aim of this study was to explore the effect of fermentation with Lactobacillus helveticus Lh191404 on the umami characteristics of Atlantic cod and to elucidate the umami mechanism of the peptide. Post-fermentation significant increases in sweet amino acids (Threonine (Thr), Serine (Ser), Proline (Pro), Glycine (Gly), Alanine (Ala)) and umami amino acids (Aspartic acid (Asp), Glutamic acid (Glu)) enhanced the palatability and umami intensity of surimi. The calculated taste activity value showed Glu as the highest at 2.598, indicating that umami was the most prominent taste after the Atlantic cod fermentation. A new approach was constructed to batch screen high abundance umami peptides. Combined with Nano Liquid Chromatography Tandem Mass Spectrometry (nLC/MS-MS), virtual screening and machine learning identified four highly abundant umami peptides (WGDL, FDDLP, VAQWR, and YGESDL). Molecular docking and molecular dynamics simulations determined that they stably bind to the receptor proteins through hydrogen bonding and electrostatic interactions, with Asp108, Arg151, and Arg277 as the key active amino acids. Sensory evaluation and electronic tongue confirmed that the screened peptides were all umami peptides and the umami intensity was consistent with the simulation results. This study not only revealed that Lactobacillus helveticus Lh191404 enhanced the taste of fermented Atlantic cod by releasing high abundance umami peptides but also provided a novel approach for identifying umami peptides from fermented marine foods.

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.

Protein profiling and assessment of amyloid beta levels in plasma in canine refractory epilepsy.

The relationship between epilepsy and cognitive dysfunction has been investigated in canines, and memory impairment was prevalent in dogs with epilepsy. Additionally, canines with epilepsy have greater amyloid-β (Aβ) accumulation and neuronal degeneration than healthy controls. The present study investigated plasma Aβ42 levels and performed proteomic profiling in dogs with refractory epilepsy and healthy dogs. In total, eight dogs, including four healthy dogs and four dogs with epilepsy, were included in the study. Blood samples were collected to analyze Aβ42 levels and perform proteomic profiling. Changes in the plasma proteomic profiles of dogs were determined by nano liquid chromatography tandem mass spectrometry. The plasma Aβ42 level was significantly higher in dogs with epilepsy (99 pg/mL) than in healthy dogs (5.9 pg/mL). In total, 155 proteins were identified, and of these, the expression of 40 proteins was altered in epilepsy. Among these proteins, which are linked to neurodegenerative diseases, 10 (25%) were downregulated in dogs with epilepsy, whereas 12 (30%) were upregulated. The expression of the acute phase proteins haptoglobin and α2-macroglobulin significantly differed between the groups. Complement factor H and ceruloplasmin were only detected in epilepsy dogs, suggesting that neuroinflammation plays a role in epileptic seizures. Gelsolin, which is involved in cellular processes and cytoskeletal organization, was only detected in healthy dogs. Gene Ontology annotation revealed that epilepsy can potentially interfere with biological processes, including cellular processes, localization, and responses to stimuli. Seizures compromised key molecular functions, including catalytic activity, molecular function regulation, and binding. Defense/immunity proteins were most significantly modified during the development of epilepsy. In Kyoto Encyclopedia of Genes and Genomes pathway analysis, complement and coagulation cascades were the most relevant signaling pathways affected by seizures. The findings suggested that haptoglobin, ceruloplasmin, α2-macroglobulin, complement factor H, and gelsolin play roles in canine epilepsy and Aβ levels based on proteomic profiling. These proteins could represent diagnostic biomarkers that, after clinical validation, could be used in veterinary practice as well as proteins relevant to disease response pathways. To determine the precise mechanisms underlying these relationships and their implications in canine epilepsy, additional research is required.

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.

Two-Dimensional-PAGE Coupled with nLC-MS/MS-Based Identification of Differentially Expressed Proteins and Tumorigenic Pathways in MCF7 Breast Cancer Cells Transfected for JTB Protein Silencing.

