Quantitative Protein Analysis Research Articles

Analysis of the effect of cations on protein conformational stability using solid-state nanopores.

The conformation of proteins is closely related to their biological functions, and it is affected by many factors, including the type of cations in solution. However, it is difficult to detect the conformational changes of a protein in situ. As a single-molecule sensing technology, nanopores can convert molecular structural information into analyzable current signals within a reasonable time range. Herein, we detect and analyze the effects of two different types of monovalent cations (Na+ and Li+) on a model protein bovine serum albumin (BSA) conformation using SiNx nanopores with different diameters. The quantitative analysis results show that the excluded volume of BSA in LiCl salt solutions is larger than the value in NaCl solution, indicating that Li+ is more prone to unfolding the proteins and making them unstable. This study demonstrated that nanopores enable the in situ detection of the structure of proteins at the single-molecule level and provide a new approach for the quantitative analysis of proteins.

#1044 Quantitative analysis of dialytic protein loss with medium cut-off and high-flux membranes

Abstract Background and Aims Hemodialysis is an effective way for eliminating uremic retention products in patients with end-stage kidney disease. Medium cut-off membranes (MCO) were designed to enhance the dialytic removal of middle-sized molecules, such as beta-2 microglobulin (β2M). However, alongside these toxins, salutary proteins could also be lost. To better understand the spectrum of proteins cleared by MCO and high-flux (HF) dialyzers, we conducted qualitative and quantitative analysis of proteins in hemodialysis ultrafiltrate (UF). Method We established an ex vivo hemodialysis simulation system by using two Fresenius 2008T machines to concurrently dialyze a 2.5-liter human plasma reservoir for simultaneous testing of two dialyzers. We compared three dialyzers, including two HF dialyzers (Fresenius Medical Care Optiflux F180NR and FX CorAL 80), and an MCO membrane (Baxter Theranova 400; TH) (Fig. 1A). The blood flow rate was 400 mL/min, the dialysate flow rate was zero, and the ultrafiltration flow rate was 13 mL/min. UF samples were collected every 10 minutes from the UF line. Upon experiment completion at one-hour, residual filtrate was collected from the dialysate compartment. All samples were stored at −80°C until analysis. In total we conducted six dialysis simulations, each involving a concurrently tested dialyzer pair (Fig. 1B). UF samples underwent protein analysis (Bradford protein assay), albumin and creatinine measurement (Horiba® Pentra C400), liquid chromatography tandem mass spectrometry (LC-MS/MS)-based proteomics (Agilent® Q-TOF 6546), and immune-based protein assay (Luminex® Magpix). We analyzed the average of four sets of data obtained from the same dialyzer at the same time point. Results The loss of total protein and albumin was significantly greater with TH compared to the two HF dialyzers (Fig. 1C). Creatinine removal was similar across all dialyzers (Fig. 1D). The overall protein loss through F180NR and CorAL was comparable, while the protein loss through TH was up to 14-fold higher (811.7 ± 149.5 mg; Fig. 1E). Similar trends were observed for albumin. Albumin comprised 62% of the protein content in UF from TH, whereas it accounts for approximately 30% for HF dialyzers. We next employed label-free LC-MS and LC-MS/MS to identify and quantify proteins in 12 UF samples collected at the 20-minute point. Using Spectrum Mill software, we identified 244 proteins in the UF, 113 were accurately semi-quantified. The protein intensity ratios of TH-to- F180NR, TH-to-CorAL, and CorAL-to-F180NR for these 113 proteins are presented in Fig. 2A-C. Each gray dot on the plot corresponds to a specific protein. In line with the total protein and albumin quantification, MS proteomics analysis consistently revealed a greater loss of every identified protein by TH compared to both HF dialyzers. The median protein intensity ratio of TH-to-F180NR and TH-to-CorAL was 9.14 and 8.25, respectively. Importantly, TH not only removed middle-molecules such as β2M, but also showed a higher loss of salutary proteins. For instance, our data indicate a 9- to 16-fold difference in the level of albumin, haptoglobin, and vitamin D-binding protein between TH and HF dialyzers. Protein loss did not differ between F180NR and CorAL. To validate the accuracy of the MS quantification, we quantified seven proteins using immune-based assays in all UF samples at the 20-minute point. The result revealed a strong consistency between MS data and other methods (Fig. 2D). Conclusion We established an ex vivo system to concurrently test two dialyzers and implemented a robust proteomics workflow for a direct comparison of protein loss in the UF. Our data comprehensively and consistently indicate a greater extent of protein loss with MCO compared to HF, including the loss of salutary proteins. Further research is warranted to explore the clinical consequences of the enhanced loss of salutary proteins.

