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 DART-ID increases single-cell proteome coverage

2018 ◽  
Author(s):  
Albert T. Chen ◽  
Alexander Franks ◽  
Nikolai Slavov
Keyword(s):  
Mass Spectrometry ◽  
Liquid Chromatography ◽  
Single Cell ◽  
Retention Time ◽  
Mammalian Cells ◽  
Statistical Power ◽  
Single Cells ◽  
Peptide Sequence ◽  
Mass Spectrometry Analysis ◽  
Spectrometry Analysis

AbstractAnalysis by liquid chromatography and tandem mass spectrometry (LC-MS/MS) can identify and quantify thousands of proteins in microgram-level samples, such as those comprised of thousands of cells. This process, however, remains challenging for smaller samples, such as the proteomes of single mammalian cells, because reduced protein levels reduce the number of confidently sequenced peptides. To alleviate this reduction, we developed Data-driven Alignment of Retention Times for IDentification (DART-ID). DART-ID implements principled Bayesian frameworks for global retention time (RT) alignment and for incorporating RT estimates towards improved confidence estimates of peptide-spectrum-matches. When applied to bulk or to single-cell samples, DART-ID increased the number of data points by 30 – 50% at 1% FDR, and thus decreased missing data. Benchmarks indicate excellent quantification of peptides upgraded by DART-ID and support their utility for quantitative analysis, such as identifying cell types and cell-type specific proteins. The additional datapoints provided by DART-ID boost the statistical power and double the number of proteins identified as differentially abundant in monocytes and T-cells. DART-ID can be applied to diverse experimental designs and is freely available at http://github.com/SlavovLab/DART-ID.Author SummaryIdentifying and quantifying proteins in single cells gives researchers the ability to tackle complex biological problems that involve single cell heterogeneity, such as the treatment of solid tumors. Mass spectrometry analysis of peptides can identify their sequence from their masses and the masses of their fragment ion, but often times these pieces of evidence are insufficient for a confident peptide identification. This problem is exacerbated when analyzing lowly abundant samples such as single cells. To identify even peptides with weak mass spectra, DART-ID incorporates their retention time – the time when they elute from the liquid chromatography used to physically separate them. We present both a novel method of aligning the retention times of peptides across experiments, as well as a rigorous framework for using the estimated retention times to enhance peptide sequence identification. Incorporating the retention time as additional evidence leads to a substantial increase in the number of samples in which proteins are confidently identified and quantified.

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 Cryopreservation of human cancers conserves tumour heterogeneity for single-cell multi-omics analysis

2021 ◽  
Vol 13 (1) ◽  
Author(s):  
Sunny Z. Wu ◽  
Daniel L. Roden ◽  
Ghamdan Al-Eryani ◽  
Nenad Bartonicek ◽  
Kate Harvey ◽  
...  
Keyword(s):  
Single Cell ◽  
Rna Sequencing ◽  
High Throughput ◽  
Single Cells ◽  
Cellular Heterogeneity ◽  
Tumour Heterogeneity ◽  
Fresh Tissue ◽  
Human Cancers ◽  
Cryopreserved Cell ◽  
Single Cell Rna Sequencing