The identification of new cancer-associated genes/proteins, the characterization of their expression variation, the interactomics-based assessment of differentially expressed genes/proteins (DEGs/DEPs), and understanding the tumorigenic pathways and biological processes involved in BC genesis and progression are necessary and possible by the rapid and recent advances in bioinformatics and molecular profiling strategies. Taking into account the opinion of other authors, as well as based on our own team's in vitro studies, we suggest that the human jumping translocation breakpoint (hJTB) protein might be considered as a tumor biomarker for BC and should be studied as a target for BC therapy. In this study, we identify DEPs, carcinogenic pathways, and biological processes associated with JTB silencing, using 2D-PAGE coupled with nano-liquid chromatography tandem mass spectrometry (nLC-MS/MS) proteomics applied to a MCF7 breast cancer cell line, for complementing and completing our previous results based on SDS-PAGE, as well as in-solution proteomics of MCF7 cells transfected for JTB downregulation. The functions of significant DEPs are analyzed using GSEA and KEGG analyses. Almost all DEPs exert pro-tumorigenic effects in the JTBlow condition, sustaining the tumor suppressive function of JTB. Thus, the identified DEPs are involved in several signaling and metabolic pathways that play pro-tumorigenic roles: EMT, ERK/MAPK, PI3K/AKT, Wnt/β-catenin, mTOR, C-MYC, NF-κB, IFN-γ and IFN-α responses, UPR, and glycolysis/gluconeogenesis. These pathways sustain cancer cell growth, adhesion, survival, proliferation, invasion, metastasis, resistance to apoptosis, tight junctions and cytoskeleton reorganization, the maintenance of stemness, metabolic reprogramming, survival in a hostile environment, and sustain a poor clinical outcome. In conclusion, JTB silencing might increase the neoplastic phenotype and behavior of the MCF7 BC cell line. The data is available via ProteomeXchange with the identifier PXD046265.

17th International Conference on Malignant Lymphoma, Palazzo dei Congressi, Lugano, Switzerland, 13 - 17 June, 2023.

