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Results for “multiplexed proteomics”

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PubMed2003

A novel experimental design for comparative two-dimensional gel analysis: two-dimensional difference gel electrophoresis incorporating a pooled internal standard.

The comparison of two-dimensional (2-D) gel images from different samples is an established method used to study differences in protein expression. Conventional methods rely on comparing images from at least 2 different gels. Due to the high variation between gels, detection and quantification of protein differences can be problematic. Two-dimensional difference gel electrophoresis (Ettan trade mark DIGE) is an emerging technique for comparative proteomics, which improves the reproducibility and reliability of differential protein expression analysis between samples. In the application of DIGE different samples are labelled with mass and charge matched spectrally resolvable fluorescent dyes and are then separated on the same 2-D gel. Using an Escherichia coli lysate "spiked" with varying amounts of four different known proteins, we have tested a novel experimental design that exploits the sample multiplexing capabilities of DIGE, by including a standard sample in each gel. The standard sample comprises equal amounts of each sample to be compared and was found to improve the accuracy of protein quantification between samples from different gels allowing accurate detection of small differences in protein levels between samples.

Electrophoresis, Gel, Two-Dimensional↗

PubMed2026

Pathogenic role of serpin B3-positive neutrophils in reinforcing thrombus stiffening during ischemic stroke.

The contribution of immune cells to thrombus architecture and mechanical properties in acute ischemic stroke (AIS) remains poorly understood. Using 3-dimensional imaging and multiplex staining, we mapped immune cells in human stroke thrombi and identified neutrophils as the dominant population. Analysis of 19 thrombi confirmed their positive correlation with collagen, increased stiffness, and poorer clinical outcomes. To preserve spatial context, we developed a laser capture-based proteomic workflow and analyzed thrombus neutrophils from 34 patients with AIS stratified by 90-day outcomes, followed by validation in an independent cohort of 22 patients. Proteomic analysis revealed serpin B3 as a neutrophil-enriched protein strongly correlated with poor prognosis. In murine models of ferric chloride-induced carotid artery thrombosis and middle cerebral artery occlusion, experiments using wild-type, neutrophil-depleted, and Serpinb3a knockout mice demonstrated that neutrophil-derived serpin B3 promotes early thrombus formation, enhances collagen deposition, and contributes to progressive thrombus stiffening. Mechanistically, serpin B3 secreted by neutrophils amplifies thrombus stiffness through upregulation of transforming growth factor β1, neutrophil extracellular traps, and COL1A1. Targeted Serpinb3a knockdown delayed vascular occlusion, improved thrombolysis efficiency, and resulted in better neurological recovery. Collectively, these findings identify a neutrophil-driven mechanism underlying thrombus stiffening and establish SERPINB3 as both a prognostic biomarker and a promising therapeutic target in AIS. This project has been registered with the Chinese Clinical Trial Registration Platform (https://www.chictr.org.cn/index.html) and has successfully passed the review process (registration number: ChiCTR2300077911).

Animals↗

PubMed2006

Proteomic strategies for individualizing therapy of acute myeloid leukemia (AML).

Acute myeloid leukemia (AML) is an aggressive hematological malignancy characterized by accumulating myeloid precursor cells in the bone marrow, with approximately 2-3 months 50% survival if left untreated. With current treatment modalities the five years overall survival hardly exceeds 50%. Cytogenetics and molecular diagnostics guide the clinician to select individualized therapy in certain subsets of AML, achieving long-term survival above 70% of these cases. However, approximately half of the AML patients have no risk stratifying features, and early reports indicate that proteomic approaches may be utilized for disease classification as well as development of novel biomarkers related to prognosis, diagnosis, and choice of therapeutic regimen. Proteomics, here defined as the analysis of all proteins in a cell, in a cell compartment or in a signaling pathway, has probably its greatest potential in investigating pathways that are easily targeted by small molecules or therapeutic antibodies. The major methodological challenges include detection sensitivity in a limited clinical material, a problem that in some cases can be solved through designated multiplexed protein assays based on single cells or cell extracts. In this review we will discuss pharmacoproteomic studies of drugs regulating leukemia specific targets like all-trans retinoic acid, histone deacetylase inhibitors, proteasome inhibitors and tyrosine kinase inhibitors, as well as studies on drug resistance and graft-versus-host studies during stem cell transplantations. These studies indicate new avenues in AML diagnostics, individualized therapy design and therapy response surveillance for the clinician.

Acute Disease↗

PubMed2006

Phosphoproteomics strategies for the functional analysis of signal transduction.