Quantitative proteomic analysis reveals unique Hsp90 cycle-dependent client interactions.

Hsp90 is an abundant and essential molecular chaperone that mediates the folding and activation of client proteins in a nucleotide-dependent cycle. Hsp90 inhibition directly or indirectly impacts the function of 10-15% of all proteins due to degradation of client proteins or indirect downstream effects. Due to its role in chaperoning oncogenic proteins, Hsp90 is an important drug target. However, compounds that occupy the ATP-binding pocket and broadly inhibit function have not achieved widespread use due to negative effects. More selective inhibitors are needed; however, it is unclear how to achieve selective inhibition. We conducted a quantitative proteomic analysis of soluble proteins in yeast strains expressing wild-type Hsp90 or mutants that disrupt different steps in the client folding pathway. Out of 2,482 proteins in our sample set (approximately 38% of yeast proteins), we observed statistically significant changes in abundance of 350 (14%) of those proteins (log2 fold change ≥ 1.5). Of these, 257/350 (∼73%) with the strongest differences in abundance were previously connected to Hsp90 function. Principal component analysis of the entire dataset revealed that the effects of the mutants could be separated into 3 primary clusters. As evidence that Hsp90 mutants affect different pools of clients, simultaneous co-expression of 2 mutants in different clusters restored wild-type growth. Our data suggest that the ability of Hsp90 to sample a wide range of conformations allows the chaperone to mediate folding of a broad array of clients and that disruption of conformational flexibility results in client defects dependent on those states.

Gastric Cancer Mesenchymal Stem Cells Trigger Endothelial Cell Functional Changes to Promote Cancer Progression.

Our previous studies have highlighted the pivotal role of gastric cancer mesenchymal stem cells (GCMSCs) in tumor initiation, progression, and metastasis. In parallel, it is well-documented that endothelial cells (ECs) undergo functional alterations in response to challenging tumor microenvironment. This study aims to elucidate whether functional changes in ECs might be induced by GCMSCs and thus influence cancer progression. Cell proliferation was assessed through CCK-8 and colony formation assays, while cell migration and invasion capabilities were evaluated by wound-healing and Transwell assays. Immunohistochemistry was employed to examine protein distribution and expression levels. Additionally, quantitative analysis of protein and mRNA expression was carried out through Western blotting and qRT-PCR respectively, with gene knockdown achieved using siRNA. Our findings revealed that GCMSCs effectively stimulate cell proliferation, migration, and angiogenesis of human umbilical vein endothelial cells (HUVECs), both in vitro and in vivo. GCMSCs promote the migration and invasion of gastric cancer cells by inducing the expression of Slit2 in HUVECs. Notably, the inhibition of phosphorylated AKT partially mitigates the aforementioned effects. In conclusion, GCMSCs may exert regulatory control over Slit2 expression in ECs via the AKT signaling pathway, thereby inducing functional changes in ECs that promote tumor progression.

SMI-FSM: Multiscale imaging and quantitative analysis of plasma membrane proteins and cortical actin

SMI-FSM: Multiscale imaging and quantitative analysis of plasma membrane proteins and cortical actin

Comparison of growth factors isolated from exosomes present in maternal blood of cleft and non-cleft infants for identification and quantification of growth factors involved in the growth and development of cartilage.