Introduction: Classic Hodgkin lymphoma (cHL) is a highly curable disease, even in advanced stages. It is controversial whether bone involvement has a negative effect on overall- and progression-free survival in patients treated with intensive chemotherapy. cHL is characterized by a unique tumor microenvironment (TME) consisting of few, scattered neoplastic Hodgkin and Reed-Sternberg (HRS) cells, embedded in an abundant background of reactive immune and stromal cells. When cHL disseminates, the new disease sites also contain both HRS cells and TME cells. Whether cases that present with bone lesions, harbour specific TME features is unknown. We investigated protein expression in lymph node biopsies from cHL patients with and without skeletal involvement at diagnosis, using nano liquid chromatography—tandem mass spectrometry (nLC-MS/MS), to identify potential markers of skeletal disease. Methods: Protein expression patterns in pre-therapeutic formalin-fixed, paraffin-embedded lymphoma samples from 69 cHL patients diagnosed at Aarhus University Hospital, Denmark between 2009 and 2018 were analysed by nLC-MS/MS. Patients were grouped according to diagnostic 18F-fluoro-2-deoxy-D-glucose (FDG) positron emission tomography (PET)/computed tomography (CT). FDG PET/CT scans were reviewed specifically for bone involvement. The study cohort included 50 patients with nodal cHL (n-cHL) and no skeletal involvement, and 19 patients with both skeletal and nodal disease (s-cHL). Results: We identified 2,298 proteins; comparison of the protein profiles between the s-cHL and n-cHL groups revealed 220 unique proteins significantly differentially expressed (p < 0.05) and with a fold change of at least 25%. Of these, 117 proteins were upregulated (fold change 1.25–3.94), and 103 were downregulated (fold change 0.44–0.80) in s-cHL. In hierarchal clustering based on an even higher significance threshold (p < 0.001), i.e., 25 significantly differentially expressed proteins, two clusters were observed: (i) a skeletal-group comprising 12 s-cHL and 4 n-cHL samples; and (ii) a nodal-group of 46 n-cHL and 7 s-cHL samples. Of particular interest among the differentially expressed proteins, we identified isocitrate dehydrogenase (IDH1) and WD repeat- and FYVE domain-containing protein 4 (WDFY4). This pattern of protein expression suggests disturbance in cytoplasmic NADPH production, antigen processing/presentation, and B-cell survival in cHL patients with skeletal involvement compared to those without. Conclusion: Our data show differential protein expression in cHL lymphoma tissues that correlate with the presence or absence of concomitant bone involvement at diagnosis. This indicates that certain biological pathways are affected among those presenting with disease disseminated to the skeletal system. The research was funded by: Department of Clinical Medicine, Aarhus University; Health Research Foundation of Central Denmark Region; Brødrene Hartmanns Foundation; Carl and Ellen Hertz Foundation; the Danish Lymphoma Group; Kong Christian den Tiendes Foundation; Torben and Alice Frimodts Foundation; Aase and Ejnar Danielsens Foundation; Foundation of 1870; Dagmar Marshalls Foundation; Merchant Einar Willumsen’s Memorial Foundation; Eva and Henry Frænkel’s Memorial Foundation; Master Carpenter Jørgen Holm and wife Elisa f. Hansen’s Memorial Grant. The Orbitrap Fusion Tribrid mass spectrometer was funded by A. P. Møller og Hustru Chastine Mc-Kinney Møllers Fond til Almene Formaal. Keywords: Genomics, Epigenomics, and Other -Omics, Hodgkin lymphoma, Microenvironment No conflicts of interests pertinent to the abstract.

Plasma Proteomic Analysis Reveals the Potential Role of Lectin and Alternative Complement Pathways in IgA Vasculitis Pathogenesis.

IgA vasculitis (IgAV) is the most common form of childhood vasculitis. A better understanding of its pathophysiology is required to identify new potential biomarkers and treatment targets. to assess the underlying molecular mechanisms in the pathogenesis of IgAV using an untargeted proteomics approach. Thirty-seven IgAV patients and five healthy controls were enrolled. Plasma samples were collected on the day of diagnosis before any treatment was initiated. We used nano-liquid chromatography-tandem mass spectrometry (nLC-MS/MS) to investigate the alterations in plasma proteomic profiles. For the bioinformatics analyses, databases including Uniprot, PANTHER, KEGG, Reactome, Cytoscape, and IntAct were used. Among the 418 proteins identified in the nLC-MS/MS analysis, 20 had significantly different expressions in IgAV patients. Among them, 15 were upregulated and 5 were downregulated. According to the KEGG pathway and function classification analysis, complement and coagulation cascades were the most enriched pathways. GO analyses showed that the differentially expressed proteins were mainly involved in defense/immunity proteins and the metabolite interconversion enzyme family. We also investigated molecular interactions in the identified 20 proteins of IgAV patients. We extracted 493 interactions from the IntAct database for the 20 proteins and used Cytoscape for the network analyses. Our results clearly suggest the role of the lectin and alternate complement pathways in IgAV. The proteins defined in the pathways of cell adhesion may serve as biomarkers. Further functional studies may lead the way to better understanding of the disease and new therapeutic options for IgAV treatment.

Mass Spectrometry based identification of site-specific proteomic alterations and potential pathways underlying the pathophysiology of schizophrenia.