Protein phosphorylation is a key regulatory mechanism of cellular signalling processes. The analysis of phosphorylated proteins and the characterisation of phosphorylation sites under different biological conditions are some of the most challenging tasks in current proteomics research. Reduction of the sample complexity is one major step for the analysis of low-abundance kinase substrates, which can be achieved by various subcellular fractionation techniques. One strategy is the enrichment of phosphorylated proteins or peptides by immunoprecipitation or chromatography, e.g. immobilised metal affinity chromatography, prior to analysis. 2-DE gels are powerful tools for the analysis of phosphoproteins when combined with new multiplexing techniques like DIGE, phosphospecific stains, autoradiography or immunoblotting. In addition, several gel-free methods combining chromatography with highly sensitive MS have been successfully applied for the analysis of complex phosphoproteomes. Recently developed approaches like KESTREL or 'chemical genetics' and also protein microarrays offer new possibilities for the identification of specific kinase targets. This review summarises various strategies for the analyses of phosphoproteins with a special focus on the identification of novel kinase substrates.

Animals↗

PubMed2003

Protein expression profiling arrays: tools for the multiplexed high-throughput analysis of proteins.

The completion of the human genome sequence has led to a rapid increase in genetic information. The invention of DNA microarrays, which allow for the parallel measurement of thousands of genes on the level of mRNA, has enabled scientists to take a more global view of biological systems. Protein microarrays have a big potential to increase the throughput of proteomic research. Microarrays of antibodies can simultaneously measure the concentration of a multitude of target proteins in a very short period of time. The ability of protein microarrays to increase the quantity of data points in small biological samples on the protein level will have a major impact on basic biological research as well as on the discovery of new drug targets and diagnostic markers. This review highlights the current status of protein expression profiling arrays, their development, applications and limitations.

Journal Article↗

PubMed2026

Plasma Proteomics Identifies Thousand-and-One-Amino Acid Kinase 3 as a Potential Biomarker of Rheumatoid Arthritis Activity and a Novel Therapeutic Target.

OBJECTIVE: Bone destruction associated with active rheumatoid arthritis (RA) remains a major therapeutic challenge, with a lack of reliable molecular markers reflecting bone injury. This study aims to identify novel biomarkers linked to bone destruction in active RA through proteomic analysis, providing new strategies for precise monitoring and targeted therapy. METHODS: Data-independent acquisition mass spectrometry was used for proteomic quantification and bioinformatic analysis on plasma samples from 160 patients with RA and 40 healthy controls. Key proteins associated with bone destruction were screened by integrating Sharp scores with synovial single-cell RNA sequencing data and subsequently validated in two independent cohorts (N1 = 50 and N2 = 10) using enzyme-linked immunosorbent assay and multiplex immunohistochemistry. Functional studies were conducted using fibroblast-like synoviocytes (FLSs) in vitro and a collagen-induced arthritis (CIA) mouse model in vivo. RESULTS: A total of 4,998 plasma proteins were identified, with 506 showing significant differential expression between active and remitted RA. Thousand-and-one-amino acid kinase 3 (TAOK3) levels were positively associated with Sharp scores and markedly elevated in patients with active RA. Combining TAOK3 with C-reactive protein improved diagnostic accuracy for active RA (area under the curve = 0.915). High TAOK3 expression was also associated with increased relapse frequency. Functional studies showed that TAOK3 knockdown suppressed the tumor-like phenotype of FLSs and down-regulated matrix metalloproteinase 1/2/3 and cathepsin K, whereas TAOK3 overexpression promoted pannus cell-mediated bone erosion, mitigated by TAOK3-targeted inhibitor. In vivo, its inhibition showed therapeutic effects in CIA mice. CONCLUSION: TAOK3 serves as a potential biomarker for bone destruction in active RA and as a therapeutic target for precision monitoring and intervention.

Arthritis, Rheumatoid↗

PubMed2026

Spatial Multiomics Reveal Insights Into ADC Efficacy.

Antibody-drug conjugates (ADCs) have transformed the therapeutic landscape of solid tumors; however, responses remain heterogeneous and complex to predict. In addition, a growing number of multiple ADC targets are either approved or in late-stage clinical development, such as NECTIN-4, HER2, or TROP2 for metastatic urothelial cancer. Spatial multiomics-representing next-generation methods that couple high-plex RNA sequencing and multiplex protein imaging with precise x-y-z coordinates within tissues-offer a direct way to correlate (ADC) antigen expression, cell state information, and micro-anatomical context with patient treatment outcomes. In this review, we highlight suitability and technological advancements in current spatial transcriptomics and proteomics approaches to decode modes of action and resistance to ADCs and extract biological insights, particularly in metastatic urothelial cancer-and propose an integrative framework that combines spatial readouts with machine and/or deep learning-driven analytics to stratify patients, forecast on- and off-target toxicities, and guide next-generation linker-payload designs or combination therapies.

