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- All Subjects: proteomics
- Creators: Guo, Jia
- Creators: Ros, Alexandra
Description
In an effort to begin validating the large number of discovered candidate biomarkers, proteomics is beginning to shift from shotgun proteomic experiments towards targeted proteomic approaches that provide solutions to automation and economic concerns. Such approaches to validate biomarkers necessitate the mass spectrometric analysis of hundreds to thousands of human samples. As this takes place, a serendipitous opportunity has become evident. By the virtue that as one narrows the focus towards "single" protein targets (instead of entire proteomes) using pan-antibody-based enrichment techniques, a discovery science has emerged, so to speak. This is due to the largely unknown context in which "single" proteins exist in blood (i.e. polymorphisms, transcript variants, and posttranslational modifications) and hence, targeted proteomics has applications for established biomarkers. Furthermore, besides protein heterogeneity accounting for interferences with conventional immunometric platforms, it is becoming evident that this formerly hidden dimension of structural information also contains rich-pathobiological information. Consequently, targeted proteomics studies that aim to ascertain a protein's genuine presentation within disease- stratified populations and serve as a stepping-stone within a biomarker translational pipeline are of clinical interest. Roughly 128 million Americans are pre-diabetic, diabetic, and/or have kidney disease and public and private spending for treating these diseases is in the hundreds of billions of dollars. In an effort to create new solutions for the early detection and management of these conditions, described herein is the design, development, and translation of mass spectrometric immunoassays targeted towards diabetes and kidney disease. Population proteomics experiments were performed for the following clinically relevant proteins: insulin, C-peptide, RANTES, and parathyroid hormone. At least thirty-eight protein isoforms were detected. Besides the numerous disease correlations confronted within the disease-stratified cohorts, certain isoforms also appeared to be causally related to the underlying pathophysiology and/or have therapeutic implications. Technical advancements include multiplexed isoform quantification as well a "dual- extraction" methodology for eliminating non-specific proteins while simultaneously validating isoforms. Industrial efforts towards widespread clinical adoption are also described. Consequently, this work lays a foundation for the translation of mass spectrometric immunoassays into the clinical arena and simultaneously presents the most recent advancements concerning the mass spectrometric immunoassay approach.
ContributorsOran, Paul (Author) / Nelson, Randall (Thesis advisor) / Hayes, Mark (Thesis advisor) / Ros, Alexandra (Committee member) / Williams, Peter (Committee member) / Arizona State University (Publisher)
Created2011
Description
Measurements of different molecular species from single cells have the potential to reveal cell-to-cell variations, which are precluded by population-based measurements. An increasing percentage of researches have been focused on proteins, for its central roles in biological processes. Immunofluorescence (IF) has been a well-established protein analysis platform. To gain comprehensive insights into cell biology and diagnostic pathology, a crucial direction would be to increase the multiplexity of current single cell protein analysis technologies.
An azide-based chemical cleavable linker has been introduced to design and synthesis novel fluorescent probes. These probes allow cyclic immunofluorescence staining which leads to the feasibility of highly multiplexed single cell in situ protein profiling. These highly multiplexed imaging-based platforms have the potential to quantify more than 100 protein targets in cultured cells and more than 50 protein targets in single cells in tissues.
This approach has been successfully applied in formalin-fixed paraffin-embedded (FFPE) brain tissues. Multiplexed protein expression level results reveal neuronal heterogeneity in the human hippocampus.
An azide-based chemical cleavable linker has been introduced to design and synthesis novel fluorescent probes. These probes allow cyclic immunofluorescence staining which leads to the feasibility of highly multiplexed single cell in situ protein profiling. These highly multiplexed imaging-based platforms have the potential to quantify more than 100 protein targets in cultured cells and more than 50 protein targets in single cells in tissues.
This approach has been successfully applied in formalin-fixed paraffin-embedded (FFPE) brain tissues. Multiplexed protein expression level results reveal neuronal heterogeneity in the human hippocampus.
ContributorsLiao, Renjie (Author) / Guo, Jia (Thesis advisor) / Borges, Chad (Committee member) / Liu, Yan (Committee member) / Arizona State University (Publisher)
Created2019
Description
Single-cell proteomics and transcriptomics analysis are crucial to gain insights of
healthy physiology and disease pathogenesis. The comprehensive profiling of biomolecules in individual cells of a heterogeneous system can provide deep insights into many important biological questions, such as the distinct cellular compositions or regulation of inter- and intracellular signaling pathways of healthy and diseased tissues. With multidimensional molecular imaging of many different biomarkers in patient biopsies, diseases can be accurately diagnosed to guide the selection of the ideal treatment.
