scholarly journals High-throughput and high-efficiency sample preparation for single-cell proteomics using a nested nanowell chip

2021 ◽  
Author(s):  
Jongmin Woo ◽  
Sarah M. Williams ◽  
Victor Aguilera-Vazquez ◽  
Ryan L. Sontag ◽  
Ronald J. Moore ◽  
...  
Keyword(s):  
Single Cell ◽  
High Efficiency ◽  
Single Cells ◽  
Protein Recovery ◽  
Processing Capacity ◽  
Protein Abundance ◽  
Proteome Coverage ◽  
Low Protein ◽  
Functional Differences ◽  
Cell Proteome

AbstractGlobal quantification of protein abundances in single cells would provide more direct information on cellular function phenotypes and complement transcriptomics measurements. However, single-cell proteomics (scProteomics) is still immature and confronts technical challenges, including limited proteome coverage, poor reproducibility, as well as low throughput. Here we describe a nested nanoPOTS (N2) chip to dramatically improve protein recovery, operation robustness, and processing throughput for isobaric-labeling-based scProteomics workflow. The N2 chip allows reducing cell digestion volume to <30 nL and increasing processing capacity to > 240 single cells in one microchip. In the analysis of ∼100 individual cells from three different cell lines, we demonstrate the N2 chip-based scProteomics platform can robustly quantify ∼1500 proteins and reveal functional differences. Our analysis also reveals low protein abundance variations (median CVs < 16.3%), highlighting the utility of such measurements, and also suggesting the single-cell proteome is highly stable for the cells cultured under identical conditions.

scholarly journals High-throughput and high-efficiency sample preparation for single-cell proteomics using a nested nanowell chip

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Jongmin Woo ◽  
Sarah M. Williams ◽  
Lye Meng Markillie ◽  
Song Feng ◽  
Chai-Feng Tsai ◽  
...  
Keyword(s):  
Single Cell ◽  
High Efficiency ◽  
Single Cells ◽  
Protein Recovery ◽  
Protein Abundance ◽  
Protein Markers ◽  
Low Protein ◽  
Tandem Mass Tag ◽  
Cellular Phenotypes ◽  
Cell Proteome

AbstractGlobal quantification of protein abundances in single cells could provide direct information on cellular phenotypes and complement transcriptomics measurements. However, single-cell proteomics is still immature and confronts many technical challenges. Herein we describe a nested nanoPOTS (N2) chip to improve protein recovery, operation robustness, and processing throughput for isobaric-labeling-based scProteomics workflow. The N2 chip reduces reaction volume to <30 nL and increases capacity to >240 single cells on a single microchip. The tandem mass tag (TMT) pooling step is simplified by adding a microliter droplet on the nested nanowells to combine labeled single-cell samples. In the analysis of ~100 individual cells from three different cell lines, we demonstrate that the N2 chip-based scProteomics platform can robustly quantify ~1500 proteins and reveal membrane protein markers. Our analyses also reveal low protein abundance variations, suggesting the single-cell proteome profiles are highly stable for the cells cultured under identical conditions.

scholarly journals Improved proteome coverage by using high efficiency cysteinyl peptide enrichment: The human mammary epithelial cell proteome

PROTEOMICS ◽  
2005 ◽  
Vol 5 (5) ◽  
pp. 1263-1273 ◽  
Author(s):  
Tao Liu ◽  
Wei-Jun Qian ◽  
Wan-Nan U. Chen ◽  
Jon M. Jacobs ◽  
Ronald J. Moore ◽  
...  
Keyword(s):  
Epithelial Cell ◽  
Mammary Epithelial Cell ◽  
High Efficiency ◽  
Human Mammary Epithelial Cell ◽  
Mammary Epithelial ◽  
Proteome Coverage ◽  
Human Mammary Epithelial ◽  
Peptide Enrichment ◽  
Cell Proteome

scholarly journals DART‐ID Increases Single‐Cell Proteome Coverage

2019 ◽  
Vol 33 (S1) ◽  
Author(s):  
Albert Tian Chen ◽  
Alexander Franks ◽  
Nikolai Slavov
Keyword(s):  
Single Cell ◽  
Proteome Coverage ◽  
Cell Proteome

Testing of solid oxide cells at high current densities

2021 ◽  
Vol 0 (0) ◽  
Author(s):  
André Weber
Keyword(s):  
Single Cell ◽  
High Performance ◽  
High Efficiency ◽  
Electrochemical Impedance ◽  
Single Cells ◽  
Operating Conditions ◽  
Solid Oxide ◽  
Current Densities ◽  
Cell Testing ◽  
The Impact

Abstract Solid Oxide Cells (SOCs) have gained an increasing interest as electrochemical energy converters due to their high efficiency, fuel flexibility and ability of reversible fuel cell/electrolysis operation. During the development process as well as in quality assurance tests, the performance of single cells and cell stacks is commonly evaluated by means of current/voltage- (CV-) characteristics. Despite of the fact that the measurement of a CV-characteristic seems to be simple compared to more complex, dynamic methods as electrochemical impedance spectroscopy or current interrupt techniques, the resulting performance strongly depends on the test setup and the chosen operating conditions. In this paper, the impact of different single cell testing environments and operating conditions on the CV-characteristic of high performance cells is discussed. The influence of cell size, contacting and current collection, contact pressure, fuel flow rate and composition on the achievable cell performance is presented and limitations arising from the test bed and testing conditions will be pointed out. As today’s high performance cells are capable of delivering current densities of several ampere per cm2 a special emphasis will be laid on single cell testing in this current range.