Humans↗

PubMed2005

High-throughput and multiplexed protein array technology: protein-DNA and protein-protein interactions.

Miniaturized protein arrays address protein interactions with various types of molecules in a high-throughput and multiplexed fashion. This review focuses on achievements in the analysis of protein-DNA and protein-protein interactions. The technological feasibility of protein arrays depends on the different factors that enable the arrayed proteins to recognize molecular partners and on the specificity of the interactions involved. Proteome-scale studies of molecular interactions require high-throughput approaches for both the production and purification of functionally active proteins. Various solutions have been proposed to avoid non-specific protein interactions on array supports and to monitor low-abundance molecules. The data accumulated indicate that this emerging technology is perfectly suited to resolve networks of protein interactions involved in complex physiological and pathological phenomena in different organisms and to develop sensitive tools for biomedical applications.

Cell-Free System↗

PubMed2002

Measuring proteins on microarrays.

A prerequisite of proteomics is the ability to quantify many selected proteins simultaneously. Immunoassays on microarrays are an attractive solution, as equipment and antibodies are available and assays are simple, scalable and reproducible. Recently, considerable progress has been made in this area as evidenced by increased sensitivity and coverage (degree of multiplexing). Routine use of antibody microarrays in research and diagnostic settings will require increased availability of binding reagents, novel signal amplification procedures, inexpensive and robust platforms for microarray production and detection, and turn-key systems for running high-throughput assays.

Animals↗

PubMed2001

Microsphere-based molecular cytometry.

Flow cytometry has recently gained attention as a powerful high-throughput technology for the analysis of molecular interactions. This recognition is mostly because of the development and use of fluorescently distinct microsphere populations. Recent applications of this technology have included detecting analytes, monitoring enzymatic activity, and genotyping SNP. This article expands the list of applications by highlighting nuclear receptor-coactivator peptide binding studies. Nuclear receptors initiate gene transcription by binding to a wide variety of accessory factors. Small molecule ligand binding by nuclear receptors modulates the interaction with some of these cofactors. Because a typical cell contains numerous cofactors, multiplexed analysis of nuclear receptor binding to coactivator peptide-coupled microspheres is a valuable approach to rapidly assess this network of complex interactions. Understanding how ligand binding regulates these interactions should help in the design of improved small molecule therapeutics. The latter part of this article has focused on the recent application of multiplexing protein:protein interactions. Developing protein interaction networks will be paramount as a greater understanding of the human genome is developed. In the future, flow cytometric analysis of fluorescent microspheres may be applied to any array of interacting molecules and may have broader applications in the area of proteomics.

Binding Sites↗

PubMedDate not supplied

Proteomic analysis of human serum by two-dimensional differential gel electrophoresis after depletion of high-abundant proteins.

Two-dimensional differential gel electrophoresis (2-D DIGE) was used to analyze human serum following the removal of albumin and five other high-abundant serum proteins. After protein removal, serum was analyzed by SDS-PAGE as a preliminary screen, and significant differences between four high-abundant protein removal methods were observed. Antibody-based albumin removal and high-abundant protein removal methods were found to be efficient and specific. To further characterize serum after protein removal, 2-D DIGE was employed, enabling multiplexed analysis of serum through the use of three fluorescent protein dyes. Comparison between crude serum and serum after removal of high-abundant proteins clearly illustrates an increase in the number of lower abundant protein spots observed. Approximately 850 protein spots were detected in crude serum whereas over 1500 protein spots were exposed following removal of six high-abundant proteins, representing a 76% increase in protein spot detection. Several proteins that showed a 2-fold increase in intensity after depletion of high-abundant proteins, as well as proteins that were depleted during abundant protein removal methods, were further characterized by mass spectrometry. This series of experiments demonstrates that high-abundant protein removal, combined with 2-D DIGE, is a practical approach for enriching and characterizing lower abundant proteins in human serum. Consequently, this methodology offers advances in proteomic characterization, and therefore, in the identification of biomarkers from human serum.

Biomarkers↗

PubMedDate not supplied

Differential radioactive proteomic analysis of microdissected renal cell carcinoma tissue by 54 cm isoelectric focusing in serial immobilized pH gradient gels.

