Nanowell-mediated two-dimensional liquid chromatography enables deep proteome profiling of <1000 mammalian cells
Multidimensional peptide separations can greatly increase the depth of coverage in proteome profiling. However, a major challenge for multidimensional separations is the requirement of large biological samples, often containing milligram amounts of protein. We have developed nanowell-mediated two-di...
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Published in | Chemical science (Cambridge) Vol. 9; no. 34; pp. 6944 - 6951 |
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Main Authors | , , , , , , , , , , , , |
Format | Journal Article |
Language | English |
Published |
England
Royal Society of Chemistry
14.09.2018
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Subjects | |
Online Access | Get full text |
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Summary: | Multidimensional peptide separations can greatly increase the depth of coverage in proteome profiling. However, a major challenge for multidimensional separations is the requirement of large biological samples, often containing milligram amounts of protein. We have developed nanowell-mediated two-dimensional (2D) reversed-phase nanoflow liquid chromatography (LC) separations for in-depth proteome profiling of low-nanogram samples. 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 HeLa digest. Furthermore, in combination with a recently developed nanowell-based sample preparation workflow, we demonstrated 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. |
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Bibliography: | ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 USDOE DP3DK110844; UC4DK104167; R33CA225248; R21EB020976; AC05-76RL01830 PNNL-SA-133217 |
ISSN: | 2041-6520 2041-6539 |
DOI: | 10.1039/c8sc02680g |