Protein Quantification Research Articles

Combination of rituximab and low-dose glucocorticoids for idiopathic refractory nephrotic syndrome with MCD/FSGS: a single-center prospective cohort study

Background The treatment of idiopathic refractory nephrotic syndrome (IRNS) remains a difficult problem in clinical practice. This study aims to determine the efficacy and safety of combining low-dose glucocorticoids with rituximab in IRNS treatment. Methods This prospective, single-center cohort study enrolled 60 patients who were diagnosed with refractory IRNS with minimal change disease (MCD) or focal segmental glomerulosclerosis (FSGS) and treated at First Affiliated Hospital of Sun Yat-sen University. All patients received a treatment regimen consisting of rituximab (375 mg/m2/week × 4) and low-dose glucocorticoids. Results 46 complete remissions and 4 partial remissions were observed within 6 months of treatment. Within 12 months of treatment, 48 patients achieved complete remission, and 4 achieved partial remission. The complete remission rate for steroid-dependent/frequently relapsing nephrotic syndrome was significantly higher than that for steroid-resistant nephrotic syndrome (88.24% vs. 33.33%, p < .01). Following up for 12 months, 16 patients relapsed, accounting for 30.76% of the total, with a mean time to relapse of 10.97 months. Compared with baseline data, 24-hour urine protein quantification, total cholesterol and triglycerides significantly decreased, while serum albumin, globulin and IgG significantly increased at 12 months after treatment. All follow-ups were without serious adverse events. Twenty-four patients experienced infusion-related adverse reactions, which could be relieved by slowing down the infusion rate or suspending the infusion. Nine patients experienced infection-related adverse reactions; six of them were relieved with antibiotic treatment, and 3 patients were controlled by symptomatic treatment. Conclusions Rituximab combined low-dose glucocorticoids therapy is effective and safe in idiopathic refractory nephrotic syndrome.

Simultaneous Protein Quantitation and Glycosylation Profiling of Antigen-Specific Immunoglobulin G1 in Large Clinical Studies.

Antibodies have a key role in the immune system, making their characterization essential to biomedical, biopharmaceutical, and clinical research questions. Antibody effector functions are mainly controlled by quantity, subclass, and Fc glycosylation. We describe an integrated method to measure these three critical dimensions simultaneously. The subclass-specific immunoglobulin G (IgG) Fc glycosylation analysis combines immunosorbance with glycopeptide-centered LC-MS detection. For integrated IgG1-specific quantitation, a commercial, stable isotope labeled IgG1 protein standard was spiked into the immunosorbent eluates. Robust quantitation was achieved, relying on a combination of a proteotypic peptide and the most abundant glycopeptides, generated through proteolytic cleavage from a mixture of natural IgG1 and the recombinant IgG1 standard. Method performance was demonstrated in a large coronavirus vaccination cohort at a throughput of 100 samples/day. LC-MS-derived, anti-SARS-CoV-2 spike protein IgG1 concentrations ranged from 100 to 10000 ng/mL and correlated well with a clinically relevant immunoassay. Technical variation was 200 times lower than biological variation; intermediate precision was 44%. In conclusion, we present a method capable of robustly and simultaneously assessing quantity, subclass, and Fc glycosylation of antigen-specific IgG in large clinical studies. This method will facilitate a broader understanding of immune responses, especially the important interplay among the three dimensions.