Hydrodynamic shuttling for deterministic high-efficiency multiple single-cell capture in a microfluidic chip

Lab on a Chip ◽  
2019 ◽  
Vol 19 (8) ◽  
pp. 1370-1377 ◽  
Author(s):  
Cheng-Kun He ◽  
Ya-Wen Chen ◽  
Ssu-Han Wang ◽  
Chia-Hsien Hsu
Keyword(s):  
Single Cell ◽  
Biomedical Research ◽  
Microfluidic Chip ◽  
High Efficiency ◽  
Single Cells ◽  
Cellular Heterogeneity ◽  
Cell Capture

A new microfluidics technique for high-efficiency paring and analyzing multiple single cells can facilitate cellular heterogeneity studies important for biological and biomedical research.

scholarly journals Global absolute quantification reveals tight regulation of protein expression in single Xenopus eggs

2014 ◽  
Vol 42 (15) ◽  
pp. 9880-9891 ◽  
Author(s):  
Arne H. Smits ◽  
Rik G.H. Lindeboom ◽  
Matteo Perino ◽  
Simon J. van Heeringen ◽  
Gert Jan C. Veenstra ◽  
...  
Keyword(s):  
Single Cell ◽  
Ribosomal Proteins ◽  
Single Cells ◽  
Transcript Level ◽  
Absolute Quantification ◽  
Protein Quantification ◽  
Protein Levels ◽  
Recent Developments ◽  
Basal Transcription Factors ◽  
Cell Proteome

Abstract While recent developments in genomic sequencing technology have enabled comprehensive transcriptome analyses of single cells, single cell proteomics has thus far been restricted to targeted studies. Here, we perform global absolute protein quantification of fertilized Xenopus laevis eggs using mass spectrometry-based proteomics, quantifying over 5800 proteins in the largest single cell proteome characterized to date. Absolute protein amounts in single eggs are highly consistent, thus indicating a tight regulation of global protein abundance. Protein copy numbers in single eggs range from tens of thousands to ten trillion copies per cell. Comparison between the single-cell proteome and transcriptome reveal poor expression correlation. Finally, we identify 439 proteins that significantly change in abundance during early embryogenesis. Downregulated proteins include ribosomal proteins and upregulated proteins include basal transcription factors, among others. Many of these proteins do not show regulation at the transcript level. Altogether, our data reveal that the transcriptome is a poor indicator of the proteome and that protein levels are tightly controlled in X. laevis eggs.

scholarly journals Single cell quantification of ribosome occupancy in early mouse development

2021 ◽  
Author(s):  
Tori Tonn ◽  
Hakan Ozadam ◽  
Crystal Han ◽  
Alia Segura ◽  
Duc Tran ◽  
...  
Keyword(s):  
Single Cell ◽  
Single Cells ◽  
Germinal Vesicle ◽  
Ribosome Profiling ◽  
Protein Abundance ◽  
Translation Efficiency ◽  
Proteomics Data ◽  
Mouse Development ◽  
High Coverage ◽  
Early Mouse

Technological limitations precluded transcriptome-wide analyses of translation at single cell resolution. To solve this challenge, we developed a novel microfluidic isotachophoresis approach, named RIBOsome profiling via IsoTachoPhoresis (Ribo-ITP), and characterized translation in single oocytes and embryos during early mouse development. We identified differential translation efficiency as a key regulatory mechanism of genes involved in centrosome organization and N6-methyladenosine modification of RNAs. Our high coverage measurements enabled the first analysis of allele-specific ribosome engagement in early development and led to the discovery of stage-specific differential engagement of zygotic RNAs with ribosomes. Finally, by integrating our measurements with proteomics data, we discovered that ribosome occupancy in germinal vesicle stage oocytes is the predominant determinant of protein abundance in the zygote. Taken together, these findings resolve the long-standing paradox of low correlation between RNA expression and protein abundance in early embryonic development. The novel Ribo-ITP approach will enable numerous applications by providing high coverage and high resolution ribosome occupancy measurements from ultra-low input samples including single cells.

