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Nanodroplet Processing in One Pot for Trace Samples (NanoPOTS)

NanoPOTS (Nanodroplet Processing in One pot for Trace Samples) enables scientists to identify more than 3,000 proteins from as few as 10 cells.
NanoPOTS (Nanodroplet Processing in One pot for Trace Samples) enables scientists to identify more than 3,000 proteins from as few as 10 cells.
Sarai Williams holds a nano pots slide.
NanoPOTS (Nanodroplet Processing in One pot for Trace Samples) enables scientists to identify more than 3,000 proteins from as few as 10 cells.

Nanodroplet Processing in One Pot for Trace Samples, or nanoPOTS, is a novel microfluidic sample preparation platform specially designed for proteomics profiling of limited biological samples, as small as a single cell. The nanoPOTS platform consists of microfabricated nanowell array chips and a nanoliter robotic pipetting system. The chips are manufactured in-house using technologies in EMSL's clean room. Proteomics sample processing is performed in nanoliter volumes with enhanced efficiency and sample recovery, enabling orders of magnitude more information from small samples than what’s available from existing technologies. Specifically, we employ flow cytometry or laser microdissection to isolate a small population or single cell into a nanoPOTS chip. 

After sample preparation with nanoPOTS, we employ our custom automated nanosampling injection system coupled with ultrasensitive liquid chromatography mass spectrometry for the high-throughput measurement of global protein expression. Currently, the nanoPOTS platform has identified and quantified over 1,000 proteins from dissociated single cells or a small number of microdissected cells from tissue sections.

Research application

Available resources

Tips for success

  • The nanoPOTS platform requires isolated single cells or tissue voxels to be inserted directly into microfabricated nanowell devices. We suggest performing all sample preparation and omics analysis at EMSL, including cell dissociation, cell isolation, protein digestion, and LC-MS analysis.
  • To get the best proteome coverage, we suggest using fresh-frozen samples. Samples should be shipped on dry ice for preservation. EMSL staff should be notified in advance to arrange for receiving.
  • Contaminations should be minimized to achieve optimal measurement results. Contaminations includes exogenous proteins, such as BSA and human keratins, and various surfactants like SDS and protease inhibitors.
  • Studies should be well designed to answer scientific questions, while keeping the scope feasible. Appropriate control samples and biological/technical replicates should be considered during study design. Prior discussion with EMSL scientists is encouraged.

Contributing teams and resources

EMSL develops and deploys capabilities for the user program by conducting original research independently or in partnership with others and by adapting/advancing science and technologies developed outside of EMSL. In some instances, EMSL directly deploys mature capabilities developed by others where there is value for the EMSL user community. The following grants/activities, PI’s and teams contributed to the development of this capability: 

NanoPOTS

  • Ryan Kelly, EB020976, National Institute of Health 
  • Richard D. Smith, P41 GM103493
  • W.J. Qian UC4 DK104167 and DP3 DK110844
  • EMSL Instrument Development Laboratory funding 

NanoPOTS autosampler for liquid chromatography-mass spectrometry

Ying Zhu, LDRD, Environmental Molecular Sciences Laboratory

Richard D. Smith, 41 GM103493, National Institute of Health

Richard D. Smith and Tao Liu, Clinical Proteomic Tumor Analysis Consortium

NanoSPLITS 

EMSL Partner proposal with Scienion and Environmental Molecular Sciences Laboratory LDRD funding 

N2 chip

Ying  Zhu, LDRD, Environmental Molecular Sciences Laboratory, EMSL Intramural program

Josh Adkins and Geremy Clair, U01 HL122703, U01 HL148860, National Institute of Health

Richard D. Smith, P41 GM103493, National Institute of Health

 

Publications

NanoPOTS

 Ying Zhu, Paul D. Piehowski, Rui Zhao, Jing Chen, Yufeng Shen, Ronald J. Moore, Anil K. Shukla, Vladislav A. Petyuk, Martha Campbell-Thompson, Clayton E. Mathews, Richard D. Smith, Wei-Jun Qian & Ryan T. Kelly. Nanodroplet processing platform for deep and quantitative proteome profiling of 10–100 mammalian cells. Nat Commun. 9, 882 (2018) https://www.nature.com/articles/s41467-018-03367-w

NanoPOTS autosampler for liquid chromatography-mass spectrometry

Sarah M. Williams, Andrey V. Liyu, Chia-Feng Tsai, Ronald J. Moore, Daniel J. Orton, William B. Chrisler, Matthew J. Gaffrey, Tao Liu, Richard D. Smith, Ryan T. Kelly, Ljiljana Pasa-Tolic, and Ying Zhu. Automated Coupling of Nanodroplet Sample Preparation with Liquid Chromatography–Mass Spectrometry for High-Throughput Single-Cell Proteomics. Anal Chem. 2020 92 (15), 10588-10596. https://pubs.acs.org/doi/10.1021/acs.analchem.0c01551

N2 chip

High-throughput and high-efficiency sample preparation for single-cell proteomics using a nested nanowell chip. Woo J, Williams SM, Markillie LM, Feng S, Tsai CF, Aguilera-Vazquez V, Sontag RL, Moore RJ, Hu D, Mehta HS, Cantlon-Bruce J, Liu T, Adkins JN, Smith RD, Clair GC, Paša-Tolić L, Zhu Y. Nat Commun. 2021 Oct 29;12(1):6246. doi: 10.1038/s41467-021-26514-2.

Contacts