Abstract A036: Next gen liquid biopsy: Comprehensive analysis from a single tube of blood

Abstract Introduction There remains a largely untapped potential for utilizing liquid biopsy as a non- invasive, real-time window into tumor biology. In this study, we report on the use of the high- multiplex protein quantitation capability of the OrionTM spatial biology platform (RareCyte®, Inc) and cell picking capability of the CyteFinder® rare cell platform, to perform a comprehensive liquid biopsy analysis of whole blood that provides a deeper understanding of samples. This novel technology was used to characterize circulating tumor cells (CTCs), perform immune profiling of white blood cells (WBCs), and perform targeted mutation profiling of CTCs and cell-free DNA (cfDNA). All of these analytes can be measured from one 7.5 mL draw of blood. This array of tests was applied to cancer samples to demonstrate clinical feasibility. MethodsBlood samples were collected into AccuCyte® blood collection tubes. Buffy coats were processed to microscope slides using AccuCyte® blood processing kits. Slides were fixed and stained with an 18-plex immuno-oncology panel and imaged using the Orion Spatial Biology platform to evaluate protein biomarker expression on CTCs and to profile WBCs. Individual CTCs and WBCs from clinical samples were isolated using the CytePicker® for downstream molecular analysis. cfDNA was isolated from plasma collected during the AccuCyte process. Picked CTCs, WBCs, and cfDNA were sequenced using the CleanPlex® OncoZoom® Cancer Hotspot Kit. ResultsCancer patient blood was tested with a high-plex immunofluorescence assay using the Orion spatial biology system. CTCs were identified and phenotypically characterized in multiple cancer types. On the same slides, immunophenotyping of WBCs was performed. T- cell, B-cell, and macrophage populations were quantified. Additional sub-populations of T-cells (regulatory and memory) and proliferating cells (Ki67+) were enumerated. Expression of checkpoint-specific markers PD-L1 and PD-1 was measured on CTCs and WBCs. Sequencing results from individual picked CTCs showed evidence of minor clones representative of tumor heterogeneity that were not detectable in the cfDNA analysis. Conclusions This novel application of a high-plex spatial biology imaging system to liquid biopsies, in conjunction with the proven capabilities of rare cell detection and genomic analysis of single cells and cfDNA, has the potential to revolutionize liquid biopsy testing. From a single blood sample, this approach allows one to ask important questions about the complex interactions between the immune system and the tumor and to follow these interactions longitudinally in real time over the course of the disease. The goal is to leverage this unprecedented depth of analysis to improve patient outcomes and better understand the biological complexity of disease. Future directions of this work are to study rare types of WBCs that indicate immune activation and response to the tumor, and the detection and longitudinal monitoring of cell therapies such as CAR T cells after infusion. Citation Format: Arturo B Ramirez, Jon Ladd, Erin Bayer, Brock Bartels, Rachel Ponting, Arista Tischner. Next gen liquid biopsy: Comprehensive analysis from a single tube of blood [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr A036.

Abstract 4117594: Fetal Synthetic Glucocorticoid (sGC) Exposure Results in Later Life Cardiac Dysfunction and Ventricular Remodeling in Male Baboon Offspring

The “theory of developmental origins of health and disease” posits that stress in critical developmental periods will program life-course health and may affect cardiovascular (CV) health. Mothers at risk for premature delivery receive synthetic glucocorticoids (sGC) to enhance fetal lung maturation and manage fetal respiratory distress, but sGC exposure is linked to long-term CV dysfunction in offspring. Four groups of male baboons were studied: sGC exposed in utero at middle age (sGC-MA, 13.8±0.2Y), older sGC exposed in utero (sGC-O, 17.1±0.4Y), middle age unexposed (CTR-MA, 13.5±0.6Y), and older, unexposed baboons (CTR-O, 17.4±0.4Y). Gated, breath-hold, cine cardiac magnetic resonance imaging was performed at 3 Tesla (Siemens TIM Trio) to assess left ventricular (LV) function and myocardial strain, analyzed using cvi42® software. Mitochondrial electron transport chain (ETC) complex activity was analyzed using LV tissue from sGC-O and CTR-O euthanized baboons. Protein quantification was used to measure mitochondrial function and RNA-seq assessed differential gene expression and gene ontology (GO) enrichment. Unpaired t-tests were used. Data are reported as mean ± SD. Stroke volume and ejection fraction were similar in the elderly groups (4 sGC-O, 8 CTR-O). However, higher end-systolic and end-diastolic sphericity indexes (SI) and shorter LV axis lengths indicated remodeling. Also, global longitudinal and radial strains were reduced (both p&lt;0.04) in sGC-O vs. CTR-O. Reduced LV cardiac output (p=0.005) was found in 5 sGC-MA animals vs 4 sGC-O animals, but not in 5 CTR-MA vs 8 CTR-O. In sGC-MA vs sGC-O, but not CTR-MA vs CTR-O, significant increases in SI (p=0.001), radial strain (p=0.02), and longitudinal (p=0.03) strain were found, but LV axis length (p=0.03) was reduced. LV mitochondrial analysis in 4 sGC-O vs 4 CTR-O myocardial samples revealed 33.5% less ETC activity (p=0.002) and 19.8% less complex I expression (p=0.048) in sGC. LV RNA-seq identified 63 upregulated and 20 downregulated genes in the sGC-O samples. GO pathway enrichment included changes in heart contraction, wounding responses, and leukocyte regulation pathways. These findings indicate that fetal sGC exposure developmentally programs offspring with LV remodeling and long-term cardiac and gene expression changes in male baboons, the closest available experimental nonhuman primate species to man. Future studies on females will probe sexual dimorphism in developmental programming.

