Label-free Liquid Chromatography-mass Spectrometry Research Articles

Characterization of the plasma proteome from healthy adult dogs.

Bloodwork is a widely used diagnostic tool in veterinary medicine, as diagnosis and therapeutic interventions often rely on blood biomarkers. However, biomarkers available in veterinary medicine often lack sensitivity or specificity. Mass spectrometry-based proteomics technology has been extensively used in the analysis of biological fluids. It offers excellent potential for a more comprehensive characterization of the plasma proteome in veterinary medicine. In this study, we aimed to identify and quantify plasma proteins in a cohort of healthy dogs and compare two techniques for depleting high-abundance plasma proteins to enable the detection of lower-abundance proteins via label-free quantification liquid chromatography-mass spectrometry. We utilized surplus lithium-heparin plasma from 30 healthy dogs, subdivided into five groups of pooled plasma from 6 randomly selected individuals each. Firstly, we used a commercial kit to deplete high-abundance plasma proteins. Secondly, we employed an in-house method to remove albumin using Blue-Sepharose. Among all the samples, some of the most abundant proteins identified were apolipoprotein A and B, albumin, alpha-2-macroglobulin, fibrinogen beta chain, fibronectin, complement C3, serotransferrin, and coagulation factor V. However, neither of the depletion techniques achieved significant depletion of highly abundant proteins. Despite this limitation, we could detect and quantify many clinically relevant proteins. Determining the healthy canine proteome is a crucial first step in establishing a reference proteome for canine plasma. After enrichment, this reference proteome can later be utilized to identify protein markers associated with different diseases, thereby contributing to the diagnosis and prognosis of various pathologies.

Label-Free Liquid Chromatography-Mass Spectrometry Quantitation of Relative N- and O-Glycan Concentrations in Human Milk in Japan.

Human milk is abundant in carbohydrates and includes human milk oligosaccharides (HMOs) and N/O-glycans conjugated to proteins. HMO compositions and concentrations vary in individuals according to the maternal secretor status based on the fucosyltransferase 2 genotype; however, the profile of N/O-glycans remains uninvestigated because of the analytical complexity. Herein, we applied a label-free chromatography-mass spectrometry (LC-MS) technique to elucidate the variation in the composition and concentration of N/O-glycans in human milk. We used label-free LC-MS to relatively quantify 16 N-glycans and 12 O-glycans in 200 samples of Japanese human milk (1-2 months postpartum) and applied high performance anion exchange chromatography with pulsed amperometric detection to absolutely quantify the concentrations of 11 representative HMOs. Cluster analysis of the quantitative data revealed that O-glycans and several HMOs were classified according to the presence or absence of fucose linked to galactose while N-glycans were classified into a different group from O-glycans and HMOs. O-glycans and HMOs with fucose linked to galactose were more abundant in human milk from secretor mothers than from nonsecretor mothers. Thus, secretor status influenced the composition and concentration of HMOs and O-glycans but not those of N-glycans in human milk.

Proteomic map of the differentially expressed proteins in the skin of Ctenopharyngodon idella against Aeromonas hydrophila infection

The skin mucus of fish is an important part of the innate immune system, which is poorly understood at the proteomic level. The study established a complete map of the proteins in the skin mucus of Ctenopharangdon idella (C. idella) and discussed the Differentially Expressed Proteins (DEPs) after Aeromonas hydrophila (A. hydrophila) infection. Using Label Free Liquid Chromatography-Mass Spectrometry (LC–MS/MS) analysis, a total of 126 proteins were identified as differentially expressed, 89 proteins of which were upregulated, and 37 proteins were downregulated. Functional annotations of DEPs showed that the upregulated proteins in the skin mucus of the treated group were mostly associated with complement system and cytoskeleton proteins, whereas downregulated proteins were associated with metabolism. The key upregulated immune proteins were transferrin variant C, lysozyme g, annexin A11, 26S proteasome non-ATPase regulatory subunit 8, hypothetical protein ROHU_000884, 60S ribosomal L7a, calpain-2 catalytic subunit-like protein, calpain-9-like protein, complement component C9, complement C3, cathepsin S, cathepsin Z, 14 kDa apolipo, heat shock protein and intelectin, whereas, leukocyte elastase inhibitor, annexin A11, C-factor-like protein, biotinidase isoform X1 and epidermal growth factor receptor substrate 15-like were the downregulated proteins. Moreover, we for the first-time report proteins such as coactosin, lamin-B2 and kelch 12, which were never reported in fish. Our study directly pointing out the possible immunological biomarkers in the skin mucus of C. idella after A. hydrophila treatment. Each of the protein we report in this study could be used as base to establish their mechanism of action during bacterial infection that may contribute to the strategies against bacterial prevention and control in fishes.