We present a proof of principle study, using laser microdissection and pressure catapulting (LMPC) of two clinical tissue samples, each containing approximately 3.8 microg renal cell carcinoma protein and 3.8 microg normal kidney protein respectively from one patient. The study involved separate radio-iodination of each sample with both (125)I and (131)I, dual inverse replicate sample loading to high resolution 54 cm "daisy chain" serial immobilized pH gradient isoelectric focusing (IPG-IEF) 2D-PAGE gels, co-electrophoretic separation of cross-labeled proteins from different samples, and precision multiplex differential radioactive imaging to obtain signals specific for each sample coelectrophoresed within single gels but labeled with different isotopes of iodine, providing extremely precise intra-gel estimates of the abundance ratio for protein spots from both samples. Twelve multiplexed analytical radioactive SDS-gels from 4 serial IPG-IEF gels provided 24 individual radioactive images for a comprehensive analytical protein multiplex quantification study. A further 12 SDS gels containing (125)I-labeled sample were coelectrophoresed with preparative protein amounts obtained from whole tissue sections for the mass spectrometric identification of comigrating proteins. This consumed <40% of the (125)I-labeled sample, and <20% of the (131)I-labeled sample from the respective original 3.8 microg samples. Twenty-nine proteins were identified by mass spectrometry with PMF scores >70 that were >2-fold differentially abundant between the samples and t-test probabilities <0.05. We conclude that this combination of technologies provides excellent quality protein multiplex data for the differential abundance analysis of large numbers of proteins from extremely small samples, and is applicable to a broad range of clinical and related applications.

Carcinoma, Renal Cell↗

PubMed2026

Engineering cold stress resilience in capsicum annuum through functional genomics and precision breeding.

This review synthesizes the molecular mechanisms of cold tolerance in pepper, integrating multi-omics data,genome editing, and precision breeding strategies to accelerate the development of cold-resilient cultivars. Cold stress is a significant environmental factor that affects the growth, productivity, and fruit quality of Capsicum annuum by impairing membrane integrity photosynthesis and cellular redox homeostasis. Although pepper has several endogenous cold-responsive regulators such as CaNAC035 and CabHLH035, along with antioxidant defense systems, its cold tolerance remains limited due to low transcriptional activation of key regulators, functional redundancy among cold-responsive genes, and the polygenicity of cold tolerance. These complexities, combined with low genetic diversity and linkage drag, have hindered the improvement of cold-resistant cultivars through conventional breeding. This review brings together the recent progress in understanding the molecular mechanisms of cold stress perception, signal transduction, transcriptional regulation, metabolic reprogramming, and phytohormone interactions in pepper. Precision Breeding 2.0 is a new innovation that combines the integration of multi-omics-based target identification with next-generation genome-editing techniques, allowing precise and multiplex engineering of complex and interconnected regulatory networks instead of single genes. We cover new approaches such as engineering the DREB/CBF pathway, allele-specific editing and targeted disruption of negative regulators to enhance the pathway(s) involved in cold response. Moreover, we propose a roadmap for integration of transcriptomics, proteomics, metabolomics, high-throughput phenomics, and speed breeding to accelerate the identification, validation, and deployment of superior alleles to boost cold tolerance. This review provides a foundation for developing climate-resilient pepper cultivars by connecting functional genomics with precision genome engineering approaches to maintain productivity under variable environmental conditions.

Capsicum↗

PubMed2003

Dual electrospray ionization source for confident generation of accurate mass tags using liquid chromatography Fourier transform ion cyclotron resonance mass spectrometry.

Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) has rapidly established a prominent role in proteomics because of its unparalleled resolving power, sensitivity and ability to achieve high mass measurement accuracy (MMA) simultaneously. However, space-charge effects must be quantitatively, routinely, and confidently corrected because they are known to profoundly influence MMA. We argue that the most effective way to account for space-charge effects is to introduce an internal mass calibrant (IMC) using a dual electrospray ionization (ESI) source where the IMC is added from a separate ESI emitter. The major disadvantage of our initial dual ESI source to achieve high MMA, and arguably the only one, was the time required to switch between the analyte emitter and IMC emitter (i.e., >300 ms). While this "switching time" was acceptable for direct infusion experiments, it did not lend itself to high-throughput applications or when conducting on-line liquid separations. In this report, we completely redesigned the dual ESI source and demonstrate several key attributes. First, the new design allows for facile alignment of ESI emitters, undetectable vibration, and the ability to extend to multiple emitters. Second, the switching time was reduced to <50 ms, which allowed the analyte and IMC to be accumulated "simultaneously" in the external ion reservoir and injected as a single ion packet into the ion cyclotron resonance cell, eliminating the need for a separate accumulation and ion injection event for the IMC. Third, by using a high concentration of the IMC, the residence time on this emitter could be reduced to approximately 80 ms, allowing for more time spent accumulating analyte ions of significantly lower concentration. Fourth, multiplexed on-line separations can be carried out providing increased throughput. Specifically, the new dual ESI source has demonstrated its ability to produce a stable ion current over a 45-min time period at 7 T resulting in mass accuracies of 1.08 ppm +/- 0.11 ppm (mean +/- confidence interval of the mean at 95% confidence; N = 160). In addition, the analysis of a tryptic digest of apomyoglobin by nanoLC-dual ESI-FT-ICR afforded an average MMA of -1.09 versus -74.5 ppm for externally calibrated data. Furthermore, we demonstrate that the amplitude of a peptide being electrosprayed at 25 nM can be linearly increased, ultimately allowing for dynamic analyte/IMC abundance modulation. Finally, we demonstrate that this source can reliably be used for multiplexing measurements from two (eventually more) flow streams.