Kidney mRNA-protein expression correlation: what can we learn from the Human Protein Atlas?

The Human Protein Atlas, with more than 10 million immunohistochemical images showing tissue- and cell-specific protein expression levels and subcellular localization information, is widely used in kidney research. The Human Protein Atlas contains comprehensive data on multi-tissue transcript and protein abundance, allowing for comparisons across tissues. However, while visual and intuitive to interpret, immunohistochemistry is limited by its semi-quantitative nature. This can lead to mismatches in protein expression measurements across different platforms. We performed a comparison of the Human Protein Atlas' kidney-specific RNA sequencing and immunohistochemistry data to determine whether the mRNA and protein abundance levels are concordant. Our study shows that there is a discordance between mRNA and protein expression in the kidney based on the Human Protein Atlas data. Using an external validation mass spectrometry dataset, we show that more than 500 proteins undetected by immunohistochemistry are robustly measured by mass spectrometry. The Human Protein Atlas transcriptome data, on the other hand, exhibit similar transcript detection levels as other kidney RNA-seq datasets. Discordance in mRNA-protein expression could be due to both biological and technical reasons, such as transcriptional dynamics, translation rates, protein half-lives, and measurement errors. This is further complicated by the heterogeneity of the kidney tissue itself, which can increase the discordance if the cell populations or tissue compartment samples do not match. As such, shedding light on the mRNA-protein relationship of the kidney-specific Human Protein Atlas data can provide context to our scientific inferences onrenal gene and protein quantification.

Determination of the Biochemical Compositions of Stored Expressed Human Breast Milk of Healthy Lactating Mothers

Aims: This study is aimed at determining the biochemical compositions and immunological components of expressed human breast milk (HBM) gotten from healthy lactating mothers stored under varying low temperatures. Place and Duration of Study: Department of Microbiology, University of Benin, Benin, Edo State; and Quick Diagnostic Centre, Umuahia, Abia State. It was a three (3) months study. Methodology: Sixty (60) samples of expressed HBM were collected from healthy lactating mothers (at varying ages) in Abia State, Nigeria. Employing basic biochemical analyses, the quantification of total protein, glucose, and triacylglycerol levels, in addition to the confirmation tests of vitamin, minerals, and immune cells etc, were carried out. Results: The findings of this study demonstrated that triacylglycerol levels were constantly higher than glucose and protein level and this clearly was manifested across the ages of the lactating mothers (15 - 25 years, 26 - 35 years, and 36 - 45 years). Under room temperature and varying refrigeration, the changes in the biochemical compositions in the expressed HBM were also evaluated. Conclusion: From our findings, expressed HBM shows good nutritive profile with various bioactive factors that are essential for supporting infant growth, and immunity. Although references demonstrated that storage conditions can impact the nutritional value of expressed HBM, the result of this study was suggestive of expressed HBM having the ability to retain good amount of its bioactive compounds when refrigerated to 24 hours, ensuring that microbial infestations were properly controlled.

