scholarly journals Automated sample preparation for high-throughput single-cell proteomics

2018 ◽  
Author(s):  
Harrison Specht ◽  
Guillaume Harmange ◽  
David H. Perlman ◽  
Edward Emmott ◽  
Zachary Niziolek ◽  
...  
Keyword(s):  
Sample Preparation ◽  
Single Cell ◽  
Mammalian Cells ◽  
Es Cells ◽  
Single Cells ◽  
Low Noise ◽  
Small Samples ◽  
Cycle Phase ◽  
Hek 293 ◽  
Mouse Es Cells

A major limitation to applying quantitative LC-MS/MS proteomics to small samples, such as single cells, are the losses incured during sample cleanup. To relieve this limitation, we developed a Minimal ProteOmic sample Preparation (mPOP) method for culture-grown mammalian cells. mPOP obviates cleanup and thus eliminates cleanup-related losses while expediting sample preparation and simplifying its automation. Bulk SILAC samples processed by mPOP or by conventional urea-based methods indicated that mPOP results in complete cell lysis and accurate relative quantification. We integrated mPOP lysis with the Single Cell ProtEomics by Mass Spectrometry (SCoPE-MS) sample preparation, and benchmarked the quantification of such samples on a Q-exactive instrument. The results demonstrate low noise and high technical reproducibility. Then, we FACS sorted single U-937, HEK-293, and mouse ES cells into 96-well plates and analyzed them by automated mPOP and SCoPE-MS. The quantified proteins enabled separating the single cells by cell-type and cell-division-cycle phase.

scholarly journals Expression Profiling of Single Mammalian Cells – Small is Beautiful

Yeast ◽  
2000 ◽  
Vol 1 (3) ◽  
pp. 211-217 ◽  
Author(s):  
Gerard Brady
Keyword(s):  
Mrna Expression ◽  
Single Cell ◽  
Mammalian Cells ◽  
Single Cell Analysis ◽  
Single Cells ◽  
Expression Patterns ◽  
Global Gene Expression ◽  
Small Samples ◽  
Biological Research ◽  
Cell Expression

Increasingly mRNA expression patterns established using a variety of molecular technologies such as cDNA microarrays, SAGE and cDNA display are being used to identify potential regulatory genes and as a means of providing valuable insights into the biological status of the starting sample. Until recently, the application of these techniques has been limited to mRNA isolated from millions or, at very best, several thousand cells thereby restricting the study of small samples and complex tissues. To overcome this limitation a variety of amplification approaches have been developed which are capable of broadly evaluating mRNA expression patterns in single cells. This review will describe approaches that have been employed to examine global gene expression patterns either in small numbers of cells or, wherever possible, in actual isolated single cells. The first half of the review will summarize the technical aspects of methods developed for single-cell analysis and the latter half of the review will describe the areas of biological research that have benefited from single-cell expression analysis.

scholarly journals Mass-spectrometry of single mammalian cells quantifies proteome heterogeneity during cell differentiation

2017 ◽  
Author(s):  
Bogdan Budnik ◽  
Ezra Levy ◽  
Guillaume Harmange ◽  
Nikolai Slavov
Keyword(s):  
Mass Spectrometry ◽  
Single Cell ◽  
Mammalian Cells ◽  
Human Cancer ◽  
Es Cells ◽  
Single Cells ◽  
Embryonic Stem ◽  
Cellular Heterogeneity ◽  
Protein Levels ◽  
Transcriptional Regulatory Mechanisms

Cellular heterogeneity is important to biological processes, including cancer and development. However, proteome heterogeneity is largely unexplored because of the limitations of existing methods for quantifying protein levels in single cells. To alleviate these limitations, we developed Single Cell ProtEomics by Mass Spectrometry (SCoPE-MS), and validated its ability to identify distinct human cancer cell types based on their proteomes. We used SCoPE-MS to quantify over a thousand proteins in differentiating mouse embryonic stem (ES) cells. The single-cell proteomes enabled us to deconstruct cell populations and infer protein abundance relationships. Comparison between single-cell proteomes and transcriptomes indicated coordinated mRNA and protein covariation. Yet many genes exhibited functionally concerted and distinct regulatory patterns at the mRNA and the protein levels, suggesting that post-transcriptional regulatory mechanisms contribute to proteome remodeling during lineage specification, especially for developmental genes. SCoPE-MS is broadly applicable to measuring proteome configurations of single cells and linking them to functional phenotypes, such as cell type and differentiation potentials.