As an urgent need to advance single-cell analysis, imaging-based technologies have been developed to detect and quantify multiple DNA, RNA and protein molecules in single cell in situ. Novel fluorescent probes have been designed and synthesized, which targets specifically either their nucleic acid counterpart or protein epitopes. These highly multiplexed imaging-based platforms have the potential to detect and quantify 100 different protein molecules and 1000 different nucleic acids in a single cell.
Using novel fluorescent probes, a large number of biomolecules have been detected and quantified in formalin-fixed paraffin-embedded (FFPE) brain tissue at single-cell resolution. By studying protein expression levels, neuronal heterogeneity has been revealed in distinct subregions of human hippocampus.
healthy physiology and disease pathogenesis. The comprehensive profiling of biomolecules in individual cells of a heterogeneous system can provide deep insights into many important biological questions, such as the distinct cellular compositions or regulation of inter- and intracellular signaling pathways of healthy and diseased tissues. With multidimensional molecular imaging of many different biomarkers in patient biopsies, diseases can be accurately diagnosed to guide the selection of the ideal treatment.
As an urgent need to advance single-cell analysis, imaging-based technologies have been developed to detect and quantify multiple DNA, RNA and protein molecules in single cell in situ. Novel fluorescent probes have been designed and synthesized, which targets specifically either their nucleic acid counterpart or protein epitopes. These highly multiplexed imaging-based platforms have the potential to detect and quantify 100 different protein molecules and 1000 different nucleic acids in a single cell.
Using novel fluorescent probes, a large number of biomolecules have been detected and quantified in formalin-fixed paraffin-embedded (FFPE) brain tissue at single-cell resolution. By studying protein expression levels, neuronal heterogeneity has been revealed in distinct subregions of human hippocampus.
ContributorsMondal, Manas (Author) / Guo, Jia (Thesis advisor) / Gould, Ian (Committee member) / Ros, Alexandra (Committee member) / Arizona State University (Publisher)
Created2018
Description
The understanding of normal human physiology and disease pathogenesis shows great promise for progress with increasing ability to profile genomic loci and transcripts in single cells in situ. Using biorthogonal cleavable fluorescent oligonucleotides, a highly multiplexed single-cell in situ RNA and DNA analysis is reported. In this report, azide-based cleavable linker connects oligonucleotides to fluorophores to show nucleic acids through in situ hybridization. Post-imaging, the fluorophores are effectively cleaved off in half an hour without loss of RNA or DNA integrity. Through multiple cycles of hybridization, imaging, and cleavage this approach proves to quantify thousands of different RNA species or genomic loci because of single-molecule sensitivity in single cells in situ. Different nucleic acids can be imaged by shown by multi-color staining in each hybridization cycle, and that multiple hybridization cycles can be run on the same specimen. It is shown that in situ analysis of DNA, RNA and protein can be accomplished using both cleavable fluorescent antibodies and oligonucleotides. The highly multiplexed imaging platforms will have the potential for wide applications in both systems biology and biomedical research. Thus, proving to be cost effective and time effective.
ContributorsSamuel, Adam David (Author) / Guo, Jia (Thesis director) / Liu, Wei (Committee member) / Wang, Xu (Committee member) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05
Description
The ability to profile proteins allows us to gain a deeper understanding of organization, regulation, and function of different biological systems. Many technologies are currently being used in order to accurately perform the protein profiling. Some of these technologies include mass spectrometry, microarray based analysis, and fluorescence microscopy. Deeper analysis of these technologies have demonstrated limitations which have taken away from either the efficiency or the accuracy of the results. The objective of this project was to develop a technology in which highly multiplexed single cell in situ protein analysis can be completed in a comprehensive manner without the loss of the protein targets. This was accomplished in the span of 3 steps which is referred to as the immunofluorescence cycle. Antibodies with attached fluorophores with the help of novel azide-based cleavable linker are used to detect protein targets. Fluorescence imaging and data storage procedures are done on the targets and then the fluorophores are cleaved from the antibodies without the loss of the protein targets. Continuous cycles of the immunofluorescence procedure can help create a comprehensive and quantitative profile of the protein. The development of such a technique will not only help us understand biological systems such as solid tumor, brain tissues, and developing embryos. But it will also play a role in real-world applications such as signaling network analysis, molecular diagnosis and cellular targeted therapies.