Effectiveness and safety of enzyme replacement therapy in the treatment of Fabry disease: a Chinese monocentric real-world study.

To assess the effectiveness and safety of enzyme replacement therapy (ERT) for treating Fabry disease in clinical practice. The clinical data of patients with Fabry disease were retrospectively collected and screened according to inclusion and exclusion criteria. The effectiveness of ERT was evaluated by analyzing the improvement in renal dysfunction (decreased estimated glomerular filtration rate (eGFR) and proteinuria), cardiac system injury (mainly increased left ventricular mass index (LVMI)), and neuropathic pain after ERT treatment. The safety of ERT was measured by summarizing the occurrence of adverse events (AE) and adverse drug reactions (ADR) before and after ERT. Sixteen patients with Fabry disease who underwent ERT treatment 2-36 times over a period of 2-89weeks were enrolled in the study. Among them, 13 received symptomatic treatment based on the involvement of various organs, 14 were treated with anti-inflammatory and anti-allergic drugs, and 16 had no AE or ADR. After ERT, there was no significant difference in (eGFR, microalbumin (mALB), 24h urinary protein quantitation (24h PRO), urinary albumin/creatinine ratio (ACR), uric acid (UA), and β2 microglobulin (β2MG) (P > 0.05), and the renal function remained stable or improved; ERT could significantly reduce left ventricular mass index (LVMI) (P = 0.043) and lactate dehydrogenase (LDH) (P = 0.031), and other cardiac function indexes had an improvement trend or remained stable, but the difference was not significant (P > 0.05). After ERT, the degree of limb pain in three of the four minor patients improved. ERT could effectively stabilize or improve renal and cardiac function and relieve neuropathic pain in patients with Fabry disease, and no AE occurred during treatment, and the clinical effectiveness and safety were satisfactory.

Celecoxib and rofecoxib have different effects on small intestinal ischemia/reperfusion injury in rats

IntroductionIntestinal ischemia/reperfusion (I/R) injury is associated with high mortality and there is an unmet need for novel therapies. The intestinal expression of cyclooxygenase-2 (COX-2) increases rapidly after mesenteric I/R, but it is still a question of debate whether selective COX-2 inhibitors can mitigate I/R-induced gut injury. Here we aimed to compare the effect of celecoxib and rofecoxib, two selective COX-2 inhibitors, on intestinal I/R-induced injury in rats.MethodsWistar rats were treated with celecoxib (10 and 100 mg/kg), rofecoxib (5 and 50 mg/kg), or vehicle for 8 days via gavage and then were subjected to sham operation or mesenteric I/R. Small intestinal inflammation and tissue damage were assessed by histology and quantification of inflammatory and tight junction proteins. The intestinal activity of COX enzymes was determined by a COX activity assay.ResultsThe higher dose of celecoxib reduced the I/R-associated increase in inflammatory mediators (myeloperoxidase, pentraxin 3, COX-2, interleukin-1β) and loss of tight junction proteins (claudin-1, occludin), whereas the lower dose of celecoxib was only marginally effective. However, even high-dose celecoxib failed to prevent the histological injury of the mucosa. In contrast to celecoxib, rofecoxib did not affect intestinal inflammation and injury at any of the tested doses. Neither celecoxib nor rofecoxib affected the I/R-induced changes of HO-1 and PPAR-γ, known off-targets of COX-inhibitors, but celecoxib increased the I/R-induced elevation of Bax/Bcl-2, a marker of apoptosis, whereas rofecoxib reduced the elevation of phospho-Akt. Importantly, high-dose celecoxib, but not rofecoxib, has already reduced intestinal COX-1 activity.ConclusionOur study provides evidence for the higher anti-inflammatory efficacy of celecoxib compared to rofecoxib in mesenteric I/R injury, which is likely due to its lower selectivity for COX-2. However, even high-dose celecoxib was unable to reduce the mucosal damage. Our results suggest that selective COX-2 inhibitors have only limited therapeutic value in intestinal I/R injury.