Proteomic profile of epidermal mucus from Labeo rohita reveals differentially abundant proteins after Aeromonas hydrophila infection

We report the proteomic profile of Epidermal Mucus (EM) from Labeo rohita and identified the differentially abundant proteins (DAPs) against Aeromonas hydrophila infection through label-free liquid chromatography-mass spectrometry (LC-MS/MS). Using discovery-based proteomics, a total of 2039 proteins were quantified in nontreated group and 1,328 proteins in the treated group, of which 114 were identified as DAPs in both the groups. Of the 114 DAPs, 68 proteins were upregulated and 46 proteins were downregulated in the treated group compared to nontreated group. Functional annotations of these DAPs shows their association with metabolism, cellular process, molecular process, cytoskeletal, stress, and particularly immune system. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis and Fisher's exact test between the two groups shows that most of the proteins were immune-related, which were significantly associated with the proteasome, phagosome, and Salmonella infection pathways. Overall, this study shows a basic and primary way for further functional research of the involvement of vitellogenin 2, alpha-2-macroglobulin-like protein, toll-like receptors (TLR-13), calpain, keratin-like proteins, and heat shock proteins against bacterial infection. Nonetheless, this first-ever comprehensive report of a proteomic sketch of EM from L. rohita after A. hydrophila infection provides systematic protein information to broadly understand the biological role of fish EM against bacterial infection.

The Proteome Profile of Olfactory Ecto-Mesenchymal Stem Cells-Derived from Patients with Familial Alzheimer's Disease Reveals New Insights for AD Study.

Alzheimer's disease (AD), the most common neurodegenerative disease and the first cause of dementia worldwide, has no effective treatment, and its pathological mechanisms are not yet fully understood. We conducted this study to explore the proteomic differences associated with Familial Alzheimer's Disease (FAD) in olfactory ecto-mesenchymal stem cells (MSCs) derived from PSEN1 (A431E) mutation carriers compared with healthy donors paired by age and gender through two label-free liquid chromatography-mass spectrometry approaches. The first analysis compared carrier 1 (patient with symptoms, P1) and its control (healthy donor, C1), and the second compared carrier 2 (patient with pre-symptoms, P2) with its respective control cells (C2) to evaluate whether the protein alterations presented in the symptomatic carrier were also present in the pre-symptom stages. Finally, we analyzed the differentially expressed proteins (DEPs) for biological and functional enrichment. These proteins showed impaired expression in a stage-dependent manner and are involved in energy metabolism, vesicle transport, actin cytoskeleton, cell proliferation, and proteostasis pathways, in line with previous AD reports. Our study is the first to conduct a proteomic analysis of MSCs from the Jalisco FAD patients in two stages of the disease (symptomatic and presymptomatic), showing these cells as a new and excellent in vitro model for future AD studies.

Comparative Label-Free Liquid Chromatography-Mass Spectrometry Milk Proteomic Profiles Highlight Putative Differences between the Autochthon Teramana and Saanen Goat Breeds.