ContributorsGupta, Aakriti (Author) / Guo, Jia (Thesis director) / Liang, Jianming (Committee member) / Computer Science and Engineering Program (Contributor) / Barrett, The Honors College (Contributor)
Created2016-12
Description
Understanding cellular processes can provide insight into disease pathogenesis and reveal critical information for prevention, diagnosis, and treatment. As key executors and signaling regulators, proteins carry relevant information not available from genomics and transcriptomics. Cell-to-cell differences significantly affect disease incidence and drug responses, generating a need for protein analysis at the single-cell level. However, quantitative protein analysis at the single-cell level remains challenging due to the low protein amount in a single cell and the proteome complexity. It requires sensitive detection techniques and appropriate sample preparation and delivery to the detection area. Here, a microfluidic platform in tandem with matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) has been developed for targeted intracellular protein analysis. The elastomeric multi-layer microfluidic platform, termed MIMAS, was designed as a series of 8.75 nL wells separated by pneumatic valves. The MIMAS platform allows cell loading, sample processing on-chip, and further in situ mass spectrometry analysis. The sample processing includes cell lysis, immunocapture, tryptic digestion and MALDI matrix solution loading for co-crystallization. This work demonstrates that the MIMAS approach is suitable for protein quantification by assessing the apoptotic protein Bcl-2 from MCF-7 breast cancer cells using an isotope-labeled peptide. The limit of detection was determined as 11.22 nM, equivalent to 5.91 x 10^7 protein molecules per well. Moreover, the MIMAS platform design was improved, allowing the successful quantification of Bcl-2 protein in small cell ensembles down to ~10 cells in 4 nL wells. Furthermore, the MIMAS platform was integrated with laser capture microdissection (LCM) for protein analysis from post-mortem human tissues. Intracellular amyloid-β peptide (Aβ), a hallmark of Alzheimer’s Disease, was assessed from human brain tissue using the LCM-MIMAS. The successful detection of Aβ from small cell ensembles (20 sliced pyramidal cells) demonstrated the LCM-MIMAS capability of assessing intracellular proteins from specific tissue cell subpopulations. The MIMAS approach is a promising tool for intracellular protein analysis from small cell ensembles, with the potential for single-cell analysis. It allows for protein analysis towards the understanding of biological phenomena for clinical and biological research.
ContributorsCruz Villarreal, Jorvani (Author) / Ros, Alexandra (Thesis advisor) / Borges, Chad R (Committee member) / Buttry, Daniel (Committee member) / Arizona State University (Publisher)
Created2022
Description
Spatial resolved detection and quantification of ribonucleic acid (RNA) molecules in single cell is crucial for the understanding of inherent biological issues, like mechanism of gene regulation or the development and maintenance of cell fate. Conventional methods for single cell RNA profiling, like single-cell RNA sequencing (scRNA-seq) or single-molecule fluorescent in situ hybridization (smFISH), suffer either from the loss of spatial information or the low detection throughput. In order to advance single-cell analysis, new approaches need to be developed with the ability to perform high-throughput detection while preserving spatial information of the subcellular location of target RNA molecules.
Novel approaches for highly multiplexed single cell in situ transcriptomic analysis were developed by our group to enable single-cell comprehensive RNA profiling in their native spatial contexts. Reiterative FISH was demonstrated to be able to detect >100 RNA species in single cell in situ, while more sophisticated approaches, consecutive FISH (C-FISH) and switchable fluorescent oligonucleotide based FISH (SFO-FISH), have the potential for whole transcriptome profiling at the single molecule sensitivity. The introduction of a cleavable fluorescent tyramide even enables sensitive RNA profiling in intact tissues with high throughput. These approaches will have wide applications in studies of systems biology, molecular diagnosis and targeted therapies.
Novel approaches for highly multiplexed single cell in situ transcriptomic analysis were developed by our group to enable single-cell comprehensive RNA profiling in their native spatial contexts. Reiterative FISH was demonstrated to be able to detect >100 RNA species in single cell in situ, while more sophisticated approaches, consecutive FISH (C-FISH) and switchable fluorescent oligonucleotide based FISH (SFO-FISH), have the potential for whole transcriptome profiling at the single molecule sensitivity. The introduction of a cleavable fluorescent tyramide even enables sensitive RNA profiling in intact tissues with high throughput. These approaches will have wide applications in studies of systems biology, molecular diagnosis and targeted therapies.
ContributorsXiao, Lu, Ph.D (Author) / Guo, Jia (Thesis advisor) / Wang, Xu (Committee member) / Borges, Chad (Committee member) / Arizona State University (Publisher)
Created2019