Background Cleft lip and palate (C.L.P.) is a common congenital condition which affects the orofacial region. The aetiology of C.L.P. is multifaceted and involves genetic and environmental factors that impact the shape and position of the nasal cartilage, resulting in an asymmetrical appearance of the nose. Growth factors are crucial in regulating cartilage synthesis and repair. Exosomes have emerged as a promising cell-free drug delivery system that facilitates intercellular communication and maintains tissue homeostasis. This study identifies and quantifies the growth factors present in exosomes derived from lactating maternal blood of cleft and non-cleft infants and compares them to evaluate the differences. The study aims to assess the potential of these exosomes as a therapeutic approach for C.L.P. management. Methods Blood samples were collected from lactating mothers at Sharad Pawar Dental College and isolating plasma using centrifugation. Exosome and total protein isolation will be conducted in the laboratory, and immunoblotting will evaluate growth factors. Quantitative and comparative protein and growth factor analysis will be conducted separately for maternal blood samples. Results The growth factors responsible for cartilage growth and development will be present in maternal exosomes, potentially leading to novel therapies for C.L.P. management. The study aims to demonstrate the therapeutic potential of exosomes, as carriers of intra-cellular factors, in transporting growth factors to the required site for managing C.L.P. Conclusions Overall, by understanding the roles played by growth factors, intercellular communication, and genetic and environmental factors in the aetiology of C.L.P., this research can contribute to creating effective treatment strategies for C.L.P. management. Finding growth factors in exosomes made from the blood of nursing mothers could be a cutting-edge therapeutic strategy for treating C.L.P.

Polycaprolactone Nanofibers Functionalized by Fibronectin/Gentamicin and Implanted Silver for Enhanced Antibacterial Properties, Cell Adhesion, and Proliferation.

Novel nanomaterials used for wound healing should have many beneficial properties, including high biological and antibacterial activity. Immobilization of proteins can stimulate cell migration and viability, and implanted Ag ions provide an antimicrobial effect. However, the ion implantation method, often used to introduce a bactericidal element into the surface, can lead to the degradation of vital proteins. To analyze the surface structure of nanofibers coated with a layer of plasma COOH polymer, fibronectin/gentamicin, and implanted with Ag ions, a new X-ray photoelectron spectroscopy (XPS) fitting method is used for the first time, allowing for a quantitative assessment of surface biomolecules. The results demonstrated noticeable changes in the composition of fibronectin- and gentamicin-modified nanofibers upon the introduction of Ag ions. Approximately 60% of the surface chemistry has changed, mainly due to an increase in hydrocarbon content and the introduction of up to 0.3 at.% Ag. Despite the significant degradation of fibronectin molecules, the biological activity of Ag-implanted nanofibers remained high, which is explained by the positive effect of Ag ions inducing the generation of reactive oxygen species. The PCL nanofibers with immobilized gentamicin and implanted silver ions exhibited very significant antipathogen activity to a wide range of Gram-positive and Gram-negative strains. Thus, the results of this work not only make a significant contribution to the development of new hybrid fiber materials for wound dressings but also demonstrate the capabilities of a new XPS fitting methodology for quantitative analysis of surface-related proteins and antibiotics.