Goat's milk is an excellent source of nutrients, with greater benefits compared to cow's milk. Limited information is available on autochthon goat breeds, which are important for biodiversity preservation. In this study, the aim of using label-free quantification was to investigate the milk proteome of two goat breeds, the autochthon Teramana and Saanen breeds, which are commonly used by the industry. Utilising label-free proteomic analysis, 749 and 666 proteins, respectively were identified and quantified from the Teramana and Saanen goat milk. Moreover, utilising statistical analysis, 29 proteins were able to discriminate the two goat breeds, with many of the identified proteins involved in complement and coagulation cascades. This work enhances our understanding of the goat milk proteome and shows differences between the two breeds, leading to an important contribution toward a more detailed molecular-view of this unique substrate. Additionally, charactersation of the milk proteins can help in guiding genetic improvements in the goat herds, and thus increasing its use in human nutrition.

Plasma proteomics approach to distinguishing PD-1 inhibitor-associated myocarditis from acute myocardial infarction in patients receiving PD-1 inhibitors treatment: A clinical and preclinical study.

e14698 Background: Immune checkpoint inhibitor (ICI)-related myocarditis is a rare but severe side effect that requires attention to distinguish it from other cardiovascular emergencies. Therefore, new biomarkers are needed for the early diagnosis of ICI-related myocarditis, especially to distinguish it from other cardiovascular emergencies. Our study aims to explore plasma biomarkers with high specificity and sensitivity using proteomic analysis for the early diagnosis of ICI-related myocarditis and to distinguish it from acute myocardial infarction (AMI). Methods: Patients with ICI-related myocarditis and AMI were selected and matched for potential confounding effects such as age, gender, smoking history, and chronic heart diseases. A label-free liquid chromatography-mass spectrometry (LC-MS) proteomics approach was used to analyze plasma samples and screen sensitive and specific myocarditis protein biomarkers. Further validation will be performed at the level of patients and animal models of ICI-related myocarditis and AMI. Results: We performed plasma proteomics on 15 plasma samples from 5 pairs of samples with PD-1 inhibitor-associated myocarditis at baseline (control group) and diagnosis (myocarditis group) and 5 cases of acute myocardial infarction (MI group) confirmed by coronary angiography in the Second Affiliated Hospital of Nanchang University. In total, we identified a total of 1521 plasma proteins and 1325 quantifiable plasma proteins across all 15 plasma samples. Further analysis of the differential proteins showed that 34 proteins with significantly differentially expressed in myocarditis group samples compared with other groups. Among them, several proteins were significantly up-regulated (MYOM3, TNNC1, MYL1, MYOM2, CA3, MYLPF, ECI1, LGALS1, PSMA7, PSMB3, PSMA4, PSMB6, CKM, ABCB6, UGP2, ALDOA, TPM2) or down-regulated (MAN1A1, SPTB, YTHDC2, CLEC11A, GSN, APOA4) in myocarditis samples. Compared with the other groups, the ICI-related myocarditis group showed molecular changes in myocardial contraction, proteasome, immunoregulation, NF-κB signaling pathway, arginine and proline metabolism, and cysteine and methionine metabolism. We then performed ELISA on additional ICI-related myocarditis and AMI patients and corresponding animal models. The Troponin C, Myom 3, UGP2, and Galectin-1 proteins related to immune or inflammation were significantly increased. Conclusions: Our study has revealed that proteins related to the immune system or inflammation may be sensitive and specific plasma biomarkers for identifying ICI-related myocarditis. However, further large sample cohorts are needed to confirm these findings.

