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. 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.
- Supporting the Biomolecular Pathways Integrated Research Platform, these resources reveal cell-type-specific pathway alterations during pathogen infection or cell/organ development.
- Supporting the Cell Signaling and Communications Integrated Research Platform, these resources reveal spatially resolved proteome variation and cell-cell communication events.
- Supporting the Rhizosphere Function Integrated Research Platform, these resources enable understanding of how individual plant cells respond to genome perturbations and abiotic stresses such as drought, salinity, and heat.
- liquid chromatography-mass spectrometry (LC-MS)
- Eclipse Orbitrap
- Lumos Orbitrap
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.