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Results for “multiplexed proteomics”

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PubMed2006

Dimethyl multiplexed labeling combined with microcolumn separation and MS analysis for time course study in proteomics.

Stable-isotope labeling coupled with liquid-phase separation and MS analysis is a powerful technique for comparative proteomics. We developed a dimethyl labeling strategy (Anal. Chem. 2003, 75, 6843-6852 and J. Proteome Res. 2005, 4, 101-108) to label peptide N-terminus and epsilon-amino groups of Lys with water-soluble formaldehyde via reductive methylation, and an isotopic pair of formaldehyde is used for binary labeling on two sets of samples. In this study, this approach is extended to a four sample labeling by combining the binary isotopic reagents of formaldehyde (d0, d2) and the binary isotopic reducing reagents, sodium cyanoborohydride (d0, d3). To ensure sufficient mass difference, this multiplexed labeling is coupled with endoproteinase Lys-C instead of trypsin for digestion, resulting in at least two labeling sites with a mass difference of 4 Da for each pair of peptide digest. Moreover, multiplex dimethyl labeling was proved to have no significant isotopic effect during RP LC elution. This method was further applied for monitoring Lys-C digestion using hemoglobin as a model. Dimethyl labeled digests derived from seven time points (1-30 h) were grouped into two sets of sample mixtures, separated by nano-LC to reduce the complexity, and then analyzed by ESI-MS/MS. The temporal study reveals that Lys-C digestion was completed in 10-15 h for all detected peptides. The multiplex dimethyl method has not only provided a simultaneous detection mean for four sample sets but has also conserved all the advantages associated with the original binary method.

Amino Acid Sequence↗

PubMed2026

Moving Beyond Morphology to Multiplexed Molecular Imaging as the Next Frontier in Diagnostic Pathology.

Diagnostic pathology has long relied on the morphologic interpretation of hematoxylin and eosin-stained tissues to guide diagnosis and assess prognostic features. Although pathologists intuitively recognize spatial patterns and architectural organization, these assessments remain largely qualitative and difficult to quantify systematically. Immunohistochemistry and immunofluorescence have introduced molecular specificity but are limited in multiplexing capacity, whereas bulk genomic and transcriptomic assays provide high molecular depth but lose spatial context by averaging signals across heterogeneous cell populations. Recent advances in spatial proteomics-including mass spectrometry-based imaging and cyclic immunofluorescence-now enable multiplexed, single-cell protein analysis within intact tissue architecture. These technologies have revealed complex immune and stromal microenvironments, spatially organized biomarkers predictive of therapeutic response, and molecular gradients underlying disease progression. By integrating histologic and molecular information, spatial proteomics bridges traditional microscopy with high-dimensional omics, allowing quantitative, spatially resolved insights into tissue organization and disease mechanisms. This review summarizes recent developments in multiplexed spatial proteomics from both scientific and pathologic perspectives, highlighting how these technologies extend beyond morphology to quantify histologic patterns, refine biomarker discovery, and facilitate clinical translation. The review also examines translational challenges and barriers to clinical implementation, including costs, standardization requirements, and workflow integration.

Humans↗

PubMed2006

Quantitative multiplexed profiling of cellular signaling networks using phosphotyrosine-specific DNA-tagged SH2 domains.

Deciphering global signaling networks is of great importance for the detailed understanding of cellular signaling processes controlling many important biological functions. Among signaling processes, tyrosine phosphorylation has a central role. At present, adequate techniques for the global characterization of the tyrosine phosphoproteome are lacking, particularly for the analysis of small amounts of protein. By combining the power of PCR amplification with the unique properties of Src homology region 2 (SH2) domains to specifically recognize tyrosine-phosphorylated proteins, we developed a new proteomic approach, termed oligonucleotide-tagged multiplex assay (OTM). For OTM, multiple SH2 domains are labeled by domain-specific oligonucleotide tags, applied as probes to complex protein mixtures in a multiplex reaction and phosphotyrosine-specific interactions are quantified by PCR. Using OTM we reproducibly quantified differential states of tyrosine phosphorylation with high sensitivity and specificity in small amounts of whole cellular extracts as demonstrated for various tumor cell lines and human leukemia samples.