Apolipoprotein C proteins in plasma non‐high‐density lipoprotein fractions and development of Alzheimer’s disease, central arterial stiffness, and cerebral small vessel disease in older adults

AbstractBackgroundPlasma non‐high‐density lipoproteins impact development of central arterial stiffness and cerebral small vessel disease (cSVD), significantly contributing to Alzheimer’s disease (AD) and related dementias (ADRDs) through an increase in cerebral amyloid beta (Aβ) burden and acceleration of atrophy in susceptible brain regions. We sought to identify proteins in three non‐HDL plasma lipoproteins (very‐low density lipoprotein [VLDL], intermediate‐density lipoprotein [IDL], low‐density lipoprotein [LDL]) and their cross‐sectional associations with brain amyloid deposition, central arterial stiffness, neuroimaging features of cSVD (e.g., white matter hyperintensity volume), brain volumes susceptible to AD and cSVD, and incidence of dementia.MethodPlasma samples from 66 participants of the Atherosclerosis Risk in Communities (ARIC) study were fractionated to isolate VLDL, IDL, and LDL and analyzed using label‐free liquid chromatography‐mass spectrometry (LC‐MS) with Data‐Independent Acquisition. Acquired MS spectra were processed using Spectronaut™ to give protein identification and relative protein abundances were calculated using R. Linear regression models were used to examine the cross‐sectional associations between protein abundances in VLDL, IDL or LDL and brain amyloid deposition (measured by amyloid PET scan), white matter hyperintensities (WMH) volume (measured by brain MRI), central arterial stiffness (carotid‐femoral and heart‐femoral pulse wave velocity), AD‐signature region, frontal, parietal, and hippocampal brain region volumes (measured by brain MRI), and incidence of dementia. Regression models were adjusted for age, sex, race, number of APOε4 alleles, and cholesterol level (if applicable). Additionally, differential analysis (student’s t‐test) was used to compare protein abundances in non‐HDL lipoproteins between participants with elevated versus normal brain amyloid deposition.ResultWe identified 50, 78, 81 proteins in VLDL, IDL, and LDL, respectively. The most significant findings were: 1) higher levels of apolipoprotein C proteins in VLDL, IDL, and LDL were associated with lower WMH volume or less central arterial stiffness; 2) lower levels of apolipoprotein C proteins in VLDL, IDL, and LDL were associated with less incidence of dementia; 3) lower levels of apolipoprotein C proteins in VLDL, IDL, and LDL for participants with elevated amyloid deposition.ConclusionThese results suggest the possible role of apolipoprotein C proteins present in non‐HDL‐lipoproteins in the development of AD, central arterial stiffness, and cSVD.

Label-Free Quantification by Liquid Chromatography-Tandem Mass Spectrometry of the Kunitz Inhibitor of Trypsin KTI3 in Soy Products.

The greater awareness of consumers regarding the sustainability of food chains has shifted part of the consumption from animal protein sources to vegetable sources. Among these, of relevance both for human food use and for animal feed, is soy. However, its high protein content is unfortunately accompanied by the presence of antinutritional factors, including Kunitz's trypsin inhibitor (KTI). Now there are few analytical methods available for its direct quantification, as the inhibitory activity against trypsin is generically measured, which however can be given by many other molecules and undergo numerous interferences. Therefore, in this work, a direct label-free liquid chromatography-mass spectrometry (LC-MS) method for the identification and quantification of trypsin Kunitz inhibitor KTI3 in soybean and derivative products has been developed. The method is based on the identification and quantification of a marker peptide, specific for the protein of interest. Quantification is achieved with an external calibration curve in the matrix, and the limit of detection and the limit of quantification of the method are 0.75 and 2.51 μg/g, respectively. The results of the LC-MS method were also compared with trypsin inhibition measured spectrophotometrically, highlighting the complementarity of these two different pieces of information.

Glomerular mTORC1 activation was associated with podocytes to endothelial cells communication in lupus nephritis

ObjectiveThis study was initiated to evaluate the mammalian target of the rapamycin (mTOR) signalling pathway involved in renal endothelial-podocyte crosstalk in patients with lupus nephritis (LN).MethodsWe compared the kidney protein...

Comparative Proteomics Analysis of Pig Muscle Exudate through Label-Free Liquid Chromatography-Mass Spectrometry.

