High-Throughput Single Cell Proteomics Enabled by Multiplex Isobaric Labeling in a Nanodroplet Sample Preparation Platform

Dou, Maowei; Clair, Gérémy; Tsai, Chia‐Feng; Xu, Kerui; Chrisler, William B.; Sontag, Ryan; Zhao, Rui; Moore, Ronald J.; Liu, Tao; Paša‐Tolić, Ljiljana; Smith, Richard; Shi, Tujin; Adkins, Joshua N.; Qian, Weijun; Kelly, Ryan; Ansong, Charles; Zhu, Ying

doi:10.1021/acs.analchem.9b03349

Cited by 168 publications

(227 citation statements)

References 71 publications

Supporting

Mentioning

218

Contrasting

Order By: Relevance

“…This design significantly enhances the detectability of the enabling comprehensive phosphoproteomic analysis of smaller samples 16 (e.g., quantification of >20,000 phosphosites from human pancreatic islet). More recently, we have also incorporated isobaric TMT labeling into our nanoPOTS workflow for enabling reliable clustering of 61 single cells from three different cell lines 17 .…”

Section: Downloaded Frommentioning

confidence: 99%

“…The Influx II cell sorter (BD Biosciences, San Jose, CA) was used to isolate single cells directly into the nanowells 11 . The procedures for microchip fabrication and single cell isolation were described previously 17 .…”

Section: Single Cell Isolation By Facsmentioning

confidence: 99%

“…The nanoPOTS-based proteomic sample preparation and TMT labeling procedures were described previously 17 , with slight modification. In brief, isolated single cells were lysed in 100 nL lysis buffer (0.1% (m/v) DDM in 50 mM TEAB and 0.5 × PBS buffer) and incubated at 70 °C for 60 mins to achieve efficient protein extraction.…”

Section: Downloaded Frommentioning

confidence: 99%

See 2 more Smart Citations

An Improved Boosting to Amplify Signal with Isobaric Labeling (iBASIL) Strategy for Precise Quantitative Single-cell Proteomics

Tsai

Zhao

Williams

et al. 2020

Molecular & Cellular Proteomics

Self Cite

133

211

View full text Add to dashboard Cite

Mass spectrometry (MS)-based proteomics has great potential for overcoming the limitations of antibody-based immunoassays for antibody-independent, comprehensive, and quantitative proteomic analysis of single cells. Indeed, recent advances in nanoscale sample preparation have enabled effective processing of single cells. In particular, the concept of using boosting/carrier channels in isobaric labeling to increase the sensitivity in MS detection has also been increasingly used for quantitative proteomic analysis of small-sized samples including single cells. However, the full potential of such boosting/carrier approaches has not been significantly explored, nor has the resulting quantitation quality been carefully evaluated. Herein, we have further evaluated and optimized our recent boosting to amplify signal with isobaric labeling (BASIL) approach, originally developed for quantifying phosphorylation in small number of cells, for highly effective analysis of proteins in single cells. This improved BASIL (iBASIL) approach enables reliable quantitative single-cell proteomics analysis with greater proteome coverage by carefully controlling the boosting-to-sample ratio (e.g. in general <100×) and optimizing MS automatic gain control (AGC) and ion injection time settings in MS/MS analysis (e.g. 5E5 and 300 ms, respectively, which is significantly higher than that used in typical bulk analysis). By coupling with a nanodroplet-based single cell preparation (nanoPOTS) platform, iBASIL enabled identification of ∼2500 proteins and precise quantification of ∼1500 proteins in the analysis of 104 FACS-isolated single cells, with the resulting protein profiles robustly clustering the cells from three different acute myeloid leukemia cell lines. This study highlights the importance of carefully evaluating and optimizing the boosting ratios and MS data acquisition conditions for achieving robust, comprehensive proteomic analysis of single cells.

show abstract

Section: Downloaded Frommentioning

confidence: 99%

Section: Single Cell Isolation By Facsmentioning

confidence: 99%

See 1 more Smart Citation

An Improved Boosting to Amplify Signal with Isobaric Labeling (iBASIL) Strategy for Precise Quantitative Single-cell Proteomics

Tsai

Zhao

Williams

et al. 2020

Molecular & Cellular Proteomics

Self Cite

133

211

View full text Add to dashboard Cite

show abstract

“…However, given recent advancements in high-throughput molecular profiling technologies, rapid progress is being made [21,25,33]. The establishment of the LungMAP program, and its success in obtaining human tissues for structural, cellular and molecular analysis has, and will continue to, facilitate this progress [22,[36][37][38]. Here, we report transcriptional analysis of newborn human lung cells, including all major cell types, and describe the molecular profile for matrix fibroblast subtypes that may represent cells at different states of maturity.…”