3T3 Cells↗

PubMed2026

PASTA: versatile tyramide-oligonucleotide amplification for multimodal spatial biology.

Spatial proteomics is limited by detection sensitivity, multiplexing and multimodal integration, leaving a gap between discovery and clinical assays. Here we present protein and nucleic acid serial tyramide amplification (PASTA), using horseradish peroxidase-mediated oligonucleotide deposition and cyclical imaging for high-plex, multimodal spatial profiling. Compatible with conjugated antibodies and in situ hybridization, PASTA enables simultaneous protein and RNA codetection from formalin-fixed, paraffin-embedded samples, providing a cost-effective bridge from discovery to clinical validation.

Tyramine↗

PubMed2000

Peptide electroextraction for direct coupling of in-gel digests with capillary LC-MS/MS for protein identification and sequencing.

An electrophoretic method has been developed for the extraction of peptides following in-gel digests of SDS-PAGE separated proteins. During electroextraction, the peptides are trapped on a strong cation-exchange microcartridge, before analysis by capillary LC--ESI-tandem mass spectrometry. The spectra obtained by tandem mass spectrometry are searched directly against a protein database for identification of the protein from which the peptide originated. By minimizing surface exposure of the peptides during electroextraction, a reduction of the detection limits for protein identification is realized. The performance of the peptide electroextraction was compared directly with the standard extraction method for in-gel protein digests, using a standard dilution series of phosphorylase B and carbonic anhydrase, separated by SDS-PAGE. The lowest gel loading in which phosphorylase B was identified using the standard extraction method was 2.5 ng or 25 fmol, and the lowest gel loading in which phosphorylase B was identified using electroextraction was 1.25 ng or 12.5 fmol. The design of the microextraction cartridge allows for direct interfacing with capillary LC, which is crucial for maintaining low detection limits. Furthermore, this method can be used for high-throughput proteomics since it can be easily multiplexed and requires only voltage control and low pressures (approximately 15 psi) for operation. We believe that peptide electroextraction is a significant advance for identification of proteins separated by one-dimensional or two-dimensional gel electrophoresis, as it can be easily automated and requires less protein than conventional methods.

Amino Acid Sequence↗

PubMedDate not supplied

Evidence-based medical perspectives: the evolving role of PSA for early detection, monitoring of treatment response, and as a surrogate end point of efficacy for interventions in men with different clinical risk states for the prevention and progression of prostate cancer.

Following FDA approval and introduction into the clinic in the mid-1980s, PSA testing has become arguably the most versatile serum tumor marker in urologic oncology with clinical use for early detection (screening) of prostate cancer (PC), risk stratification for clinical staging, prognosis, intermediate biomarker for monitoring tumor recurrence, and more recently as an intermediate biomarker for assessing therapeutic response to antiandrogens, radiation therapy, and chemotherapy. PSA now routinely guides health care providers for the clinical management of PC over a wide range of clinical risk states for men at risk of PC, after local definitive therapy and after systemic therapy to prevent progression to metastatic bone disease, and to palliate men with hormone refractory prostate cancer (HRPC). To further assess the evidence that supports these clinical applications, this commentary reviews and critically evaluates the emerging body of new data focusing on several recently published seminal articles by D'Amico et al and Thompson et al, the new National Comprehensive Cancer Network 2004 recommendations for starting PSA testing at the age of 40 years old, the latest results from 2 phase 3 randomized, controlled trials of taxane-based regimens showing improved survival for men with HRPC, and the recent US FDA Public Workshop on Clinical Trial Endpoints in Prostate Cancer that helped to distill and synthesize the current state of the art and the progress toward validation of PSA metrics (eg, PSA velocity) as a surrogate end point (SE) for treatment efficacy with taxane-based regimens. Furthermore, several randomized, controlled chemoprevention trials in progress evaluating agents such as selenium and vitamin E in high-risk cohorts are well poised to confirm the validity of PSA as an SE for clinical efficacy for the prevention and progression of PC. Although there continues to be a need to validate better biomarkers before diagnosis of PC (more sensitive and specific) and after diagnosis to discern between indolent and aggressive forms of PC, it is very likely that some metric of PSA as a biomarker alone or as part of a panel of other serum proteomic markers or tissue-derived multiplex gene expression arrays will be around for years to come as a useful tool for risk stratification, early detection, prognosis, prediction, and as an SE of efficacy for prevention and treatment of PC.