Schizophrenia (SZ) is a complex multifactorial disorder that affects 1% of the population worldwide with no available effective treatment. Although proteomic alterations are reported in SZ however proteomic expression aberrations among different brain regions are not fully determined. Therefore, the present study aimed spatial differential protein expression profiling of three distinct regions of SZ brain and identification of associated affected biological pathways in SZ progression. Comparative protein expression profiling of three distinct autopsied human brain regions (i.e., substantia nigra, hippocampus and prefrontal cortex) of SZ was performed with respective healthy controls. Using two-dimensional electrophoresis (2DE)-based nano liquid chromatography tandem mass spectrometry (Nano-LC MS /MS) analysis, 1443 proteins were identified out of which 58 connote to be significantly dysregulated, representing 26 of substantia nigra,14 of hippocampus and 18 of prefrontal cortex. The 58 differentially expressed proteins were further analyzed using Ingenuity pathway analysis (IPA). The IPA analysis provided protein-protein interaction networks of several proteins including nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kb), extracellular signal regulated kinases 1/2 (ERK1/2), alpha serine / Threonine-protein kinase (AKT1), cellular tumor antigen p53 (TP53) and amyloid precursor protein (APP), holding prime positions in networks and interacts with most of the identified proteins and their closely interacting partners. These findings provide conceptual insights of novel SZ related pathways and the cross talk of co and contra regulated proteins. This spatial proteomic analysis will further broaden the conceptual framework for schizophrenia research in future.

Identification and In Silico Simulation on Inhibitory Platelet-Activating Factor Acetyl Hydrolase Peptides from Dry-Cured Pork Coppa

The unique processing technology of dry-cured meat products leads to strong proteolysis, which produces numerous peptides. The purpose of this investigation was the systematic isolation, purification, and identification of potentially cardioprotective bioactive peptides from dry-cured pork coppa during processing. According to the results of anti-platelet-activating factor acetyl hydrolase activity and radical scavenging ability in vitro, the inhibitory effect of M1F2 in purified fractions on cardiovascular inflammation was higher than that of M2F2. The peptide of M1F2 was identified by nano-liquid chromatography-tandem mass spectrometry. A total of 30 peptides were identified. Based on bioinformatics methods, including in silico analysis and molecular docking, LTDKPFL, VEAPPAKVP, KVPVPAPK, IPVPKK, and PIKRSP were identified as the most promising potential platelet-activating factor acetyl hydrolase inhibitory peptides. Overall, bioactive peptides produced during dry-cured pork coppa processing demonstrate positive effects on human health.

Pilot Study of the Ex Vivo Blood Leukocytes’ Proteomic Response to Prednisone Stimulation in Corticosteroid-responsive Asthma

Background: Leukocytes are key cellular effectors of inflammation in asthma and understanding their function in this disease is of crucial importance. Blood leukocytes reflect the actions of their counterparts in the lungs and they can be obtained through minimal invasive procedures as part of the peripheral blood. Objective: The aim of the study was to identify proteins in blood leukocyte proteomes that respond to ex vivo treatment by prednisone in order to pinpoint candidates for predictive biomarkers in corticosteroid- responsive asthma. Methods: The study included five children diagnosed with asthma and five healthy children. After the ex vivo treatment of blood samples with prednisone, lysis of erythrocytes was performed and proteins were extracted from the remaining leukocytes by ultrasonic disintegration. Protein extracts were analyzed by reversed phase nano-liquidchromatography–tandem mass spectrometry (LC–MS/MS). Results: The stimulation of asthmatics' leukocytes with prednisone has led to an increase in the levels of FYB (fold change 3.4) and LYZ (fold change 2.2) with a statistical significance of p&lt;0.005. The two proteins with expressions significantly altered upon the prednisone treatment should be further explored as tools to evaluate the patient's response before therapy administration, especially when lung function measurements are not possible, as is the case with young pediatric patients. Conclusion: The approach that entails ex vivo response of blood leukocytes to therapeutics can facilitate asthma management and help overcome the need for therapeutic adjustments in a clinical setting.