In Situ and Label-Free Quantification of Membrane Protein–Ligand Interactions Using Optical Imaging Techniques: A Review

Membrane proteins are crucial for various cellular processes and are key targets in pharmacological research. Their interactions with ligands are essential for elucidating cellular mechanisms and advancing drug development. To study these interactions without altering their functional properties in native environments, several advanced optical imaging methods have been developed for in situ and label-free quantification. This review focuses on recent optical imaging techniques such as surface plasmon resonance imaging (SPRi), surface plasmon resonance microscopy (SPRM), edge tracking approaches, and surface light scattering microscopy (SLSM). We explore the operational principles, recent advancements, and the scope of application of these methods. Additionally, we address the current challenges and explore the future potential of these innovative optical imaging strategies in deepening our understanding of biomolecular interactions and facilitating the discovery of new therapeutic agents.

Thapsigargin: An Unlikely Drug in the Battle Against Viral Pandemics

Antiviral resistance is an increasingly categorized phenomenon, and with the high pandemic potential of emergent pathogens such as coronaviruses, the need for novel therapeutic agents is dire. Thapsigargin, a potent endoplasmic reticulum stress inducer found in the Mediterranean region, has shown significant promise in combatting replication of several viruses including coronaviruses by harnessing the host's innate immune system, even being more efficacious than some antivirals currently used. There is a major gap in knowledge regarding the exact mechanism of action that permits Thapsigargin to be an incredible endoplasmic reticulum stress inducer and an potent antiviral. We hypothesize that interferon 1 production and SERCA-inhibition caused by thapsigargin mediates the antiviral effects observed. This review outlines what is currently known about this novel compound, the gap in research, and details methodologies including gene expression analyses, protein quantification, and mammalian cell culturing, to address the gap and answer the hypothesis.

Infrared Laser Ablation and Capture of Formalin-Fixed Paraffin-Embedded Tissue.

Formalin-fixed paraffin-embedded (FFPE) tissue is a ubiquitous and invaluable resource for biomedical research and clinical applications. However, FFPE tissue proteomics is challenging due to protein cross-linking and chemical modification. Laser ablation sampling allows precise removal of material from tissue sections with high spatial control and reproducibility for offline proteomics by liquid chromatography coupled with tandem mass spectrometry. In this work, we used a pulsed mid-infrared laser for microsampling of rat liver tissue for subsequent identification and quantification of proteins. It was found that more proteins were identified by FFPE tissue laser ablation sampling compared to fresh frozen (FF) tissue laser ablation sampling and that more proteins were identified by laser ablation than by manual dissection of FFPE tissue. In contrast to previous studies, no loss of hydrophilic proteins due to residual cross-linking was observed. The efficient capture of proteins by laser ablation microsampling is attributed to efficient laser breakup of the tissue which facilitates downstream processing of the proteins.

Protein aggregation in plant mitochondria lacking Lon1 inhibits translation and induces unfolded protein responses.