Biomarkers, Tumor↗

PubMed2026

High-Plex Tissue Imaging with Conventional Immunofluorescence Platforms and Open-Source Software via Iterative Bleaching Extends Multiplexity (IBEX).

Iterative bleaching extends multiplexity (IBEX) is an easy-to-use, highly multiplex immunofluorescent tissue imaging method that employs widely available microscopy platforms, commercial reagents, and open-source software. In this article, we describe how to implement this method in a laboratory that has minimal experience with immunohistochemistry.

Software↗

PubMed2006

Multiplexed protein array platforms for analysis of autoimmune diseases.

Several proteomics platforms have emerged in the past decade that show great promise for filling in the many gaps that remain from earlier studies of the genome and from the sequencing of the human genome itself. This review describes applications of proteomics technologies to the study of autoimmune diseases. We focus largely on biased technology platforms that are capable of analyzing a large panel of known analytes, as opposed to techniques such as two-dimensional gel electrophoresis (2DIGE) or mass spectroscopy that represent unbiased approaches (as reviewed in 1). At present, the main analytes that can be systematically studied in autoimmunity include autoantibodies, cytokines and chemokines, components of signaling pathways, and cell-surface receptors. We review the most commonly used platforms for such studies, citing important discoveries and limitations that exist. We conclude by reviewing advances in biomedical informatics that will eventually allow the human proteome to be deciphered.

Autoantibodies↗

PubMed2003

PubMatrix: a tool for multiplex literature mining.

BACKGROUND: Molecular experiments using multiplex strategies such as cDNA microarrays or proteomic approaches generate large datasets requiring biological interpretation. Text based data mining tools have recently been developed to query large biological datasets of this type of data. PubMatrix is a web-based tool that allows simple text based mining of the NCBI literature search service PubMed using any two lists of keywords terms, resulting in a frequency matrix of term co-occurrence. RESULTS: For example, a simple term selection procedure allows automatic pair-wise comparisons of approximately 1-100 search terms versus approximately 1-10 modifier terms, resulting in up to 1,000 pair wise comparisons. The matrix table of pair-wise comparisons can then be surveyed, queried individually, and archived. Lists of keywords can include any terms currently capable of being searched in PubMed. In the context of cDNA microarray studies, this may be used for the annotation of gene lists from clusters of genes that are expressed coordinately. An associated PubMatrix public archive provides previous searches using common useful lists of keyword terms. CONCLUSIONS: In this way, lists of terms, such as gene names, or functional assignments can be assigned genetic, biological, or clinical relevance in a rapid flexible systematic fashion. http://pubmatrix.grc.nia.nih.gov/

Cell Line, Tumor↗

PubMed2005

Novel isothermal, linear nucleic acid amplification systems for highly multiplexed applications.

BACKGROUND: Global analysis of the genome, transcriptome, and proteome is facilitated by the recent development of tools for large-scale, highly parallel analysis. We describe a novel nucleic acid amplification system that generates products by several methods. 3'-Ribo-SPIA primes cDNA synthesis at the 3' polyA tail, and whole transcript (WT)-Ribo-SPIA primes cDNA synthesis across the full length of the transcripts and thus provides whole-transcriptome amplification, independent of the 3' polyA tail. METHODS: We developed isothermal linear nucleic acid amplification systems, which use a single chimeric primer, for amplification of DNA (SPIA) and RNA (Ribo-SPIA). The latter allows mRNA amplification from as little as 1 ng of total RNA. Amplification efficiency was calculated based on the delta threshold cycle between nonamplified cDNA targets and amplified cDNA. The amounts and quality of total RNA and amplification products were determined after purification of the amplification products. GeneChip array gene expression profiling and real-time PCR were used to test the accuracy and reproducibility of the method. Quantification of cDNA products (before and after amplification) at the 2 loci along the transcripts was used to assess product length (for evaluation of the 3'-initiated Ribo-SPIA) and equal representation throughout the length of the transcript (for evaluation of the whole transcript amplification system, WT-Ribo-SPIA). RESULTS: Ribo-SPIA-based global RNA amplification exhibited linearity over 6 orders of magnitude of transcript abundance and generated microgram amounts of amplified cDNA from as little as 1 ng of total RNA. CONCLUSIONS: The described methods enable comprehensive gene expression profiling and analysis from limiting biological samples. The WT-Ribo-SPIA procedure, which enables amplification of non-polyA-tailed RNA, is suitable for amplification and gene expression analysis of both eukaryotic and prokaryotic biological samples.