Yak bone collagen-derived anti-inflammatory bioactive peptides alleviate lipopolysaccharide-induced inflammatory by inhibiting the NF-κB signaling pathway and nitric oxide production

Anti-inflammatory peptides that derived from food attract more and more attention due to their wide range of sources and easy absorption. In this study, the aim was to identify and characterize anti-inflammatory peptides from yak bone collagen. The prepared yak bone collagen peptides (YBCPs) was separated, purified and identified through ultrafiltration, reverse-phase high-performance liquid chromatography (RP-HPLC), and nano-liquid chromatography–tandem mass spectrometry (nano LC-MS/MS) successively. Finally, a total of 115 peptides were identified. After that, 12 peptides were screened out based on the predicted biological activity score. Molecular docking results indicated that 6 peptides (GPAGPSGPAGK, GPAGPSGPAGKDGR, GPSGPQGIR, GPAGPQGPR, GEAGPAGPAGPAGPR, and GEGGPQGPR) could successfully interact with the key factors in the nuclear factor kappa-B (NF-κB) signaling pathway and nitric oxide (NO) production due to form various bonds such as salt bridge, conventional hydrogen bond, and carbon-hydrogen bond. The anti-inflammatory effects of these 6 peptides were further verified by in vitro cell test. Results indicated that the 6 peptides might play an anti-inflammatory role by regulating different proteins in the NF-κB signaling pathway and NO production. This study would further promote the application of yak bone collagen in health food.

Multidimensional separation and analysis of alpha-1-acid glycoprotein N-glycopeptides using high-field asymmetric waveform ion mobility spectrometry (FAIMS) and nano-liquid chromatography tandem mass spectrometry.

Bottom-up nLC-MS/MS-based glycoprotein mass spectrometry workflows rely on the generation of a mixture of non-glycosylated and glycosylated peptides via proteolysisof glycoproteins. Such methods are challenged by suppression of hydrophilic glycopeptide ions by more abundant, hydrophobic, and readily ionizable non-glycosylated peptides. Commercially available high-field asymmetric waveform ion mobility spectrometry (FAIMS) devices have recently been introduced and present a potential benefit for glycoproteomic workflows by enabling orthogonal separation of non-glycosylated peptides and glycopeptides following chromatographic separation, and prior to MS/MS analysis. However, knowledge is lacking regarding optimal FAIMS conditions for glycopeptide analyses. Here, we document optimal FAIMS compensation voltages for the transmission and analysis of human alpha-1-acid glycoprotein (AGP) tryptic N-glycopeptide ions. Further, we evaluate the effect of FAIMS on AGP glycopeptide assignment confidence by comparing the number of assigned glycopeptides at different confidence levels using a standard nLC-MS/MS method or an otherwise identical method employing FAIMS. Optimized methods will potentiate glycoproteomic analyses by increasing the number of unique glycopeptide identifications and the confidence of glycopeptide assignments. Data are available via ProteomeXchange with identifier PXD036667. Analysis of alpha-1-acid glycoprotein (AGP) tryptic digests via nLC-FAIMS-MS/MS (top) led to the establishment of ideal FAIMS voltages for the analysis of AGP N-glycopeptides (bottom), suggesting that FAIMS can improve the depth of glycoproteome characterization. Pairs of CV magnitudes are shown along the x-axis.

Multiplexed Detection of Pancreatic Cancer by Combining a Nanoparticle-Enabled Blood Test and Plasma Levels of Acute-Phase Proteins.