Loss of Lon1 led to stunted plant growth and accumulation of nuclear-encoded mitochondrial proteins including Lon1 substrates. However, an in-depth label-free proteomics quantification of mitochondrial proteins in lon1 revealed that the majority of mitochondrial-encoded proteins decreased in abundance. Additionally, we found that lon1 mutants contained protein aggregates in the mitochondrial that were enriched in metabolic enzymes, ribosomal subunits and PPR-containing proteins of the translation apparatus. These mutants exhibited reduced general mitochondrial translation as well as deficiencies in RNA splicing and editing. These findings support the role of Lon1 in maintaining a functional translational apparatus for mitochondrial-encoded gene translation. Transcriptome analysis of lon1 revealed a mitochondrial unfolded protein response reminiscent of the mitochondrial retrograde signalling dependent on the transcription factor ANAC017. Notably, lon1 mutants exhibited transiently elevated ethylene production, and the shortened hypocotyl observed in lon1 mutants during skotomorphogenesis was partially alleviated by ethylene inhibitors. Furthermore, the short root phenotype was partially ameliorated by introducing a mutation in the ethylene receptor ETR1. Interestingly, the upregulation of only a select few target genes was linked to ETR1-mediated ethylene signalling. Together this provides multiple steps in the link between loss of Lon1 and signalling responses to restore mitochondrial protein homoeostasis in plants.

A Workflow for Accurate and Consistent Quantitation of Host Cell Proteins by SWATH LC-MS/MS Analysis to Support Process Development

A Workflow for Accurate and Consistent Quantitation of Host Cell Proteins by SWATH LC-MS/MS Analysis to Support Process Development

Berberine inhibits the malignant cell phenotype by inactivating PI3K/AKT/mTOR signaling in laryngeal squamous cell carcinoma.

Berberine is an active compound found in different herbs used in Chinese medicine and is well-known for its potential anticancer properties. The study aimed to figure out the role of berberine in regulating the malignant behavior of laryngeal squamous cell carcinoma (LSCC) cells. LSCC cell lines (SNU-899 and AMC-HN-8) were treated with different concentrations of berberine (0-200 μM) to determine its cytotoxicity. The migration, invasion, and apoptosis of LSCC cells were measured by wound healing assays, Transwell assays, and flow cytometry. Western blot was performed for the quantification of proteins involved in PI3K/AKT/mTOR signaling. The viability of LSCC cells was dose-dependently reduced by berberine. Berberine dampened LSCC cell migration and invasion while augmenting cell apoptosis, as evidenced by a reduced wound closure rate, a decrease in invaded cell number, and a surge in cell apoptosis in the context of berberine stimulation. Importantly, the effects of berberine on the cancer cell process were enhanced by LY294002 (an inhibitor for PI3K) treatment. Moreover, the protein levels of phosphorylated PI3K, AKT, and mTOR were markedly reduced in response to berberine treatment. Berberine inhibits cell viability, migration, and invasion but augments cell apoptosis by inactivating PI3K/AKT/mTOR signaling in LSCC.

Tuning the properties of peptide imprinted nanoparticles for protein immunoprecipitation using magnetic streptavidin beads

Maximizing the binding properties of thermoresponsive molecularly imprinted nanoparticles (MIN) was aimed to explore their feasibility as antibody substitutes in protein immunoprecipitation (IPP) with magnetic streptavidin beads (MSB). Thermoresponsive MIN targeting the cannabinoid CB1 receptor were produced by epitope imprinting through solid-phase synthesis. It was intended to determine how different variables influenced physicochemical features, binding behaviour and immunoprecipitation of the target recombinant glutathione S-transferase tagged fusion protein (GST-CTer). Such variables included the cross-linking degree of MIN, and variables like pH, temperature or the use of Tween-20 for binding and IPP experiments. The cross-linker (CL) amount influenced the coil-to-globule transition of thermoresponsive MIN, making the lower critical solution temperature (LCST) decrease from 37.2 °C using 5% of CL, to 29.0 °C using 25%, also suggesting higher plasticity on the former. Temperature influence on size was corroborated by dynamic light scattering, observing size reductions from 250–450 nm (RT) to 70–100 nm (> LCST) for MIN produced with 5–15% of CL. However, binding behaviour did not clearly improve for more than 10% CL. Further experiments revealed that temperature and pH control were critical for efficient binding and release, selecting 40 °C and pH 5 as appropriate. Following binding experiments, the GST-CTer-MIN complex was successfully immunoprecipitated using MSB, achieving an IPP efficiency of 11.48% over the initial input protein concentration, which was calculated after SDS-PAGE separation and Western blot analysis. The methodology may be exploited for selective protein extraction and quantification from complex tissue homogenates.Graphical