scholarly journals Mass-spectrometry of single mammalian cells quantifies proteome heterogeneity during cell differentiation

2017 ◽  
Author(s):  
Bogdan Budnik ◽  
Ezra Levy ◽  
Nikolai Slavov
Keyword(s):  
Mass Spectrometry ◽  
Single Cell ◽  
Mammalian Cells ◽  
Human Cancer ◽  
Es Cells ◽  
Single Cells ◽  
Embryonic Stem ◽  
Cellular Heterogeneity ◽  
Protein Levels ◽  
Transcriptional Regulatory Mechanisms

Cellular heterogeneity is important to biological processes, including cancer and development. However, proteome heterogeneity is largely unexplored because of the limitations of existing methods for quantifying protein levels in single cells. To alleviate these limitations, we developed Single Cell ProtEomics by Mass Spectrometry (SCoPE-MS), and validated its ability to identify distinct human cancer cell types based on their proteomes. We used SCoPE-MS to quantify over a thousand proteins in differentiating mouse embryonic stem (ES) cells. The single-cell proteomes enabled us to deconstruct cell populations and infer protein abundance relationships. Comparison between single-cell proteomes and transcriptomes indicated coordinated mRNA and protein covariation. Yet many genes exhibited functionally concerted and distinct regulatory patterns at the mRNA and the protein levels, suggesting that post-transcriptional regulatory mechanisms contribute to proteome remodeling during lineage specification, especially for developmental genes. SCoPE-MS is broadly applicable to measuring proteome configurations of single cells and linking them to functional phenotypes, such as cell type and differentiation potentials.

scholarly journals Mass-spectrometry of single mammalian cells quantifies proteome heterogeneity during cell differentiation

2017 ◽  
Author(s):  
Bogdan Budnik ◽  
Ezra Levy ◽  
Nikolai Slavov
Keyword(s):  
Mass Spectrometry ◽  
Single Cell ◽  
Mammalian Cells ◽  
Human Cancer ◽  
Es Cells ◽  
Single Cells ◽  
Embryonic Stem ◽  
Cellular Heterogeneity ◽  
Protein Levels ◽  
Transcriptional Regulatory Mechanisms

Cellular heterogeneity is important to biological processes, including cancer and development. However, proteome heterogeneity is largely unexplored because of the limitations of existing methods for quantifying protein levels in single cells. To alleviate these limitations, we developed Single Cell ProtEomics by Mass Spectrometry (SCoPE-MS), and validated its ability to identify distinct human cancer cell types based on their proteomes. We used SCoPE-MS to quantify over a thousand proteins in differentiating mouse embryonic stem (ES) cells. The single-cell proteomes enabled us to deconstruct cell populations and infer protein abundance relationships. Comparison between single-cell proteomes and transcriptomes indicated coordinated mRNA and protein covariation. Yet many genes exhibited functionally concerted and distinct regulatory patterns at the mRNA and the protein levels, suggesting that post-transcriptional regulatory mechanisms contribute to proteome remodeling during lineage specification, especially for developmental genes. SCoPE-MS is broadly applicable to measuring proteome configurations of single cells and linking them to functional phenotypes, such as cell type and differentiation potentials.

scholarly journals Expression Profiling of Single Mammalian Cells – Small is Beautiful

Yeast ◽  
2000 ◽  
Vol 1 (3) ◽  
pp. 211-217
Author(s):  
Gerard Brady
Keyword(s):  
Mrna Expression ◽  
Single Cell ◽  
Mammalian Cells ◽  
Single Cell Analysis ◽  
Single Cells ◽  
Expression Patterns ◽  
Global Gene Expression ◽  
Small Samples ◽  
Biological Research ◽  
Cell Expression

Increasingly mRNA expression patterns established using a variety of molecular technologies such as cDNA microarrays, SAGE and cDNA display are being used to identify potential regulatory genes and as a means of providing valuable insights into the biological status of the starting sample. Until recently, the application of these techniques has been limited to mRNA isolated from millions or, at very best, several thousand cells thereby restricting the study of small samples and complex tissues. To overcome this limitation a variety of amplification approaches have been developed which are capable of broadly evaluating mRNA expression patterns in single cells. This review will describe approaches that have been employed to examine global gene expression patterns either in small numbers of cells or, wherever possible, in actual isolated single cells. The first half of the review will summarize the technical aspects of methods developed for single-cell analysis and the latter half of the review will describe the areas of biological research that have benefited from single-cell expression analysis.