Quantitative label-free proteomic analysis of excretory-secretory proteins in different developmental stages of Trichinella spiralis

Trichinella spiralis (T. spiralis) is a zoonotic parasitic nematode with a unique life cycle, as all developmental stages are contained within a single host. Excretory-secretory (ES) proteins are the main targets of the interactions between T. spiralis and the host at different stages of development and are essential for parasite survival. However, the ES protein profiles of T. spiralis at different developmental stages have not been characterized. The proteomes of ES proteins from different developmental stages, namely, muscle larvae (ML), intestinal infective larvae (IIL), preadult (PA) 6 h, PA 30 h, adult (Ad) 3 days post-infection (dpi) and Ad 6 dpi, were characterized via label-free mass spectrometry analysis in combination with bioinformatics. A total of 1217 proteins were identified from 9341 unique peptides in all developmental stages, 590 of which were quantified and differentially expressed. GO classification and KEGG pathway analysis revealed that these proteins were important for the growth of the larvae and involved in energy metabolism. Moreover, the heat shock cognate 71 kDa protein was the centre of protein interactions at different developmental stages. The results of this study provide comprehensive proteomic data on ES proteins and reveal that these ES proteins were differentially expressed at different developmental stages. Differential proteins are associated with parasite survival and the host immune response and may be potential early diagnostic antigen or antiparasitic vaccine candidates.

Detection and Validation of A2 Milk Suitable for Consumers Having Milk Intolerance by ELISA Method

Casein proteins, which make up 80% of the total proteins in cow's milk, consist of mainly A1 and A2 genetic types which differ by a mutation that causes conversion from proline to histidine. Histidine-containing A1 protein undergoes proteolytic degradation in the gastrointestinal system, while this is not observed during the digestion of A2 protein. A1 milk consumption causes bloating, gas, discomfort and symptoms confused with lactose intolerance. Studies showed that A1 milk consumption may cause diabetes, coronary heart disease, arteriosclerosis, sudden infant death, and is associated with autism and schizophrenia. With an increasing trend in the world, A2A2 milk (milk without A1 protein) production is becoming widespread with consumer preferences; and, A2 milk takes its place on the market shelves. With the onset of this trend, the need for a new analysis on food safety became evident. It will be required by food control laboratories to test the absence of A1 protein in milk to be labeled as A2 milk. In this study, the quantitative analysis and validation of β-casein A1 and A2 proteins in cow's milk by EnzymeLinked ImmunoSorbent Analysis (ELISA) method was investigated. The methods have detection limits of 1.8 and 0.8 ppm, and quantitation limits of 17 and 2.4 ppm for A1 and A2, respectively.

Quantitative proteomics based on TMT revealed the response of PK15 cells infected PEDV wild strain

Porcine epidemic diarrhea (PED), caused by porcine epidemic diarrhea virus (PEDV), is an acute and highly contagious enteric disease with a high mortality rate in suckling piglets. Identification of proteins associated with PEDV infection may provide insights into the pathogenesis of this viral disease. In this study, we employed tandem mass tag (TMT) quantitative protein analysis to investigate proteomic changes in PK15 cells following PEDV infection, and differential protein expression profiles were obtained at 0 h, 24 h, and 48 h post-infection. Overall, a total of 6330 proteins were identified. Applying criteria for fold change >1.5 < 0.67 and p-values <0.05 resulted in the identification of 59 up-regulated proteins and 103 down-regulated proteins that exhibited significant alterations in the H24 group compared to the H0 group. The H48 group demonstrated significant upregulation of 110 proteins and downregulation of 144 proteins compared to the H0 group; additionally, there were also 10 upregulated and 30 downregulated proteins in the H48 group when compared to the H24 group. These differentially expressed proteins (DEPs) were involved in immune response regulation, signal transduction, lipid transport and metabolism processes as well as cell apoptosis pathways. Based on these DEPs, we propose that PEDV may disrupt signal transduction pathways along with lipid transport and metabolism processes leading to maximal viral replication, it may also trigger inflammatory cascades accordingly. These findings could provide valuable information for elucidating specific pathogenesis related to PEDV infection while contributing towards developing new antiviral strategies.

Integrated Advanced Molecular Tools Predict In Situ cVOC Degradation Rates: Field Demonstration.

