Nanodroplet processing platform for deep and quantitative proteome profiling of 10–100 mammalian cells
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The document discusses advancements in micro-proteomics aimed at analyzing small cell populations, highlighting the need for sensitive measurement technologies. It details the development of a nanopots platform allowing for effective processing of minimal samples, achieving detection of 1500 to 3000 proteins from just a few cells. Challenges in protein analysis, including amplification and post-translational modifications, are addressed along with the platform's capabilities in enhancing analytical sensitivity.
Nanodroplet processing platform for deep and quantitative proteome profiling of 10–100 mammalian cells
- 1. NATURE COMMUNICATIONS 28 February, 2018 DOI: 10.1038/s41467-018-03367-w Journal Club Presenter: Gul Muneer Dated: 20191225
- 2. Genomics/Transcriptomics to Proteomics ― WHAT and WHY? TranscriptomicsGenomics Single-cell isolation Rare cell populations Specific cells Clinical tissues Needle aspiration biopsies Genome Transcriptome Proteome Phenotype Genomics & Transcriptomics INDIRECTcharacterizationofPhenotypes DIRECT Biomedical field is in critical need of highly sensitive technologies for proteome measurement of small number of cells.
- 3. (macro)Proteomics to (micro)Proteomics ― WHY? Single cell analysis Measure each cell individually to understand biological system Track original spatial organization Locate trace components Clinical tissues Challenges in measurement sensitivity and throughput Bulk analysis A lot of material, but…. Measure an average of the constituents Lost information are: • Composition (e.g., trace components) • Spatial organization
- 4. Micro-proteomics ― A Dream of Many and Its Challenges Unlike DNA/RNA, proteins don’t benefit from Amplification. Post Translational Modification (PTM) adds another dimension of difficulty. If no amplification then bring more sample to achieve deep coverage. Thus, ‘dreamers’ changed their focus to enhance “analytical sensitivity”.
- 5. Micro-proteomics ― Efforts and Achievements Mass detection limit 10 zmol (10⁻²¹ moles) for MS and 50 zmol for tandem MS This level is sufficient to detect many proteins at levels expressed in single cells.
- 6. Micro-proteomics ― Efforts and Achievements ~600 proteins were reported using only 100 cells. “Single cell” has been reported for proteome profiling of large cells (e.g., blastomeres). ~800 proteins were identified from ~20 ng tryptic peptides of single blastomeres. “One-pot” digestion and low binding sample tubes has been previously used to attempt Micro-proteomics.
- 7. Rationale of the Study Urea DTT Trypsin Desalting Collection Concentration Homogenization Extraction Sample Amount decreased but not reaction volume. Non-specific adsorption of peptides/proteins to surface of reaction vessels To advance sample processing efficiency for nanoscale biological samples
- 8. Nanowell-based sample preparation for small cell populations Platform Name: NanoPOTs (Nanodroplet Processing in One pot for Trace samples). Automated dispensing system (robotic arm) Reaction volume: < 200nL. LC-MS Platform for ultra small samples: 30-μ, i.d., 70-cm, 60nL/min Aim of the Study
- 9. Photomask designed with AutoCAD® Direct-write Lithography Chen’s Lab Photomask Light source Condenser lens Photomask Projector lens Photoresist Understanding of NanoPOTs Platform Design and Fabrication
- 10. NanoPOTs Platform Design and Fabrication
- 13. 50 nL cell suspension 50 nL Rapigest + DTT 70ᵒC, 30 minutes 50 nL IAA 50 nL Enzyme (Lys-C) 50 nL Enzyme (Trypsin) 50 nL Formic acid 1 hours incubation
- 14. Sensitivity for (processing and analyzing) small numbers of cells
- 15. Proteome coverage for small numbers of cells ~1500 proteins from 10 to 14 cells ~3000 proteins from 140 cells. With MBR, ~3000 proteins from 10 cells ~300 proteins from 10 cells ~2000 proteins from 140 cells Nanoliter-based platform is beneficial for ultra small samples.
- 16. Assessment of Absolute sensitivity of nanoPOTs plaform Protein copy numbers determined per HeLa cell we can confidently conclude the detection limit of nanoPOTS for protein is <5 × 10⁵ copies, or <830 zmol.
- 17. 2892 proteins matched the proteins in 10-14 samples. . Assessment of Absolute sensitivity of nanoPOTs plaform
- 18. Label-free Quantification Reproducibility ~13000 peptides quantifiable for 10-14 cells ~2600 proteins were quantifiable for same.
- 19. Schematic Workflow for analysis of Pancreatic Islet Sections An average of 2676 and a total of 3219 proteins were identified for the 18 single islet sections.
- 20. Correlation analysis and Comparison of protein abundances
- 21. Conclusion and Discussion A sophisticated and beautiful platform for small cell population proteome analysis. Extremely low processing volume <200 nL to minimize surface losses. Identification of ~1500 to ~3000 proteins from ~10 to ~140 cells With MBR algorithm, >3000 proteins from ~10 cells. ~2400 quantified proteins from single human pancreatic islet thin sections. 200 nL is still ~10⁵ times larger than a single cell (PicoPOTs?) With MBR algorithm, whether it could be said that >3000 proteins form ~10 cells? Delivery to analytical platform is not automated yet. Mis-cleavage 23% to 28% (in-efficient digestion?)
- 22. Thank you