Section: Discussionmentioning

confidence: 99%

Single cell transcriptomic profiling identifies molecular phenotypes of newborn human lung cells

Bhattacharya¹,

Myers

Baker

et al. 2020

Preprint

View full text Add to dashboard Cite

While animal model studies have extensively defined mechanisms controlling cell diversity in the developing mammalian lung, the limited data available from late stage human lung development represents a significant knowledge gap. The NHLBI Molecular Atlas of Lung Development Program (LungMAP) seeks to fill this gap by creating a structural, cellular and molecular atlas of the human and mouse lung. Single cell RNA sequencing generated transcriptional profiles of 5500 cells obtained from two newborn human lungs from the LungMAP Human Tissue Core Biorepository. Frozen single cell isolates were captured, and library preparation was completed on the Chromium 10X system. Data was analyzed in Seurat, and cellular annotation was performed using the ToppGene functional analysis tool. Single cell sequence data from an additional 32000 postnatal day 1 through 10 mouse lung cells generated by the LungMAP Cincinnati Research Center was integrated with the human data. Transcriptional interrogation of newborn human lung cells identified distinct clusters representing multiple populations of epithelial, endothelial, fibroblasts, pericytes, smooth muscle, and immune cells and signature genes for each of these population. Computational integration of newborn human and postnatal mouse lung development cellular transcriptomes facilitated the identification of distinct epithelial lineages including AT1, AT2 and ciliated epithelial cells. Integration of the newborn human and mouse cellular transcriptomes also demonstrated cell type-specific differences in maturation states of newborn human lung cells.In particular, newborn human lung matrix fibroblasts could be separated into those representative of younger cells (n=393), or older cells (n=158). Cells with each molecular profile were spatially resolved within newborn human lung tissue. This is the first comprehensive molecular map of the cellular landscape of neonatal human lung, including biomarkers for cells at distinct states of maturity.

show abstract

“…Moreover, ScoPE-MS enabled identification of cell-to-cell heterogeneity during the differentiation process of embryonic stem cells [119]. The same principle was then implemented with the nanoPOTS approach which improved the proteome coverage further [120]. In this work, the proteome amount to be used as carrier was revisited, and 50-times the amount estimated for a single cell was found to be the maximum to ensure quantitative accuracy.…”

Section: A New Venue For Ms-based Proteomics: Single-cell Analysismentioning

confidence: 99%

Mass Spectrometry Advances and Perspectives for the Characterization of Emerging Adoptive Cell Therapies

Lombard‐Banek

Schiel

2020

Molecules

View full text Add to dashboard Cite

Adoptive cell therapy is an emerging anti-cancer modality, whereby the patient’s own immune cells are engineered to express T-cell receptor (TCR) or chimeric antigen receptor (CAR). CAR-T cell therapies have advanced the furthest, with recent approvals of two treatments by the Food and Drug Administration of Kymriah (trisagenlecleucel) and Yescarta (axicabtagene ciloleucel). Recent developments in proteomic analysis by mass spectrometry (MS) make this technology uniquely suited to enable the comprehensive identification and quantification of the relevant biochemical architecture of CAR-T cell therapies and fulfill current unmet needs for CAR-T product knowledge. These advances include improved sample preparation methods, enhanced separation technologies, and extension of MS-based proteomic to single cells. Innovative technologies such as proteomic analysis of raw material quality attributes (MQA) and final product quality attributes (PQA) may provide insights that could ultimately fuel development strategies and lead to broad implementation.

show abstract

High-Throughput Single Cell Proteomics Enabled by Multiplex Isobaric Labeling in a Nanodroplet Sample Preparation Platform

Cited by 168 publications

References 71 publications

An Improved Boosting to Amplify Signal with Isobaric Labeling (iBASIL) Strategy for Precise Quantitative Single-cell Proteomics

An Improved Boosting to Amplify Signal with Isobaric Labeling (iBASIL) Strategy for Precise Quantitative Single-cell Proteomics

Single cell transcriptomic profiling identifies molecular phenotypes of newborn human lung cells

Mass Spectrometry Advances and Perspectives for the Characterization of Emerging Adoptive Cell Therapies

Contact Info

Product

Resources

About