Chlorinated volatile organic compound (cVOC) degradation rate constants are crucial information for site management. Conventional approaches generate rate estimates from the monitoring and modeling of cVOC concentrations. This requires time series data collected along the flow path of the plume. The estimates of rate constants are often plagued by confounding issues, making predictions cumbersome and unreliable. Laboratory data suggest that targeted quantitative analysis of Dehalococcoides mccartyi (Dhc) biomarker genes (qPCR) and proteins (qProt) can be directly correlated with reductive dechlorination activity. To assess the potential of qPCR and qProt measurements to predict rates, we collected data from cVOC-contaminated aquifers. At the benchmark study site, the rate constant for degradation of cis-dichloroethene (cDCE) extracted from monitoring data was 11.0 ± 3.4 yr-1, and the rate constant predicted from the abundance of TceA peptides was 6.9 yr-1. The rate constant for degradation of vinyl chloride (VC) from monitoring data was 8.4 ± 5.7 yr-1, and the rate constant predicted from the abundance of TceA peptides was 5.2 yr-1. At the other study sites, the rate constants for cDCE degradation predicted from qPCR and qProt measurements agreed within a factor of 4. Under the right circumstances, qPCR and qProt measurements can be useful to rapidly predict rates of cDCE and VC biodegradation, providing a major advance in effective site management.

Proteomic analysis of N-glycosylation of the human placenta between preeclampsia and normal pregnancies

Protein N-glycosylation plays critical roles in modulating placental function, but little is known about N-glycoproteins in the human placenta and modifications in preeclampsia (PE). Here, we show a large, site-specific N-glycoproteome profiling study of PE and normal placenta using quantitative N-glycoproteomics based on mass spectrometry. The study identified disease signatures of altered N-glycoproteins and N-glycosylation site occupancy in PE and provided a system-level view of human placental N-glycoproteins and in vivo N-glycosylation sites. The study led to the discovery of a roster of glycoproteins with aberrant N-glycosylation levels associated with PE, including CD34, ENPP1 (ectonucleotide pyrophosphatase/phosphodiesterase family member 1), insulin-like growth factor binding protein (IGFBP3), and HYOU1 (hypoxia up-regulated 1). An emerging phenomenon that N-glycosylation is involved in several PE pathways, including cell adhesion molecules, PI3K-Akt signaling, pyrimidine metabolism, and metabolic pathways was revealed by pathway analysis of PE-associated aberrant glycoproteins. After enzymolysis, the proteins in each group were enriched with N-glycosylated peptides by lectin, the glycochain was excised by peptide N-glycosidase F (PNGase F) in H218O, and the glycosylated sites were analyzed by LC-MS/MS to achieve large-scale qualitative and quantitative analysis of N-glycosylated proteins. Our findings highlight the role of N-glycosylation in the pathogenesis of PE and provide new molecular and system-level insights for understanding and treating this disease.

TMT-based quantitative proteomics reveals the nutritional and stress resistance functions of anaerobic fungi in yak rumen during passage at different time intervals

ObjectivesAnaerobic fungi are critical for nutrient digestion in the yak rumen. Although studies have reported the effects of passage at different time intervals on the community structure of yak rumen anaerobic fungi, it is unknown whether passage culture at different time intervals affects the microbial proteins of rumen anaerobic fungi and their functions. MethodsMycelium was obtained using the anaerobic continuous batch culture (CBC) of yak rumen fluid at intervals of 3 d, 5 d and 7 d. Quantitative analysis of fungal proteins and functional analysis was performed using tandem mass tagging (TMT) and bioinformatics. ResultsA total of 56 differential proteins (DPs) were found in 5 d vs. 3 d and 7 d vs. 3 d. Gene ontology (GO) enrichment indicated that the up-regulated proteins were mainly involved in biological regulation, cellular process, metabolic process, macromolecular complex, membrane, cell part, organelle, binding, catalytic activity and transporter activity. The downregulated proteins were mainly enriched in metabolic process, cell part, binding and catalytic activity. Furthermore, the downregulated proteins in 7 d vs. 3 d were related to membrane and organelle. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment results indicated that DPs were enriched in 14 pathways in 5 d vs. 3 d and 7 d vs. 3 d, mainly including terpenoid backbone biosynthesis, alaine, aspartate and glutamate metabolism, arginine biosynthesis, hypotaurine, cyanoamino acid, glutathione, β-alanine, pyrimidine, purine, galactose and propanate metabolism, steroid biosynthesis, ribosome biogenesis in eukaryotes and aminoacyl tRNA biosynthesis. The DPs were enriched in only 2 pathways in 5 d vs 3 d, lysine biosynthesis and cysteine and methionine metabolism. N-glycan biosynthesis and retinol metabolism are only found in the metabolism of DPs in 7 d vs 3 d. ConclusionsYak rumen anaerobic fungal proteins are involved in nutrition and stress tolerance during passage at different time intervals.