Gene Expression Profiling↗

PubMed2004

AutoGenomics, Inc.

AutoGenomics has created an automated multiplexing microarray platform to make genomic and proteomic analyses routine and efficient for clinical and research laboratories. While the emergence of microarrays has advanced genomic analyses, a number of underlying issues, such as cross-hybridization, poor spot morphology and intrinsic fluorescence of the solid substrate, have yet to be fully resolved. Current methods use discrete instrumentation, are manual and require highly skilled labor, which leads to inconsistent results. AutoGenomics' automated platform uses a three-dimensional BioFilmChip microarray to circumvent these issues, providing optimal spot morphology and utilizing solution-based hybridization with allele-specific primer extension to improve single-base discrimination. AutoGenomics is developing applications for the early detection and management of complex disease states in oncology, cardiology, and mental disorders. Customers include clinical reference laboratories, hospitals, academic institutions, and pharmaceutical and biotech companies. Founded in 1999, the company is headquartered in Carlsbad, California, USA.

Drug Industry↗

PubMed2006

Multiplex detection of surface molecules on colorectal cancers.

A technique of fluorescence multiplexing is described for analysis of the plasma membrane proteome of colorectal cancer cells from surgically resected specimens, enabling detection and immunophenotyping when the cancer cells are in the minority. A single-cell suspension was prepared from a colorectal tumour, and the mixed population of cells was captured on a CD antibody microarray. The cancer cells were detected using a fluorescently tagged antibody for carcinoembryonic antigen (CEA-Alexa647) or epithelial cell adhesion marker (EpCAM-Alexa488). Using this multiplexing procedure, dot patterns from colorectal cancers were distinct from those of adjacent normal tissue. Subtraction of the expression levels for each antigen from normal tissue from those for the cancer shows differential expression in the cancer of CD66c, CD15s, CD55, CD45, CD71, CD45RO, CD11b and CEA, in descending order. Cells captured on the same microarray were also labelled with fluorescent CD3-phycoerythrin antibody revealing the presence of tumour-infiltrating lymphocytes. The immunophenotypes of T lymphocytes from the tumour samples showed differential expression of HLA-DR, TCR alpha/beta, CD49d, CD52, CD49e, CD5, CD95, CD28, CD38 and CD71, in descending order. Fluorescence multiplexing of mixed cell populations captured on a single antibody microarray enables expression profiling of multiple sub-populations of cells within a tumour sample.

CD4 Antigens↗

PubMed2006

Antibody microarrays: current status and key technological advances.

Antibody-based microarrays are among the novel classes of rapidly evolving proteomic technologies that holds great promise in biomedicine. Miniaturized microarrays (< 1 cm2) can be printed with thousands of individual antibodies carrying the desired specificities, and with biological sample (e.g., an entire proteome) added, virtually any specifically bound analytes can be detected. While consuming only minute amounts (< microL scale) of reagents, ultra- sensitive assays (zeptomol range) can readily be performed in a highly multiplexed manner. The microarray patterns generated can then be transformed into proteomic maps, or detailed molecular fingerprints, revealing the composition of the proteome. Thus, protein expression profiling and global proteome analysis using this tool will offer new opportunities for drug target and biomarker discovery, disease diagnostics, and insights into disease biology. Adopting the antibody microarray technology platform, several biomedical applications, ranging from focused assays to proteome-scale analysis will be rapidly emerging in the coming years. This review will discuss the current status of the antibody microarray technology focusing on recent technological advances and key issues in the process of evolving the methodology into a high-performing proteomic research tool.