Tagmentation-based single-cell genomics

2021 ◽  
Vol 31 (10) ◽  
pp. 1693-1705 ◽  
Author(s):  
Andrew C. Adey
Keyword(s):  
Single Cell ◽  
High Throughput Sequencing ◽  
High Efficiency ◽  
Single Cells ◽  
Molecular Techniques ◽  
Molecular Properties ◽  
Diverse Range ◽  
Single Cell Genomics ◽  
Sequencing Library ◽  
Regulatory Landscape

It has been just over 10 years since the initial description of transposase-based methods to prepare high-throughput sequencing libraries, or “tagmentation,” in which a hyperactive transposase is used to simultaneously fragment target DNA and append universal adapter sequences. Tagmentation effectively replaced a series of processing steps in traditional workflows with one single reaction. It is the simplicity, coupled with the high efficiency of tagmentation, that has made it a favored means of sequencing library construction and fueled a diverse range of adaptations to assay a variety of molecular properties. In recent years, this has been centered in the single-cell space with a catalog of tagmentation-based assays that have been developed, covering a substantial swath of the regulatory landscape. To date, there have been a number of excellent reviews on single-cell technologies structured around the molecular properties that can be profiled. This review is instead framed around the central components and properties of tagmentation and how they have enabled the development of innovative molecular tools to probe the regulatory landscape of single cells. Furthermore, the granular specifics on cell throughput or richness of data provided by the extensive list of individual technologies are not discussed. Such metrics are rapidly changing and highly sample specific and are better left to studies that directly compare technologies for assays against one another in a rigorously controlled framework. The hope for this review is that, in laying out the diversity of molecular techniques at each stage of these assay platforms, new ideas may arise for others to pursue that will further advance the field of single-cell genomics.

scholarly journals CLONAL GROWTH IN VITRO OF HUMAN CELLS WITH FIBROBLASTIC MORPHOLOGY

1957 ◽  
Vol 106 (1) ◽  
pp. 145-158 ◽  
Author(s):  
Theodore T. Puck ◽  
Steven J. Cieciura ◽  
Harold W. Fisher
Keyword(s):  
Single Cell ◽  
Cell Lines ◽  
High Efficiency ◽  
Single Cells ◽  
Previous History ◽  
Good Growth ◽  
Epithelioid Cells ◽  
Cultivation In Vitro ◽  
Fibroblastic Cells

A methodology has been described for reliable cultivation in vitro of dispersed fibroblastic cells obtained from normal human organs. The procedure has permitted establishment of stable cell lines from almost every sample taken, among which the following organs were represented: skin, spleen, amnion, lung, liver, bone marrow, brain, muscle, and heart. Equally good growth has been achieved with cells from embryonic or adult tissues. The methods previously developed whereby single cells plated in Petri dishes grow into isolated macroscopic colonies can successfully be applied to the plating of human fibroblastic stocks. Plating efficiencies in the neighborhood of 50 to 60 per cent are readily achieved with such strains. The resulting colonies can be picked and clonal stocks established. Fibroblastic morphology is maintained in the colonies arising from every single cell of such clonal stocks. All of the single cells from epithelioid clonal strains also maintain their integrity throughout repeated subculture. Since the difference between clonal stocks of these two types is always maintained whenever the respective single cells are plated in the same medium, regardless of the previous history of these stocks, it may be concluded that a true genetic difference exists in these cell lines. In addition to the morphological differences between epithelioid and fibroblastic cell strains, the latter have more demanding nutritional requirements for single cell growth. Thus, single cells of fibroblastic lines almost never produce colonies with high efficiency unless the growth medium which is sufficient for epithelioid cells is supplemented with embryo extract, or a cell feeder layer. Fibroblastic cells are also more resistant to tryptic digestion of the bond uniting the cells to glass surfaces. By use of differential media, growth of both fibroblastic and epithelioid cells, respectively, has been obtained, from dispersed single cells obtained by trypsinization of a specimen of human embryonic lung.

scholarly journals Single-cell metabolomics reveals the metabolic heterogeneity among microbial cells

2021 ◽  
Author(s):  
Xuanlin Meng ◽  
Fei Tao ◽  
Ping Xu
Keyword(s):  
Single Cell ◽  
High Efficiency ◽  
Single Cells ◽  
Proteome Analysis ◽  
Population Heterogeneity ◽  
Microbial Cells ◽  
Batch Correction ◽  
Multiple Dimensions ◽  
Metabolic Heterogeneity ◽  
Cell Metabolomics

In microbial research, the heterogeneity phenomenon is closely associated with microbial physiology in multiple dimensions. For now, A few studies were proposed in transcriptome and proteome analysis to discover the heterogeneity among single cells. However, microbial single cell metabolomics has not been possible yet. Herein, we developed a method, RespectM, based on discontinuous mass spectrometry imaging, which can detect more than 700 metabolites at a rate of 500 cells per hour. While ensuring the high throughput of RespectM, it integrates matrix sublimation, QC-based peak filtering, and batch correction strategies to improve accuracy. The results show that RespectM can distinguish single microbial cells from the blank matrix with an accuracy of 98.4%, depending on classification algorithms. Furthermore, to verify the accuracy of RespectM for distinguishing different single cells, we performed a classification test on Chlamydomonas reinhardtii single cells among allelic strains. The results showed an accuracy of 93.1%, which provides RespectM with enough confidence to perform microbial single cell metabolomics analysis. As we expected, untreated microbial cells will spontaneously undergo metabolic grouping coherence with genetic and biochemical similarities. Interestingly, the pseudo-time analysis also provided intuitive evidence on the metabolic dimension, indicating the cell grouping is based on microbial population heterogeneity. We believe that the RespectM can offer a powerful tool in the microbial study. Researchers can now directly analyze the changes in microbial metabolism at a single-cell level with high efficiency.

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