Abstract C039: Cancer labeling should be retained for prostate cancer with a Gleason score of 3+3=6 - based on genomic, transcriptomic, and proteomic aspects

Abstract Purpose: The decision to remove the cancer label for prostate cancer with a Gleason score (GS) of 3+3 relies on pathology and clinical manifestations. However, evidence at the molecular level, especially for comparing the Excluding 3 group (GS4+4, GS4+5, GS5+4 and GS5+5) and the Gleason Grade Group 1 (GG1) (GS3+3), is lacking. To address this gap, we aimed to explore the differences between these two groups at the genomic, transcriptomic, and proteomic levels. Methods: We compared the GG1 group (n=82) and Excluding 3 group (n=148) using data from the TCGA in terms of genomic alterations, including gene mutations, chromosomal structure variants, and copy number variants. Pathways were revealed by using GSEA enrichment analysis at the transcriptome level. We also conducted a quantitative whole protein analysis of prostate cancer and paraneoplastic tissues in 6 pairs of GG1 and 11 pairs of Excluding 3 groups to compare the differences at the protein level and explored the enriched pathways between them by GSEA enrichment analysis. Finally, we validated the accuracy of DIA quantification by performing immunohistochemical experiments on radical resection pathology specimens from 29 patients diagnosed with GG1 and 29 patients diagnosed with Excluding 3 group. Results: At the genomic level, we found that the mutation frequencies of GG1 and Excluding 3 group were comparable. But, the mutation frequencies of TP53, FOXA1 and PTEN were significantly higher in the Excluding 3 group than in the GG1 group , while the TMPRSS2-ERG fusion was not significantly different between the two groups. And the incidence of the p53 signaling pathway, WNT pathway, PI3K pathway, and DNA damage was significantly higher in the Excluding 3 group than in the GG1 group. For the transcriptomic level, GSEA enrichment analysis revealed that both groups were enriched for multiple tumor-related pathways. At the proteome level, with protein quantitative testing by mass spectrometry, we identified that androgen-response pathway was inhibited in the GG1 group, while DNA repair and PPAR signaling pathway was activated in the Excluding 3 group. Six differential proteins were identified which had important roles in cancer development. And immunohistochemical experiments confirmed the accuracy of quantification analysis for 3 representative proteins (DDX21, NOLC1, and TIMP1). Conclusion: The GG1 and Excluding 3 groups showed similar oncogene mutation frequencies and chromosomal structural variations. However, shared and distinct pathway alterations were observed in genomic and transcriptomic analyses, indicating common driving factors and differential influences of biological factors. Proteomic analysis identified differentially expressed proteins and enriched cancer-related pathways associated with tumor characteristics. These findings support the malignancy features of GG1 prostate cancer. Citation Format: Sijie Wen, Yinzhao Wang, Lin Qi, Hongling Yin, Xiaomei Gao, Minfeng Chen, Yi Cai. Cancer labeling should be retained for prostate cancer with a Gleason score of 3+3=6 - based on genomic, transcriptomic, and proteomic aspects [abstract]. In: Proceedings of the 16th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2023 Sep 29-Oct 2;Orlando, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2023;32(12 Suppl):Abstract nr C039.