Capital-driven animal husbandry systems undertaken in the last century led to the abandoning of many pig breeds that were not profitable. These local pig breeds and their respective production systems have great potential as they are able to respond to the high criteria and needs of modern society concerning some environmental aspects, animal-welfare, healthiness, etc. This is the case of the black pigs of Italy. The Apulo-Calabrese is a breed of black pig, known by many other names such as Nero d'Abruzzo. In order to further understand the biological differences between different types of porcine genetics (Nero d'Abruzzo and commercial-hybrid) we used a label-free LC-MS strategy and Western-blot to characterize the proteomes of muscle-exudate collected from these pigs. This proteomics approach identified 1669 proteins of which 100 changed significantly in abundance between breeds. Bioinformatics functional analysis indicated that differentially expressed proteins were involved in several biological processes related to energy-metabolism and response to oxidative stress, suggesting that these functions might distinguish between these pigs. Fatty-acid synthase, catalase and glutathione-peroxidase, involved in enzymatic activity were found to be more represented in samples obtained from the Nero d'Abruzzo. This biological information can potentially provide new biological factors that could determine the different production performances of these pigs, distinguished by their different genetic backgrounds.

Use of Affinity Purification-Mass Spectrometry to Identify Phosphorylated Tau Interactors in Alzheimer's Disease.

Phosphorylated tau is the main protein present in neurofibrillary tangles, the presence of which is a key neuropathological hallmark of Alzheimer's disease (AD). The toxic effects of phosphorylated tau are likely mediated by interacting proteins; however, methods to identify these interacting proteins comprehensively in human brain tissue are limited. Here, we describe a method that enables the efficient identification of hundreds of proteins that interact with phosphorylated tau (pTau), using affinity purification-mass spectrometry (AP-MS) on human, fresh-frozen brain tissue from donors with AD. Tissue is homogenized using a gentle technique that preserves protein-protein interactions, and co-immunoprecipitation of pTau and its interacting proteins is performed using the PHF1 antibody. The resulting protein interactors are then identified using label-free quantitative liquid chromatography-mass spectrometry (LC-MS)/MS. The Significance Analysis of INTeractome (SAINT) algorithm is used to determine which proteins significantly interact with pTau. This approach enables the detection of an abundance of all 6 isoforms of tau, 23 phosphorylated residues on tau, and 125 significant pTau protein interactors, in human AD brain tissue.

Label-free LC-MS based assay to characterize small molecule compound binding to cells.

Study of small molecule binding to live cells provides important information on the characterization of ligands pharmacologically. Here we developed and validated a label-free, liquid chromatography-mass spectrometry (LC-MS) based cell binding assay, using centrifugation to separate binders from non-binders. This assay was applied to various target classes, with particular emphasis on those for which protein-based binding assay can be difficult to achieve. In one example, to study a G protein coupled receptor (GPCR), we used one antagonist as probe and multiple other antagonists as competitor ligands. Binding of the probe was confirmed to be specific and saturable, reaching a fast equilibrium. Competition binding analysis by titration of five known ligands suggested a good correlation with their inhibition potency. In another example, this assay was applied to an ion channel target with its agonists, of which the determined binding affinity was consistent with functional assays. This versatile method allows quantitative characterization of ligand binding to cell surface expressed targets in a physiologically relevant environment.

Proteomics Profiling of Osteoporosis and Osteopenia Patients and Associated Network Analysis.