Animals↗

PubMed2006

Differential proteomic alterations between localised and metastatic prostate cancer.

Molecular alterations in the prostate cancer proteome mediate the functional and phenotypic transformation from clinically localised to metastatic cancer, a transition that drives patient's mortality and challenges therapeutic intervention. A first approximation of differential proteomic alterations stratified by disease stage has yielded repertoires of potential diagnostic and prognostic markers, multiplex signatures of predictive value, and yield fundamental insight into molecular commonalities in cancer progression. Deciphering these causative proteomic alterations from the molecular noise will continue to mature our understanding of tumour biology and drive new computational and integrative approaches to model a system's view that accommodates the heterogeneity of prostate cancer progression.

Biomarkers, Tumor↗

PubMed2003

Analysis of protein interactions using fluorescence technologies.

Biophotonics techniques, especially those involving fluorescence, are widely used in proteomics to characterize the in vitro interactions between proteins in high-throughput mode. On the other hand, fluorescence-based imaging studies often show that protein activity is regulated through large protein complexes that transiently form at specific sites in the cell. One could therefore argue that a systematic functional analysis of the human proteome requires technologies that are capable of time and spatially resolved, multiplexed analysis of protein interactions within cells.

Fluorescence Resonance Energy Transfer↗

PubMed2002

The use of accurate mass tags for high-throughput microbial proteomics.

We describe and review progress towards a global strategy that aims to extend the sensitivity, dynamic range, comprehensiveness, and throughput of proteomic measurements for microbial systems based upon the use of polypeptide accurate mass tags (AMTs) produced by global protein enzymatic digestions. The two-stage strategy exploits high accuracy mass measurements using Fourier transform ion cyclotron resonance mass spectrometry (FTICR) to validate polypeptide AMTs for a specific organism, from potential mass tags tentatively identified using tandem mass spectrometry (MS/MS), providing the basis for subsequent measurements without the need for routine MS/MS. A high-resolution capillary liquid chromatography separation combined with high sensitivity, and high-resolution accurate FTICR measurements is shown to be capable of characterizing polypeptide mixtures of more than 10(5) components, sufficient for broad protein identification using AMTs. Advantages of the approach include the high confidence of protein identification, its broad proteome coverage, and the capability for stable-isotope labeling methods for precise relative protein abundance measurements. The strategy has been initially evaluated using the microorganisms Saccharomyces cerevisiae and Deinococcus radiodurans. Additional developments, including the use of multiplexed-MS/MS capabilities and methods for dynamic range expansion of proteome measurements that promise to further extend the quality of proteomics measurements, are also described.

Amino Acid Sequence↗

PubMed2025

Single-cell proteomics using mass spectrometry.

Over the past 2 to 3 years, mass-spectrometry-based single-cell proteomics (SCP) has experienced transformative improvements in microfluidic and robotic sample preparation, innovative MS1- and MS2-based multiplexing strategies, and specialized hardware (e.g., timsTOF Ultra 2, Astral), which have dramatically boosted sensitivity, throughput, and proteome coverage from picogram-level protein inputs. Concurrently, tailored computational workflows that encompass normalization, imputation, and no-code platforms have addressed pervasive missing data challenges and standardized analyses, collectively enabling high-throughput, reproducible profiling of cellular heterogeneity. This minireview summarizes the latest progress in SCP technology and software solutions, highlighting how the closer integration of analytical, computational, and experimental strategies will facilitate a deeper and broader coverage of single-cell proteomes.

Single-Cell Analysis↗

PubMed2006

Multiplexed protein measurement: technologies and applications of protein and antibody arrays.

The ability to measure the abundance of many proteins precisely and simultaneously in experimental samples is an important, recent advance for static and dynamic, as well as descriptive and predictive, biological research. The value of multiplexed protein measurement is being established in applications such as comprehensive proteomic surveys, studies of protein networks and pathways, validation of genomic discoveries and clinical biomarker development. As standards do not yet exist that bridge all of these applications, the current recommended best practice for validation of results is to approach study design in an iterative process and to integrate data from several measurement technologies. This review describes current and emerging multiplexed protein measurement technologies and their applications, and discusses the remaining challenges in this field.

Animals↗