Solid-State Nanopore Sensors with Enhanced Sensitivity through Nucleic Acid Amplification.

Solid-state nanopores have wide applications in DNA sequencing, energy conversion and storage, seawater desalination, sensors, and reactors due to their high stability, controllable geometry, and a variety of pore-forming materials. Solid-state nanopore sensors can be used for qualitative and quantitative analyses of ions, small molecules, proteins, and nucleic acids. The combination of nucleic acid amplification and solid-state nanopores to achieve trace detection of analytes is gradually attracting attention. This review outlines nucleic acid amplification strategies for enhancing the sensitivity of solid-state nanopore sensors by summarizing the articles published in the past 10 years. The future development prospects and challenges of nucleic acid amplification in solid-state nanopore sensors are discussed. This review helps readers better understand the field of solid-state nanopore sensors. We believe that solid-state nanopore sensors will break through the bottleneck of traditional detection and become a powerful single-molecule detection platform.

Label-free quantitative proteomic analysis of milk fat globule membrane proteins in porcine colostrum and mature milk.

Milk fat globule membrane (MFGM) proteins are nutritional components with various biological functions. This study aimed to analyze and compare MFGM proteins in porcine colostrum (PC) and porcine mature milk (PM), via label-free quantitative proteomics. In total, 3917 and 3966 MFGM proteins were identified in PC and PM milk, respectively. A total of 3807 common MFGM proteins were found in both groups, including 303 significant differentially expressed MFGM proteins. Gene Ontology (GO) analysis revealed that the differentially expressed MFGM proteins were mainly related to the cellular process, cell, and binding. The dominant pathway of the differentially expressed MFGM proteins was related to the phagosome according to Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. These results reveal crucial insights into the functional diversity of MFGM proteins in porcine milk during lactation and provide theoretical guidance for the development of MFGM proteins in the future.

Untargeted metabolomics and quantitative label-free proteomics analysis of whole milk protein from Chinese native buffaloes

ABSTRACT This study aimed to analyze metabolite abundances and proteome differences between buffalo milk from Binglangjiang (BBM) and Dehong (DBM). Untargeted UPLC-MS/MS, label-free proteomics approach, and bioinformatics analyses including GO, KEGG, and PPI were performed. The results revealed that catechol, L-proline, γ-aminobutyric acid, hippuric acid, creatinine, and betaine were detected in BBM and DBM as the major metabolites. One hundred thirteen proteins, mainly within the molecular weight range of 20–80 kDa, were identified, and the differential protein numbers of BBM were higher than those of DBM. These proteins were primarily involved in cholesterol metabolism, Staphylococcus aureus and Salmonella infections, the peroxisome proliferator-activated receptor signaling pathway, and coagulation cascades. Relative abundances of α-s1-casein, α-s2-casein, and β-casein were plentiful in BBM, while κ-casein, albumin, and α-lactalbumin were in DBM. PPI network analysis indicated that the identified proteins were highly interconnected. These findings promote a better understanding of quality biomarkers in dairy product improvements.

Employing the Anchor DSPE-PEG as a Redox Probe for Ratiometric Electrochemical Detection of Surface Proteins on Extracellular Vesicles with Aptamers.