Gene Targeting

1999 ◽  
Keyword(s):  
Homologous Recombination ◽  
Gene Targeting ◽  
Mammalian Cells ◽  
Es Cells ◽  
Practical Approach ◽  
Simple Method ◽  
Mouse Es Cells ◽  
Cell Clones ◽  
Previous Edition ◽  
Pros And Cons

Since the publication of the first edition of Gene Targeting: A Practical Approach in 1993 there have been many advances in gene targeting and this new edition has been thoroughly updated and rewritten to include all the major new techniques. It provides not only tried-and-tested practical protocols but detailed guidance on their use and applications. As with the previous edition Gene Targeting: A Practical Approach 2e concentrates on gene targeting in mouse ES cells, but the techniques described can be easily adapted to applications in tissue culture including those for human cells. The first chapter covers the design of gene targeting vectors for mammalian cells and describes how to distinguish random integrations from homologous recombination. It is followed by a chapter on extending conventional gene targeting manipulations by using site-specific recombination using the Cre-loxP and Flp-FRT systems to produce 'clean' germline mutations and conditionally (in)activating genes. Chapter 3 describes methods for introducing DNA into ES cells for homologous recombination, selection and screening procedures for identifying and recovering targeted cell clones, and a simple method for establishing new ES cell lines. Chapter 4 discusses the pros and cons or aggregation versus blastocyst injection to create chimeras, focusing on the technical aspects of generating aggregation chimeras and then describes some of the uses of chimeras. The next topic covered is gene trap strategies; the structure, components, design, and modification of GT vectors, the various types of GT screens, and the molecular analysis of GT integrations. The final chapter explains the use of classical genetics in gene targeting and phenotype interpretation to create mutations and elucidate gene functions. Gene Targeting: A Practical Approach 2e will therefore be of great value to all researchers studying gene function.

scholarly journals 1D1400 Single-cell-based analysis of differentiation of mouse ES cells(Development, Differentiation, Neuroscience,Oral Presentation,The 50th Annual Meeting of the Biophysical Society of Japan)

2012 ◽  
Vol 52 (supplement) ◽  
pp. S25
Author(s):  
Atsushi Maruyama ◽  
Yuichi Wakamoto ◽  
Shogo Nakamura ◽  
Tatsuo Michiue ◽  
Shin-ichi Sakai ◽  
...  
Keyword(s):  
Single Cell ◽  
Annual Meeting ◽  
Oral Presentation ◽  
Es Cells ◽  
Mouse Es Cells ◽  
Biophysical Society

scholarly journals Global cDNA Amplification Combined with Real-Time RT–PCR: Accurate Quantification of Multiple Human Potassium Channel Genes at the Single Cell Level

Yeast ◽  
2000 ◽  
Vol 1 (3) ◽  
pp. 201-210 ◽  
Author(s):  
A. Al-Taher ◽  
A. Bashein ◽  
T. Nolan ◽  
M. Hollingsworth ◽  
Brady G.
Keyword(s):  
Potassium Channel ◽  
Single Cell ◽  
Real Time ◽  
Quantitative Pcr ◽  
Single Cells ◽  
Initial Step ◽  
Small Samples ◽  
Rt Pcr ◽  
Real Time Quantitative Pcr ◽  
Global Amplification

