Single Cell Proteomics: the Potential of Laser Capture Microdissection-Quanterix SIMOA Immunoassays & Protein Simple Milo Single Cell Multiplex Westerns
Matthew Davison*, Marty Kramer, Jean Cavallo, David Chain1, Jason Stafford, Kelly Gardner2 1Translational Sciences, Sanofi Genzyme, Framingham, MA 01701, USA 2Milo Single Cell Western Team, Protein Simple, San Jose, CA, USA
The cell is the fundamental unit of biology and protein expression/modifications regulate cell functions. Monitoring and quantifying the proteins at the single cell level requires new ultra sensitive, broad dynamic range technologies able to detect both low abundanceproteins~ 5,000 molecules per cell up to high abundance proteins with > 100 million molecules per cell, median ~ 170,000 (1). We performed proof of principle experiments with two promising new technologies.
Firstly, we coupled extraction of low numbers epithelial cells from human prostate tissue slices by laser capture microdissection (LCM) with a Quanterix single molecule array (SIMOA)(2,3) immunoassay for prostate specific antigen (PSA). We accurately measured PSA levels from lysates from only 5 cells using only 1/100th of the total lysate, which indicates single cell sensitivity. We calculated PSA was present at about 12 million molecules per cell. We were able to quantitate PSA down to 137,000 molecules per assay replicate. Additional data from single cells will be present.
Secondly, we used the Protein Simple Milo single cell multiplex western technology (4) to try to ‘immunoprofile’ by quantitating 4 proteins (CD3, CD4, CCR7, FoxP3) in single cells from normal human PBMC. This technology can analyze 1,000 single cells per assay chip. FOXP3 was successfully detected in 8% of stimulated PBMC with a ten-fold range of signal intensity, and housekeeping β-tubulin in all cells. Additional data on the other 3 markers in the multiplex will be presented.
1. Li JJ et al. (2014) PeerJ2:e270
2. Rissin DM et al. (2011) Analytical Chem 83:2279-2285
3. Schubert SM et al. (2016) Analytical Chem 88:2952-2957
4. Hughes AJ et al. (2014) Nature Methods 11:749-760
Credits: None available.
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