Suspension Bead Loading (SBL): An Economical Protein Delivery Platform to Study URM1’s Behavior in Live Cells

AbstractUniquely modified synthetic proteins are difficult to produce in large quantities, which could limit their use in various in vitro settings and in cellular studies. In this study, we developed a method named “suspension bead loading” (SBL), to deliver protein molecules into suspended living cells using glass beads, which significantly reduces the amount of protein required for effective delivery. We investigated the delivery efficiency of functionally different proteins and evaluated the cytotoxic effect of our method and the chemical and functional integrity of the delivered protein. We utilized SBL to address questions related to ubiquitin‐related modifier 1 (URM1). Employing minimal protein quantities, SBL has enabled us to study its behavior within live cells under different redox conditions, including subcellular localization and conjugation patterns. We demonstrate that oxidative stress alters both the localization and conjugation pattern of URM1 in cells, highlighting its possible role in cellular response to such extreme conditions.

Strategies to verify equimolar peptide release in mass spectrometry-based protein quantification exemplified for apolipoprotein(a).

Quantitative protein mass spectrometry (MS) is ideally suited for precision diagnostics and for reference standardization of protein analytes. At the Leiden Apolipoprotein Reference Laboratory we apply MS strategies to obtain detailed insight into the protein-to-peptide conversion in order to verify that quantifier peptides are not partly concealed in miscleaved protein backbone. Apolipoprotein(a) (apo(a)) was digested in a non-optimal manner to enhance the number of miscleaved peptides that were identified by high resolution liquid chromatography tandem-MS measurements. The protein-to-peptide conversion was carefully mapped with specific attention for miscleaved peptides that contain an apo(a) quantifier peptide. Four different isotopologues of each apo(a)-quantifier peptide were applied to evaluate linearity of internal peptide standards during measurement of specific real-life samples. Two apo(a) quantifier peptides that were concealed in two different miscleaved peptides were included into a multiple reaction monitoring list in our targeted MS-based apo(a) quantifications to alert for potential protein digestion discrepancies. The presence of miscleaved peptides could be ruled out when applying our candidate reference measurement procedure (RMP) for apo(a) quantification. These data further corroborate the validity of our apo(a) candidate RMP as higher order method for certification of commercial Lp(a) tests that is endorsed by theInternational Federation of Clinical Chemistry and Laboratory Medicine. MS-based molecular detection and quantification of heterogeneous apo(a) proteoforms will allow manufacturers' transitioning from confounded lipoprotein(a) [Lp(a)] mass levels into accurate molar apo(a) levels.

Preparation and characterization of bovine dental pulp-derived extracellular matrix hydrogel for regenerative endodontic applications: an in vitro study