We have developed a sensitive quantitative RT–PCR procedure suitable for the analysis of small samples, including single cells, and have used it to measure levels of potassium channel mRNAs in a panel of human tissues and small numbers of cells grown in culture. The method involves an initial global amplification of cDNA derived from all added polyadenylated mRNA followed by quantitative RT–PCR of individual genes using specific primers. In order to facilitate rapid and accurate processing of samples, we have adapted the approach to allow use ofTaqMan™real-time quantitative PCR. We demonstrate that the approach represents a major improvement over existing conventional and real-time quantitative PCR approaches, since it can be applied to samples equivalent to a single cell, is able to accurately measure expression levels equivalent to less than 1/100th copy/cell (one specific cDNA molecule present amongst108total cDNA molecules). Furthermore, since the initial step involves a global amplification of all expressed genes, a permanent cDNA archive is generated from each sample, which can be regenerated indefinitely for further expression analysis.

scholarly journals mDia2 formin selectively interacts with catenins and not E-cadherin to regulate Adherens Junction formation

2019 ◽  
Author(s):  
Yuqi Zhang ◽  
Krista M. Pettee ◽  
Kathryn N. Becker ◽  
Kathryn M. Eisenmann
Keyword(s):  
Single Cell ◽  
Actin Polymerization ◽  
Plasma Membranes ◽  
Adherens Junctions ◽  
Single Cells ◽  
Critical Role ◽  
Cell Junctions ◽  
Hek 293 ◽  
E Cadherin ◽  
Cell Cell

AbstractBackgroundEpithelial ovarian cancer (EOC) cells disseminate within the peritoneal cavity, in part, via the peritoneal fluid as single cells, clusters, or spheroids. Initial single cell egress from a tumor can involve disruption of cell-cell adhesions as cells are shed from the primary tumor into the peritoneum. In epithelial cells, Adherens Junctions (AJs) are characterized by homotypic linkage of E-cadherins on the plasma membranes of adjacent cells. AJs are anchored to the intracellular actin cytoskeletal network through a complex involving E-cadherin, p120 catenin, β-catenin, and αE-catenin. However, the specific players involved in the interaction between the junctional E-cadherin complex and the underlying F-actin network remains unclear. Recent evidence indicates that mammalian Diaphanous-related (mDia) formins plays a key role in epithelial cell AJ formation and maintenance through generation of linear actin filaments. Binding of αE-catenin to linear F-actin inhibits association of the branched-actin nucleator Arp2/3, while favoring linear F-actin bundling. We previously demonstrated that loss of mDia2 was associated with invasive single cell egress from EOC spheroids through disruption of junctional F-actin.ResultsIn the current study, we now show that mDia2 has a role at adherens junctions (AJs) in EOC OVCA429 cells and human embryonic kidney (HEK) 293 cells through its association with αE-catenin and β-catenin. mDia2 depletion in EOC cells leads to reduction in actin polymerization and disruption of cell-cell junctions with decreased interaction between β-catenin and E-cadherin.ConclusionsOur results support a necessary role for mDia2 in AJ stability in EOC cell monolayers and indicate a critical role for mDia formins in regulating EOC AJs during invasive transitions.

scholarly journals Preparation of Tissues and Heterogeneous Cellular Samples for Single-Cell Analysis

2021 ◽  
Author(s):  
E. Celeste Welch ◽  
Anubhav Tripathi
Keyword(s):  
Sample Preparation ◽  
Single Cell ◽  
Single Cell Analysis ◽  
Single Cells ◽  
Cell Analysis ◽  
Processing Times ◽  
Tissue Dissociation ◽  
Emerging Trends ◽  
Recent Developments ◽  
Preparation Techniques

While sample preparation techniques for the chemical and biochemical analysis of tissues are fairly well advanced, the preparation of complex, heterogenous samples for single-cell analysis can be difficult and challenging. Nevertheless, there is growing interest in preparing complex cellular samples, particularly tissues, for analysis via single-cell resolution techniques such as single-cell sequencing or flow cytometry. Recent microfluidic tissue dissociation approaches have helped to expedite the preparation of single cells from tissues through the use of optimized, controlled mechanical forces. Cell sorting and selective cellular recovery from heterogenous samples have also gained traction in biosensors, microfluidic systems, and other diagnostic devices. Together, these recent developments in tissue disaggregation and targeted cellular retrieval have contributed to the development of increasingly streamlined sample preparation workflows for single-cell analysis technologies, which minimize equipment requirements, enable lower processing times and costs, and pave the way for high-throughput, automated technologies. In this chapter, we survey recent developments and emerging trends in this field.

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