Abstract Background High throughput single-cell RNA sequencing (scRNA-Seq) has emerged as a powerful tool for exploring cellular heterogeneity among complex human cancers. scRNA-Seq studies using fresh human surgical tissue are logistically difficult, preclude histopathological triage of samples, and limit the ability to perform batch processing. This hindrance can often introduce technical biases when integrating patient datasets and increase experimental costs. Although tissue preservation methods have been previously explored to address such issues, it is yet to be examined on complex human tissues, such as solid cancers and on high throughput scRNA-Seq platforms. Methods Using the Chromium 10X platform, we sequenced a total of ~ 120,000 cells from fresh and cryopreserved replicates across three primary breast cancers, two primary prostate cancers and a cutaneous melanoma. We performed detailed analyses between cells from each condition to assess the effects of cryopreservation on cellular heterogeneity, cell quality, clustering and the identification of gene ontologies. In addition, we performed single-cell immunophenotyping using CITE-Seq on a single breast cancer sample cryopreserved as solid tissue fragments. Results Tumour heterogeneity identified from fresh tissues was largely conserved in cryopreserved replicates. We show that sequencing of single cells prepared from cryopreserved tissue fragments or from cryopreserved cell suspensions is comparable to sequenced cells prepared from fresh tissue, with cryopreserved cell suspensions displaying higher correlations with fresh tissue in gene expression. We showed that cryopreservation had minimal impacts on the results of downstream analyses such as biological pathway enrichment. For some tumours, cryopreservation modestly increased cell stress signatures compared to freshly analysed tissue. Further, we demonstrate the advantage of cryopreserving whole-cells for detecting cell-surface proteins using CITE-Seq, which is impossible using other preservation methods such as single nuclei-sequencing. Conclusions We show that the viable cryopreservation of human cancers provides high-quality single-cells for multi-omics analysis. Our study guides new experimental designs for tissue biobanking for future clinical single-cell RNA sequencing studies.

Transplantation of embryonic stem cells improves cardiac function in postinfarcted rats

2002 ◽  
Vol 92 (1) ◽  
pp. 288-296 ◽  
Author(s):  
Jiang-Yong Min ◽  
Yinke Yang ◽  
Kimber L. Converso ◽  
Lixin Liu ◽  
Qin Huang ◽  
...  
Keyword(s):  
Cardiac Function ◽  
Cell Transplantation ◽  
Troponin I ◽  
Es Cells ◽  
Single Cells ◽  
Embryonic Stem ◽  
Control Group ◽  
Positive Staining ◽  
Es Cell ◽  
Intramyocardial Injection

Massive loss of cardiac myocytes after myocardial infarction (MI) is a common cause of heart failure. The present study was designed to investigate the improvement of cardiac function in MI rats after embryonic stem (ES) cell transplantation. MI in rats was induced by ligation of the left anterior descending coronary artery. Cultured ES cells used for cell transplantation were transfected with the marker green fluorescent protein (GFP). Animals in the treated group received intramyocardial injection of ES cells in injured myocardium. Compared with the MI control group injected with an equivalent volume of the cell-free medium, cardiac function in ES cell-implanted MI animals was significantly improved 6 wk after cell transplantation. The characteristic phenotype of engrafted ES cells was identified in implanted myocardium by strong positive staining to sarcomeric α-actin, cardiac α-myosin heavy chain, and troponin I. GFP-positive cells in myocardium sectioned from MI hearts confirmed the survival and differentiation of engrafted cells. In addition, single cells isolated from cell-transplanted MI hearts showed rod-shaped GFP-positive myocytes with typical striations. The present data demonstrate that ES cell transplantation is a feasible and novel approach to improve ventricular function in infarcted failing hearts.

scholarly journals Transformative opportunities for single-cell proteomics

2018 ◽  
Author(s):  
Harrison Specht ◽  
Nikolai Slavov
Keyword(s):  
Mass Spectrometry ◽  
Stem Cell ◽  
Cell Differentiation ◽  
Single Cell ◽  
Single Cells ◽  
Biological Systems ◽  
Stem Cell Differentiation ◽  
New Age ◽  
Technological Developments

Many pressing medical challenges - such as diagnosing disease, enhancing directed stem cell differentiation, and classifying cancers - have long been hindered by limitations in our ability to quantify proteins in single cells. Mass-spectrometry (MS) is poised to transcend these limitations by developing powerful methods to routinely quantify thousands of proteins and proteoforms across many thousands of single cells. We outline specific technological developments and ideas that can increase the sensitivity and throughput of single cell MS by orders of magnitude and usher in this new age. These advances will transform medicine and ultimately contribute to understanding biological systems on an entirely new level.

scholarly journals Target frequency and integration pattern for insertion and replacement vectors in embryonic stem cells