Simple SummaryIn this study, a multiplexed strategy based on the combination of a nanoparticle-enabled blood test and serum levels of acute-phase proteins proved to be able to distinguish pancreatic cancer patients from healthy controls with a good and sex-dependent prediction ability. This study suggests a possible role of acute-phase proteins as pancreatic cancer biomarkers and paves the way for the development of multiplexed technologies for early cancer detection.The development of new tools for the early detection of pancreatic ductal adenocarcinoma (PDAC) represents an area of intense research. Recently, the concept has emerged that multiplexed detection of different signatures from a single biospecimen (e.g., saliva, blood, etc.) may exhibit better diagnostic capability than single biomarkers. In this work, we develop a multiplexed strategy for detecting PDAC by combining characterization of the nanoparticle (NP)-protein corona, i.e., the protein layer that surrounds NPs upon exposure to biological fluids and circulating levels of plasma proteins belonging to the acute phase protein (APPs) family. As a first step, we developed a nanoparticle-enabled blood (NEB) test that employed 600 nm graphene oxide (GO) nanosheets and human plasma (HP) (5% vol/vol) to produce 75 personalized protein coronas (25 from healthy subjects and 50 from PDAC patients). Isolation and characterization of protein corona patterns by 1-dimensional (1D) SDS-PAGE identified significant differences in the abundance of low-molecular-weight corona proteins (20–30 kDa) between healthy subjects and PDAC patients. Coupling the outcomes of the NEB test with the circulating levels of alpha 2 globulins, we detected PDAC with a global capacity of 83.3%. Notably, a version of the multiplexed detection strategy run on sex-disaggregated data provided substantially better classification accuracy for men (93.1% vs. 77.8%). Nanoliquid chromatography tandem mass spectrometry (nano-LC MS/MS) experiments allowed to correlate PDAC with an altered enrichment of Apolipoprotein A-I, Apolipoprotein D, Complement factor D, Alpha-1-antichymotrypsin and Alpha-1-antitrypsin in the personalized protein corona. Moreover, other significant changes in the protein corona of PDAC patients were found. Overall, the developed multiplexed strategy is a valid tool for PDAC detection and paves the way for the identification of new potential PDAC biomarkers.

Magnetic Levitation Patterns of Microfluidic-Generated Nanoparticle-Protein Complexes.

Magnetic levitation (MagLev) has recently emerged as a powerful method to develop diagnostic technologies based on the exploitation of the nanoparticle (NP)–protein corona. However, experimental procedures improving the robustness, reproducibility, and accuracy of this technology are largely unexplored. To contribute to filling this gap, here, we investigated the effect of total flow rate (TFR) and flow rate ratio (FRR) on the MagLev patterns of microfluidic-generated graphene oxide (GO)–protein complexes using bulk mixing of GO and human plasma (HP) as a reference. Levitating and precipitating fractions of GO-HP samples were characterized in terms of atomic force microscopy (AFM), bicinchoninic acid assay (BCA), and one-dimensional sodium dodecyl sulfate–polyacrylamide gel electrophoresis (1D SDS-PAGE), and nanoliquid chromatography–tandem mass spectrometry (nano-LC-MS/MS). We identified combinations of TFR and FRR (e.g., TFR = 35 μL/min and FRR (GO:HP) = 9:1 or TFR = 3.5 μL/min and FRR (GO:HP) = 19:1), leading to MagLev patterns dominated by levitating and precipitating fractions with bulk-like features. Since a typical MagLev experiment for disease detection is based on a sequence of optimization, exploration, and validation steps, this implies that the optimization (e.g., searching for optimal NP:HP ratios) and exploration (e.g., searching for MagLev signatures) steps can be performed using samples generated by bulk mixing. When these steps are completed, the validation step, which involves using human specimens that are often available in limited amounts, can be made by highly reproducible microfluidic mixing without any ex novo optimization process. The relevance of developing diagnostic technologies based on MagLev of coronated nanomaterials is also discussed.

