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Technology / AssayTechnology
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Nanowell-mediated 2D liquid chromatography separations for in-depth proteome profiling

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Description
The nanowell platform reduces the amount of protein required for multidimensional peptide separation, increasing the possible depth of coverage. Peptides are first separated using high-pH LC and the effluent is concatenated into 4 or 12 nanowells. The contents of each nanowell are reconstituted in LC buffer and collected for subsequent separation and analysis by low-pH nanoLC-MS/MS. The nanowell platform minimizes peptide losses to surfaces in offline 2D LC fractionation, enabling >5800 proteins to be confidently identified from just 50 ng of cell digest. As proof of principle, the platform was combined with a recently developed nanowell-based sample preparation workflow to do deep proteome profiling of >6000 protein groups from small populations of cells, including ∼650 HeLa cells and 10 single human pancreatic islet thin sections (∼1000 cells) from a pre-symptomatic type 1 diabetic donor.
Contributors
NameOrganizationConsortiumContact
Jing ChenUniversity of FloridaCMAI Contact
Mark AtkinsonUniversity of FloridaCBDS, HPAC, OPP Contact
Clayton MathewsUniversity of FloridaCMAI, CBDS, OPP Contact
Wei-Jun QianPacific Northwest National LaboratoryCBDS, OPP Contact
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