BackgroundThe use of biological scaffolds in regenerative endodontics has gained much attention in recent years. The search for a new biomimetic scaffold that contains tissue-specific cell homing factors could lead to more predictable tissue regeneration. The aim of this study was to prepare and characterize decellularized bovine dental pulp-derived extracellular matrix (P-ECM) hydrogels for regenerative endodontic applications.MethodsFreshly extracted bovine molar teeth were collected. Bovine dental pulp tissues were harvested, and stored at -40º C. For decellularization, a 5-day protocol was implemented incorporating trypsin/EDTA, deionized water and DNase treatment. Decellularization was evaluated by DNA quantification and histological examination to assess collagen and glycosaminoglycans (GAGs) content. This was followed by the preparation of P-ECM hydrogel alone or combined with hyaluronic acid gel (P-ECM + HA). The fabricated scaffolds were then characterized using protein quantification, hydrogel topology and porosity, biodegradability, and growth factor content using Enzyme-linked immunosorbent assay (ELISA): transforming growth factor beta-1(TGF-β1), basic fibroblast growth factor (bFGF), bone morphogenetic protein 2 (BMP-2) and vascular endothelial growth factor (VEGF).ResultsDecellularization was histologically confirmed, and DNA content was below (50 ng/mg tissue). P-ECM hydrogel was prepared with a final ECM concentration of 3.00 mg/ml while P-ECM + HA hydrogel was prepared with a final ECM concentration of 1.5 mg/ml. Total protein content in P-ECM hydrogel was found to be (439.0 ± 123.4 µg/µl). P-ECM + HA showed sustained protein release while the P-ECM group showed gradual decreasing release. Degradation was higher in P-ECM + HA which had a significantly larger fiber diameter, while P-ECM had a larger pore area percentage. ELISA confirmed the retention and release of growth factors where P-ECM hydrogel had higher BMP-2 release, while P-ECM + HA had higher release of TGF-β1, bFGF, and VEGF.ConclusionsBoth P-ECM and P-ECM + HA retained their bioactive properties demonstrating a potential role as functionalized scaffolds for regenerative endodontic procedures.

Metal-Organic Framework Sub-Nanochannels within the Confined Micropipettes: Precise Construction Makes It a Universal Aptamer-Based Sensing Platform.

It is crucial to precisely construct metal-organic framework (MOF) sub-nanochannels at the tip of micro/nanopipettes for fundamental research and sensing applications. The quality of the MOF modification plays a significant role in influencing subsequent research, particularly in sensing applications. In this work, we present a precise method of constructing MOF sub-nanochannels at the tip of glass micropipettes, which serve as a universal aptamer-based sensing platform for the selective detection of proteins. In situ scanning electron microscopy (SEM), energy-dispersive spectroscopy (EDS) mapping, and fluorescence microscopy results demonstrate that the synthesized MOF (UiO-66) nanocrystals fully block the orifice of glass micropipettes (UiO-66-GMs) without forming any nanometer-scale cracks and remain confined within the geometric boundaries of the orifice. The terminal phosphate-modified aptamer readily binds to the surface of UiO-66-GMs through metal (Zr)-phosphate coordination, ultimately forming the aptamer sensor (Apt-UiO-66-GMs). The selective quantification of proteins is achieved via a decrease in current resulting from protein binding to the aptamer. Our results indicate that the precisely constructed Apt-UiO-66-GMs sensor enables highly selective and sensitive detection of SARS-CoV-2 nucleocapsid protein and holds potential for real sample detection. Furthermore, given the sharp tip of the micropipets and the external sensing interface we have constructed, our aptamer-based sensing platform also opens avenues for single-cell analysis and in vivo sensing.

A Proteomics Pipeline for Generating Clinical Grade Biomarker Candidates from Data-Independent Acquisition Mass Spectrometry (DIA-MS) Discovery.

Clinical biomarker development has been stymied by inaccurate protein quantification from mass spectrometry (MS) discovery data and a prolonged validation process. To mitigate these issues, we created the Targeted Extraction Assessment of Quantification (TEAQ) software package that uses data-independent acquisition analysis from a discovery cohort to select precursors, peptides, and proteins that adhere to analytical criteria required for established targeted assays. TEAQ was applied to DIA-MS data from plasma samples acquired on a new high resolution accurate mass (HRAM) mass spectrometry platform where precursors were evaluated for linearity, specificity, repeatability, reproducibility, and intra-protein correlation based on 8- or 11-point loading curves at three throughputs. This data can be used as a general resource for developing other targeted assays. TEAQ analysis of data from a case and control cohort for inflammatory bowel disease (n=492) identified 1110 signature peptides for 326 quantifiable proteins from the 1179 identified proteins. Applying TEAQ analysis to discovery data will streamline targeted assay development and the transition to validation and clinical studies.