1991 ◽  
Vol 11 (9) ◽  
pp. 4509-4517
Author(s):  
P Hasty ◽  
J Rivera-Pérez ◽  
C Chang ◽  
A Bradley
Keyword(s):  
Homologous Recombination ◽  
Gene Targeting ◽  
Mammalian Cells ◽  
Germ Line ◽  
Es Cells ◽  
Embryonic Stem ◽  
Homologous Sequence ◽  
Reciprocal Recombination ◽  
Replacement Vector ◽  
Insertion Vector

Gene targeting has been used to direct mutations into specific chromosomal loci in murine embryonic stem (ES) cells. The altered locus can be studied in vivo with chimeras and, if the mutated cells contribute to the germ line, in their offspring. Although homologous recombination is the basis for the widely used gene targeting techniques, to date, the mechanism of homologous recombination between a vector and the chromosomal target in mammalian cells is essentially unknown. Here we look at the nature of gene targeting in ES cells by comparing an insertion vector with replacement vectors that target hprt. We found that the insertion vector targeted up to ninefold more frequently than a replacement vector with the same length of homologous sequence. We also observed that the majority of clones targeted with replacement vectors did not recombine as predicted. Analysis of the recombinant structures showed that the external heterologous sequences were often incorporated into the target locus. This observation can be explained by either single reciprocal recombination (vector insertion) of a recircularized vector or double reciprocal recombination/gene conversion (gene replacement) of a vector concatemer. Thus, single reciprocal recombination of an insertion vector occurs 92-fold more frequently than double reciprocal recombination of a replacement vector with crossover junctions on both the long and short arms.

scholarly journals NudCL2 regulates cell migration by stabilizing both myosin-9 and LIS1 with Hsp90

2020 ◽  
Vol 11 (7) ◽  
Author(s):  
Wenwen Chen ◽  
Wei Wang ◽  
Xiaoxia Sun ◽  
Shanshan Xie ◽  
Xiaoyang Xu ◽  
...  
Keyword(s):  
Cell Migration ◽  
Single Cell ◽  
Mammalian Cells ◽  
Ectopic Expression ◽  
Heat Shock Protein 90 ◽  
Underlying Mechanism ◽  
Collective Cell Migration ◽  
Interacting Protein ◽  
Protein Levels ◽  
Protein Instability

Abstract Cell migration plays pivotal roles in many biological processes; however, its underlying mechanism remains unclear. Here, we find that NudC-like protein 2 (NudCL2), a cochaperone of heat shock protein 90 (Hsp90), modulates cell migration by stabilizing both myosin-9 and lissencephaly protein 1 (LIS1). Either knockdown or knockout of NudCL2 significantly increases single-cell migration, but has no significant effect on collective cell migration. Immunoprecipitation–mass spectrometry and western blotting analyses reveal that NudCL2 binds to myosin-9 in mammalian cells. Depletion of NudCL2 not only decreases myosin-9 protein levels, but also results in actin disorganization. Ectopic expression of myosin-9 efficiently reverses defects in actin disorganization and single-cell migration in cells depleted of NudCL2. Interestingly, knockdown of myosin-9 increases both single and collective cell migration. Depletion of LIS1, a NudCL2 client protein, suppresses both single and collective cell migration, which exhibits the opposite effect compared with myosin-9 depletion. Co-depletion of myosin-9 and LIS1 promotes single-cell migration, resembling the phenotype caused by NudCL2 depletion. Furthermore, inhibition of Hsp90 ATPase activity also reduces the Hsp90-interacting protein myosin-9 stability and increases single-cell migration. Forced expression of Hsp90 efficiently reverses myosin-9 protein instability and the defects induced by NudCL2 depletion, but not vice versa. Taken together, these data suggest that NudCL2 plays an important role in the precise regulation of cell migration by stabilizing both myosin-9 and LIS1 via Hsp90 pathway.

Sign in / Sign up

Export Citation Format

Share Document