Three Diverse Granule Preparation Methods for Proteomic Analysis of Mature Rice (Oryza sativa L.) Starch Grain

Starch is the primary form of reserve carbohydrate storage in plants. Rice (Oryza sativa L.) is a monocot whose reserve starch is organized into compounded structures within the amyloplast, rather than a simple starch grain (SG). The mechanism governing the assembly of the compound SG from polyhedral granules in apposition, however, remains unknown. To further characterize the proteome associated with these compounded structures, three distinct methods of starch granule preparation (dispersion, microsieve, and flotation) were performed. Phase separation of peptides (aqueous trypsin-shaving and isopropanol solubilization of residual peptides) isolated starch granule-associated proteins (SGAPs) from the distal proteome of the amyloplast and the proximal ‘amylome’ (the amyloplastic proteome), respectively. The term ‘distal proteome’ refers to SGAPs loosely tethered to the amyloplast, ones that can be rapidly proteolyzed, while proximal SGAPs are those found closer to the remnant amyloplast membrane fragments, perhaps embedded therein—ones that need isopropanol solvent to be removed from the mature organelle surface. These two rice starch-associated peptide samples were analyzed using nano-liquid chromatography–tandem mass spectrometry (Nano-HPLC-MS/MS). Known and novel proteins, as well as septum-like structure (SLS) proteins, in the mature rice SG were found. Data mining and gene ontology software were used to categorize these putative plastoskeletal components as a variety of structural elements, including actins, tubulins, tubulin-like proteins, and cementitious elements such as reticulata related-like (RER) proteins, tegument proteins, and lectins. Delineating the plastoskeletal proteome begins by understanding how each starch granule isolation procedure affects observed cytoplasmic and plastid proteins. The three methods described herein show how the technique used to isolate SGs differentially impacts the subsequent proteomic analysis and results obtained. It can thus be concluded that future investigations must make judicious decisions regarding the methodology used in extracting proteomic information from the compound starch granules being assessed, since different methods are shown to yield contrasting results herein. Data are available via ProteomeXchange with identifier PXD032314.

Multi-Omic Analysis to Characterize Metabolic Adaptation of the E. coli Lipidome in Response to Environmental Stress.

As an adaptive survival response to exogenous stress, bacteria undergo dynamic remodelling of their lipid metabolism pathways to alter the composition of their cellular membranes. Here, using Escherichia coli as a well characterised model system, we report the development and application of a ‘multi-omics’ strategy for comprehensive quantitative analysis of the temporal changes in the lipidome and proteome profiles that occur under exponential growth phase versus stationary growth phase conditions i.e., nutrient depletion stress. Lipidome analysis performed using ‘shotgun’ direct infusion-based ultra-high resolution accurate mass spectrometry revealed a quantitative decrease in total lipid content under stationary growth phase conditions, along with a significant increase in the mol% composition of total cardiolipin, and an increase in ‘odd-numbered’ acyl-chain length containing glycerophospholipids. The inclusion of field asymmetry ion mobility spectrometry was shown to enable the enrichment and improved depth of coverage of low-abundance cardiolipins, while ultraviolet photodissociation-tandem mass spectrometry facilitated more complete lipid structural characterisation compared with conventional collision-induced dissociation, including unambiguous assignment of the odd-numbered acyl-chains as containing cyclopropyl modifications. Proteome analysis using data-dependent acquisition nano-liquid chromatography mass spectrometry and tandem mass spectrometry analysis identified 83% of the predicted E. coli lipid metabolism enzymes, which enabled the temporal dependence associated with the expression of key enzymes responsible for the observed adaptive lipid metabolism to be determined, including those involved in phospholipid metabolism (e.g., ClsB and Cfa), fatty acid synthesis (e.g., FabH) and degradation (e.g., FadA/B,D,E,I,J and M), and proteins involved in the oxidative stress response resulting from the generation of reactive oxygen species during β-oxidation or lipid degradation.

Classic Hodgkin Lymphoma Refractory for ABVD Treatment Is Characterized by Pathologically Activated Signal Transduction Pathways as Revealed by Proteomic Profiling.