Bone mass reduction due to an imbalance in osteogenesis and osteolysis is characterized by low bone mineral density (LBMD) and is clinically classified as osteopenia (ON) or osteoporosis (OP), which is more severe. Multiple biomarkers for diagnosing OP and its progression have been reported; however, most of these lack specificity. This cohort study aimed to investigate sensitive and specific LBMD-associated protein biomarkers in patients diagnosed with ON and OP. A label-free liquid chromatography-mass spectrometry (LC-MS) proteomics approach was used to analyze serum samples. Patients’ proteomics profiles were filtered for potential confounding effects, such as age, sex, chronic diseases, and medication. A distinctive proteomics profile between the control, ON, and OP groups (Q2 = 0.7295, R2 = 0.9180) was identified, and significant dysregulation in a panel of proteins (n = 20) was common among the three groups. A comparison of these proteins showed that the levels of eight proteins were upregulated in ON, compared to those in the control and the OP groups, while the levels of eleven proteins were downregulated in the ON group compared to those in the control group. Interestingly, only one protein, myosin heavy chain 14 (MYH14), showed a linear increase from the control to the ON group, with the highest abundance in the OP group. A significant separation in the proteomics profile between the ON and OP groups (Q2 = 0.8760, R2 = 0.991) was also noted. Furthermore, a total of twenty-six proteins were found to be dysregulated between the ON and the OP groups, with fourteen upregulated and twelve downregulated proteins in the OP, compared to that in the ON group. Most of the identified dysregulated proteins were immunoglobulins, complement proteins, cytoskeletal proteins, coagulation factors, and various enzymes. Of these identified proteins, the highest area under the curve (AUC) in the receiver operating characteristic (ROC) analysis was related to three proteins (immunoglobulin Lambda constant 1 (IGLC1), RNA binding protein (MEX3B), and fibulin 1 (FBLN1)). Multiple reaction monitoring (MRM), LC-MS, was used to validate some of the identified proteins. A network pathway analysis of the differentially abundant proteins demonstrated dysregulation of inflammatory signaling pathways in the LBMD patients, including the tumor necrosis factor (TNF), toll-like receptor (TL4), and interferon-γ (IFNG) signaling pathways. These results reveal the existence of potentially sensitive protein biomarkers that could be used in further investigations of bone health and OP progression.

Quantitative Acetylomics Uncover Acetylation-Mediated Pathway Changes Following Histone Deacetylase Inhibition in Anaplastic Large Cell Lymphoma.

Histone deacetylases (HDACs) target acetylated lysine residues in histone and non-histone proteins. HDACs are implicated in the regulation of genomic stability, cell cycle, cell death and differentiation and thus critically involved in tumorigenesis. Further, HDACs regulate T-cell development and HDAC inhibitors (HDACis) have been approved for clinical use in some T-cell malignancies. Still, the exact targets and mechanisms of HDAC inhibition in cancer are understudied. We isolated tumor cell lines from a transgenic mouse model of anaplastic large cell lymphoma (ALCL), a rare T-cell lymphoma, and abrogated HDAC activity by treatment with the HDACis Vorinostat and Entinostat or Cre-mediated deletion of Hdac1. Changes in overall protein expression as well as histone and protein acetylation were measured following Hdac1 deletion or pharmacological inhibition using label-free liquid chromatography mass spectrometry (LC-MS/MS). We found changes in overall protein abundance and increased acetylation of histones and non-histone proteins, many of which were newly discovered and associated with major metabolic and DNA damage pathways. For non-histone acetylation, we mapped a total of 1204 acetylated peptides corresponding to 603 proteins, including chromatin modifying proteins and transcription factors. Hyperacetylated proteins were involved in processes such as transcription, RNA metabolism and DNA damage repair (DDR). The DDR pathway was majorly affected by hyperacetylation following HDAC inhibition. This included acetylation of H2AX, PARP1 and previously unrecognized acetylation sites in TP53BP1. Our data provide a comprehensive view of the targets of HDAC inhibition in malignant T cells with general applicability and could have translational impact for the treatment of ALCL with HDACis alone or in combination therapies.

Plant-Based Titanium Dioxide Nanoparticles Trigger Biochemical and Proteome Modifications in Triticum aestivum L. under Biotic Stress of Puccinia striiformis.

