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  Loss-less Nano-fractionator for High Sensitivity, High Coverage Proteomics

Kulak, N. A., Geyer, P. E., & Mann, M. (2017). Loss-less Nano-fractionator for High Sensitivity, High Coverage Proteomics. Molecular and Cellular Proteomics, 16(4), 694-705. doi:10.1074/mcp.O116.065136.

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Mol Cell Proteomics-2017-Kulak-694-705.pdf (Publisher version), 2MB
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Mol Cell Proteomics-2017-Kulak-694-705.pdf
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Author's Choice—Final version free via Creative Commons CC-BY license.

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http://www.mcponline.org/content/16/4/694/suppl/DC1 (Supplementary material)
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 Creators:
Kulak, Nils A.1, Author              
Geyer, Philipp E.1, Author              
Mann, Matthias1, Author              
Affiliations:
1Mann, Matthias / Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, Max Planck Society, ou_1565159              

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Free keywords: IMPROVED PROTEIN IDENTIFICATION; STRONG-CATION-EXCHANGE; LIQUID-CHROMATOGRAPHY; MASS-SPECTROMETRY; HIGH PH; PEPTIDE FRACTIONATION; PHASE CHROMATOGRAPHY; CELL-LINES; SEPARATION; HPLCBiochemistry & Molecular Biology;
 Abstract: Recent advances in mass spectrometry (MS)-based proteomics now allow very deep coverage of cellular proteomes. To achieve near-comprehensive identification and quantification, the combination of a first HPLC-based peptide fractionation orthogonal to the on-line LC-MS/MS step has proven to be particularly powerful. This first dimension is typically performed with milliliter/min flow and relatively large column inner diameters, which allow efficient pre-fractionation but typically require peptide amounts in the milligram range. Here, we describe a novel approach termed spider fractionator in which the post-column flow of a nanobore chromatography system enters an eight-port flow-selector rotor valve. The valve switches the flow into different flow channels at constant time intervals, such as every 90 s. Each flow channel collects the fractions into autosampler vials of the LC-MS/MS system. Employing a freely configurable collection mechanism, samples are concatenated in a loss-less manner into 2-96 fractions, with efficient peak separation. The combination of eight fractions with 100 min gradients yields very deep coverage at reasonable measurement time, and other parameters can be chosen for even more rapid or for extremely deep measurements. We demonstrate excellent sensitivity by decreasing sample amounts from 100 g into the sub-microgram range, without losses attributable to the spider fractionator and while quantifying close to 10,000 proteins. Finally, we apply the system to the rapid automated and in-depth characterization of 12 different human cell lines to a median depth of 11,472 different proteins, which revealed differences recapitulating their developmental origin and differentiation status. The fractionation technology described here is flexible, easy to use, and facilitates comprehensive proteome characterization with minimal sample requirements.

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Language(s): eng - English
 Dates: 2017
 Publication Status: Published in print
 Pages: 12
 Publishing info: -
 Table of Contents: -
 Rev. Type: -
 Identifiers: ISI: 000398811800013
DOI: 10.1074/mcp.O116.065136
 Degree: -

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Title: Molecular and Cellular Proteomics
Source Genre: Journal
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Publ. Info: Bethesda, MD : American Society for Biochemistry and Molecular Biology
Pages: - Volume / Issue: 16 (4) Sequence Number: - Start / End Page: 694 - 705 Identifier: ISSN: 1535-9476
CoNE: https://pure.mpg.de/cone/journals/resource/111035577487002