Simple SummaryClassic Hodgkin lymphoma (cHL) patients refractory to standard ABVD chemo-therapy are known to have a dismal prognosis. This has led to the hypothesis that ABVD treatment-sensitive and ABVD treatment-refractory tumours are biologically distinct. In this study, cHL patients refractory to standard ABVD treatment show subtle but significant differences in protein expression that enable clustering of the two response groups, thus indicating differences between ABVD sensitive and refractory patients at the molecular level, and thereby strengthening the hypothesis that ABVD sensitive and ABVD refractory tumours may be biologically distinct.In classic Hodgkin lymphoma (cHL), the tumour microenvironment (TME) is of major pathological relevance. The paucity of neoplastic cells makes it important to study the entire TME when searching for prognostic biomarkers. Cure rates in cHL have improved markedly over the last several decades, but patients with primary refractory disease still show inferior survival. We performed a proteomic comparison of pretreatment tumour tissue from ABVD treatment-refractory versus ABVD treatment-sensitive cHL patients, in order to identify biological differences correlating with treatment outcome. Formalin-fixed paraffin-embedded tumour tissues from 36 patients with cHL, 15 with treatment-refractory disease, and 21 with treatment-sensitive disease, were processed for proteomic investigation. Label-free quantification nano liquid chromatography tandem mass spectrometry was performed on the tissues. A total of 3920 proteins were detected and quantified between the refractory and sensitive groups. This comparison revealed several subtle but significant differences in protein expression which could identify subcluster characteristics of the refractory group. Bioinformatic analysis of the biological differences indicated that a number of pathologically activated signal transduction pathways are disturbed in ABVD treatment-refractory cHL.

Comprehensive Proteomic Analysis Revealed a Large Number of Newly Identified Proteins in the Small Extracellular Vesicles of Milk from Late-Stage Lactating Cows.

Simple SummaryBovine milk provides excellent nutrition for infants and contains significant bioactive compounds, including small extracellular vesicles. Small extracellular vesicles are nanosized particles that have long been recognized to carry a variety of cargos, including proteins, nucleic acids, lipids, and other biomolecules. Previous research has shown that the proteomic profile in milk small extracellular vesicles differed in accordance with the lactation stages of cows, such as early- and mid-stage lactation. However, the protein profile in milk small extracellular vesicles from late-stage lactation is mostly unknown. In this study, a comprehensive proteomic analysis was performed to explore the proteins in milk small extracellular vesicles from late-stage lactating cows. The present study identified a total of 2225 proteins in milk small extracellular vesicles from late-stage lactating cows. Notably, a total of 429 proteins were newly identified after comparing with the previously published bovine milk small extracellular vesicles proteomic datasets. These findings indicate that the newly identified proteins could be helpful in better understanding the proteomic dynamics in milk small extracellular vesicles during the late-stage lactating cows.Bovine milk contains small extracellular vesicles (sEVs) that provide proteins, miRNAs, mRNAs, DNAs, and lipids to target cells and play a role in intracellular communications. Previous studies have characterized proteins in milk sEVs from early- and mid-stage lactation. However, the proteins in milk sEVs from late-stage lactation are mostly unexplored. The aim of this study was to determine the proteomic profile of milk sEVs from late-stage lactating cows. A comprehensive nanoliquid chromatography–tandem mass spectrometry (nanoLC-MS/MS) approach was carried out to reveal the proteins in milk sEVs. Additionally, bioinformatics analysis was carried out to interpret the molecular signatures of newly identified proteins in milk sEVs from three late-stage lactating cows. NanoLC-MS/MS analysis revealed a total of 2225 proteins in milk sEVs from cows. Notably, after comparing these identified proteins with previously deposited datasets of proteins in bovine milk sEVs, 429 proteins were detected as newly identified. Bioinformatic analysis indicated that these newly identified proteins in milk sEVs were engaged in a diverse range of molecular phenomena relevant to mammary gland physiology, milk production, immunity, and immune response. These findings suggest that the newly identified proteins could expand the inventory application of molecular cargos, nutritional status, and immune modulation of sEVs in milk during the late-stage lactation.