In this study, we evaluated bioinspired titanium dioxide nanoparticles (TiO2 NPs) that elicited biochemical and proteome modifications in wheat plants under the biotic stress caused by Puccinia striiformis f. sp. tritici (Pst). Biosynthesis of TiO2 NPs was confirmed using UV–Vis spectrophotometry, energy dispersive X-ray spectroscopy (EDX), scanning electron microscopy (SEM), and Fourier transform infrared (FTIR) spectroscopy. We found that the nanoparticles with crystalline nature were smaller than 100 nm. The results of FTIR analysis showed the presence of potential functional groups exhibiting O-H, N-H, C-C, and Ti-O stretching. The TiO2 NPs of different concentrations (20, 40, 60, and 80 mg L−1) were exogenously applied to wheat plants under the biotic stress caused by Pst, which is responsible for yellow stripe rust disease. The results of the assessment of disease incidence and percent disease index displayed time- and dose-dependent responses. The 40 mg L−1 TiO2 NPs were the most effective in decreasing disease severity. The bioinspired TiO2 NPs were also evaluated for enzymatic (superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT)), and nonenzymatic metabolites (total proline, phenolic, and flavonoid contents) in wheat plants under stripe rust stress. The 40 mg L−1 TiO2 NPs were effective in eliciting biochemical modifications to reduce biotic stress. We further evaluated the effects of TiO2 NPs through gel- and label-free liquid chromatography-mass spectrometry (LC-MS) proteome analysis. We performed proteome analysis of infected wheat leaves and leaves treated with 40 mg L−1 TiO2 NPs under stripe rust stress. The functional classification of the proteins showed downregulation of proteins related to protein and carbohydrate metabolism, as well as of photosynthesis in plants under biotic stress. An upregulation of stress-related proteins was observed, including the defense mechanisms and primary metabolic pathways in plants treated with 40 mg L−1 TiO2 NPs under stress. The experimental results showed the potential of applying biogenic TiO2 NPs to combat fungal diseases of wheat plants and provided insight into the protein expression of plants in response to biotic stress.

Squaric Ester-Based Nanogels Induce No Distinct Protein Corona but Entrap Plasma Proteins into their Porous Hydrogel Network.

After intravenous administration of nanocarriers, plasma proteins may rapidly adsorb onto their surfaces. This process hampers the prediction of the nanocarriers' pharmacokinetics as it determines their physiological identity in a complex biological environment. Toward clinical translation it is therefore an essential prerequisite to investigate the nanocarriers' interaction with plasma proteins. Here, this work evaluates a highly "PEGylated" squaric ester-based nanogel with inherent prolonged blood circulation properties. After incubation with human blood plasma, the nanogels are isolated by asymmetrical flow-field flow fractionation. Multiangle light scattering measurements confirm the absence of significant size increases as well as aggregation upon plasma incubation. However, proteomic analyses by gel electrophoresis find minor absolute amounts of proteins (3 wt%), whereas label-free liquid chromatography mass spectrometry identify 65 enriched proteins. Interestingly, the relative abundance of these proteins is almost similar to their proportion in pure native plasma. Due to the nanogels' hydrated and porous network morphology, it is concluded that the detected proteins rather result from passive diffusion into the nanogel network than from specific interactions at the plasma particle interface. Consequently, these results do not indicate a classical surface protein corona but rather reflect the highly outer and inner stealth-like behavior of the porous hydrogel network.

Proteomic profiling of kidney samples in patients with pure membranous and proliferative lupus nephritis.