Quantitative analysis of surface proteins on extracellular vesicles (EVs) has been considered to be a crucial approach for reflecting the status of diseases. Due to the diverse composition of surface proteins on EVs and the interference from nonvesicular proteins, accurately detecting the expression of surface proteins on EVs remains a challenging task. While membrane affinity molecules have been widely employed as EVs capture probes to address this issue, their inherent biochemical properties have not been effectively harnessed. In this paper, we found that the electrochemical redox activity of the DSPE-PEG molecule was diminished upon its insertion into the membrane of EVs. This observation establishes the DSPE-PEG molecule modified on the Au electrode surface as a capture and a redox probe for the electrochemical detection of EVs. By utilizing methylene blue-labeled aptamers, the targeted surface proteins of EVs can be detected by recording the ratio of the oxidation peak current of methylene blue and DSPE-PEG. Without complicated signal amplification, the detection limit for EVs is calculated to be 8.11 × 102 particles/mL. Using this platform, we directly analyzed the expression of CD63 and HER2 proteins on the surface of EVs in human clinical plasma samples, demonstrating its significant potential in distinguishing breast cancer patients from healthy individuals.

Proteomic analysis of lysine 2-hydroxyisobutyryl in SLE reveals protein modification alteration in complement and coagulation cascades and platelet activation Pathways

BackgroundPost-translational modifications (PTMs) are considered to be an important factor in the pathogenesis of Systemic lupus erythematosus (SLE). Lysine 2-hydroxyisobutyryl (Khib), as an emerging post-translational modification of proteins, is involved in some important biological metabolic activities. However, there are poor studies on its correlation with diseases, especially SLE.ObjectiveWe performed quantitative, comparative, and bioinformatic analysis of Khib proteins in Peripheral blood mononuclear cells (PBMCs) of SLE patients and PBMCs of healthy controls. Searching for pathways related to SLE disease progression and exploring the role of Khib in SLE.MethodsKhib levels in SLE patients and healthy controls were compared based on liquid chromatography tandem mass spectrometry, then proteomic analysis was conducted.ResultsCompared with healthy controls, Khib in SLE patients was up-regulated at 865 sites of 416 proteins and down-regulated at 630 sites of 349 proteins. The site abundance, distribution and function of Khib protein were investigated further. Bioinformatics analysis showed that Complement and coagulation cascades and Platelet activation in immune-related pathways were significantly enriched, suggesting that differentially modified proteins among them may affect SLE.ConclusionKhib in PBMCs of SLE patients was significantly up- or down-regulated compared with healthy controls. Khib modification of key proteins in the Complement and coagulation cascades and Platelet activation pathways affects platelet activation and aggregation, coagulation functions in SLE patients. This result provides a new direction for the possible significance of Khib in the pathogenesis of SLE patients.

Development of a Microfluidic Flow Cytometer with a Uniform Optical Field (Uni-μFCM) Enabling Quantitative Analysis of Single-Cell Proteins and Its Applications in Leukemia Gating, Tumor Classification, and Hierarchy of Cancer Stem Cells.

Fast and quantitative estimation of single-cell proteins with various distribution patterns remains a technical challenge. Here, a microfluidic flow cytometer with a uniform optical field (Uni-μFCM) was developed, which enabled the translation of multicolor fluorescence signals of bound antibodies into targeted protein numbers with arbitrary distributions of biological cells. As the core of Uni-μFCM, a uniform optical field for optical excitation and fluorescence detection was realized by adopting a microfabricated metal window to shape the optical beam for excitation, which was modeled and validated by both numerical simulation and experimental characterization. After the validation of Uni-μFCM in single-cell protein quantification by measuring single-cell expressions of three transcriptional factors from four cell lines of variable sizes and origins, Uni-μFCM was applied to (1) quantify membrane and cytoplasmic markers of myeloid and lymphocytic leukocytes to classify cell lines and normal and patient blood samples; (2) measure single-cell expressions of key cytokines affiliated with gene stabilities, differentiating paired oral and colon tumor cell lines with varied malignancies, and (3) quantify single-cell stemming markers of liver tumor cell lines, cell subtypes, and liver patient samples to determine a variety of lineage hierarchy. By quantitatively assessing complex cellular phenotypes, Uni-μFCM substantially expanded the phenotypic space accessible to single-cell applications in leukemia gating, tumor classification, and hierarchy determination of cancer stem cells.