Amino Acid Sequence↗

PubMed2007

A novel quantitative proteomics strategy to study phosphorylation-dependent peptide-protein interactions.

Phosphorylation-dependent protein-protein interactions provide the mechanism for a large number of intracellular signal transduction pathways. One of the goals of signal transduction research is to understand more precisely the nature of these phosphorylation-dependent interactions. Here, we report a novel strategy based on quantitative proteomics that allows for the rapid analysis of peptide-protein interactions with more than one phosphorylation site involved. The phosphorylation of two tyrosine residues, Y342 and Y346, within the linker B region of the protein-tyrosine kinase Syk is important for optimal signaling from the B cell receptor for antigen. We employed four amino-specific, isobaric reagents to differentially label proteins interacting in vitro with four Syk peptides containing none, one, or two phosphates on tyrosine residues Y342 and Y346, respectively. In total, 76 proteins were identified and quantified, 11 of which were dependent on the phosphorylation of individual tyrosine residues. One of the proteins, peroxiredoxin 1, preferably bound to phosphorylated Y346, which was further verified by Western blotting results. Thus, we demonstrate that the use of 4-fold multiplexing allows for relative protein measurements simultaneously for the identification of interacting proteins dependent on the phosphorylation of specific residues.

Algorithms↗

PubMed2004

Protein profiling of human postmortem brain using 2-dimensional fluorescence difference gel electrophoresis (2-D DIGE).

Two-dimensional gel electrophoresis (2-D GE) is a key tool for comparative proteomics research. With its ability to separate complex protein mixtures with high resolution, 2-D GE is a technique commonly employed for protein profiling studies. Significant improvements have been made in 2-D GE technology with the development of two-dimensional fluorescence difference gel electrophoresis (2-D DIGE), where proteins are first labelled with one of three spectrally resolvable fluorescent cyanine dyes before being separated over first and second dimensions according to their charge and size, respectively. When used in conjunction with automated analysis packages, this multiplexing approach can accurately and reproducibly quantify protein expression for control and experimental groups. Differentially expressed proteins can be subsequently identified by mass spectrometric methods. Here, we describe the successful application and optimisation of 2-D DIGE technology for human postmortem brain studies. This technology, especially when coupled with other functional genomics approaches, such as transcriptomics and metabolomics studies, will enhance our current understanding of human disease and lead to new therapeutic and diagnostic possibilities.

Brain Chemistry↗

PubMed2005

High throughput production of mouse monoclonal antibodies using antigen microarrays.

Recent advances in proteomics research underscore the increasing need for high-affinity monoclonal antibodies, which are still generated with lengthy, low-throughput antibody production techniques. Here we present a semi-automated, high-throughput method of hybridoma generation and identification. Monoclonal antibodies were raised to different targets in single batch runs of 6-10 wk using multiplexed immunisations, automated fusion and cell-culture, and a novel antigen-coated microarray-screening assay. In a large-scale experiment, where eight mice were immunized with ten antigens each, we generated monoclonal antibodies against 68 of the targets (85%), within 6 wk of the primary immunization.

Animals↗

PubMed2002

Suspension array technology: evolution of the flat-array paradigm.

Suspension arrays of microspheres analyzed using flow cytometry offer a new approach to multiplexed assays for large-scale screening applications. By optically encoding micron-sized polymer particles, suspension microarrays can be created to enable highly multiplexed analysis of complex samples. Each element in the array is comprised of a subpopulation of particles with distinct optical properties and each array element bears a different surface receptor. Nucleic acids, proteins, lipids or carbohydrates can serve as receptors to support the analysis of a wide range of biomolecular assemblies, and applications in genomic and proteomic research are being developed. Coupled with recent innovations for rapid serial analysis of samples, molecular analysis with microsphere arrays holds significant potential as a general analysis platform for both research and clinical applications.

Directed Molecular Evolution↗