Renal injury in lupus nephritis (LN) does not manifest as one uniform entity. The clinical presentation, management, and prognosis of membranous LN (MLN) differ from that of the proliferative LN (PLN). Differentiating the molecular mechanisms involved in MLN and PLN and discovering the reliable biomarkers for early diagnosis and target therapy are important. We compared the kidney protein expression patterns of 11 pure MLN and 12 pure PLN patients on formalin-fixed paraffin-embedded (FFPE) kidney tissues using label-free liquid chromatography-mass spectrometry (LC-MS) for quantitative proteomics analysis. FunRich software was used to identify proteins in differentially expressed pathways. Quantitative comparisons of differentially expressed proteins in each patient were further analyzed based on protein intensity levels determined by LC-MS. The protein-protein interaction (PPI) network of the differentially expressed genes (DEGs) was established through Search Tool for the Retrieval of Interacting Genes database (STRING) website, visualized by Cytoscape. A total of 5112 proteins were identified. In total, 12 significantly upregulated (fold change ≥2, p < 0.05) proteins were identified in the MLN group and 220 proteins (fold change ≥2, p < 0.05) were upregulated in the PLN group. Further analysis showed that the most significant upregulated pathway involved in MLN was histone deacetylase (HDAC) class I pathway, and the three most significant upregulated pathways in PLN were interferon signaling, interferon gamma signaling, and the immune system. Next, we selected sirtuin-2 (SIRT2) in MLN, and vascular cell adhesion protein 1 (VCAM1) and Bcl-xl in PLN for further mass spectrometry (MS) intensity and PPI analysis. SIRT2 expression was significantly increased in the MLN group compared with the PLN group, and VCAM1, Bcl-xl expression was significantly increased in the PLN group compared with the MLN group, based on MS intensity. These results may help to improve our understanding of the underlying molecular mechanisms of MLN and PLN and provide potential targets for the diagnosis and treatment of different subclasses of LN.

Target engagement of ginsenosides in mild cognitive impairment using mass spectrometry-based drug affinity responsive target stability

BackgroundMild cognitive impairment (MCI) is a transitional condition between normality and dementia. Ginseng is known to have effects on attenuating cognitive deficits in neurogenerative diseases. Ginsenosides are the main bioactive component of ginseng, and their protein targets have not been fully understood. Furthermore, no thorough analysis is reported in ginsenoside-related protein targets in MCI. MethodsThe candidate protein targets of ginsenosides in brain tissues were identified by drug affinity responsive target stability (DARTS) coupled with label-free liquid chromatography-mass spectrometry (LC–MS) analysis. Network pharmacology approach was used to collect the therapeutic targets for MCI. Based on the above-mentioned overlapping targets, we built up a protein–protein interaction (PPI) network in STRING database and conducted gene ontology (GO) enrichment analysis. Finally, we assessed the effects of ginseng total saponins (GTS) and different ginsenosides on mitochondrial function by measuring the activity of the mitochondrial respiratory chain complex and performing molecular docking. ResultsWe screened 2526 MCI-related protein targets by databases and 349 ginsenoside-related protein targets by DARTS. On the basis of these 81 overlapping genes, enrichment analysis showed the mitochondria played an important role in GTS-mediated MCI pharmacological process. Mitochondrial function analysis showed GTS, protopanaxatriol (PPT), and Rd increased the activities of complex I in a dose-dependent manner. Molecular docking also predicted the docking pockets between PPT or Rd and mitochondrial respiratory chain complex I. ConclusionThis study indicated that ginsenosides might alleviate MCI by targeting respiratory chain complex I and regulating mitochondrial function, supporting ginseng's therapeutic application in cognitive deficits.

Temperature-Responsive Nanoparticles Enable Specific Binding of Apolipoproteins from Human Plasma.

Apolipoproteins are an important class of proteins because they provide a so-called stealth effect to nanoparticles. The stealth effect on nanocarriers leads to a reduced unspecific uptake into immune cells and thereby to a prolonged blood circulation time. Herein, a novel strategy to bind apolipoproteins specifically on nanoparticles by adjusting the temperature during their incubation in human plasma is presented. This specific binding, in turn, allows a control of the stealth behavior of the nanoparticles. Nanoparticles with a well-defined poly(N-isopropylacrylamide) shell are prepared, displaying a reversible change of hydrophobicity at a temperature around 32°C. It is shown by label-free quantitative liquid chromatography-mass spectrometry that the nanoparticles are largely enriched with apolipoprotein J (clusterin) at 25°C while they are enriched with apolipoprotein A1 and apolipoprotein E at 37°C. The temperature-dependent protein binding is found to significantly influence the uptake of the nanoparticles by RAW264.7 and HeLa cells. The findings imply that the functionalization of nanoparticles with temperature-responsive materials is a suitable method for imparting stealth properties to nanocarriers for drug-delivery.