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  A Double-Barrel Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) System to Quantify 96 Interactomes per Day

Hosp, F., Scheltema, R. A., Eberl, H. C., Kulak, N. A., Keilhauer, E. C., Mayr, K., et al. (2015). A Double-Barrel Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) System to Quantify 96 Interactomes per Day. MOLECULAR & CELLULAR PROTEOMICS, 14(7), 2030-2041. doi:10.1074/mcp.O115.049460.

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Mol Cell Proteomics-2015-Hosp-2030-41.pdf (Any fulltext), 722KB
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 Creators:
Hosp, Fabian1, Author              
Scheltema, Richard A.1, Author              
Eberl, H. Christian1, Author              
Kulak, Nils A.1, Author              
Keilhauer, Eva C.1, Author              
Mayr, Korbinian1, 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: PROTEIN-PROTEIN INTERACTIONS; HIGH-THROUGHPUT PROTEOMICS; SACCHAROMYCES-CEREVISIAE; MS PLATFORM; CELL-LINE; COMPLEXES; QUANTIFICATION; EXTRACTION; IDENTIFICATION; NETWORK
 Abstract: The field of proteomics has evolved hand-in-hand with technological advances in LC-MS/MS systems, now enabling the analysis of very deep proteomes in a reasonable time. However, most applications do not deal with full cell or tissue proteomes but rather with restricted subproteomes relevant for the research context at hand or resulting from extensive fractionation. At the same time, investigation of many conditions or perturbations puts a strain on measurement capacity. Here, we develop a high-throughput workflow capable of dealing with large numbers of low or medium complexity samples and specifically aim at the analysis of 96-well plates in a single day (15 min per sample). We combine parallel sample processing with a modified liquid chromatography platform driving two analytical columns in tandem, which are coupled to a quadrupole Orbitrap mass spectrometer (Q Exactive HF). The modified LC platform eliminates idle time between measurements, and the high sequencing speed of the Q Exactive HF reduces required measurement time. We apply the pipeline to the yeast chromatin remodeling landscape and demonstrate quantification of 96 pulldowns of chromatin complexes in about 1 day. This is achieved with only 500 mu g input material, enabling yeast cultivation in a 96-well format. Our system retrieved known complex-members and the high throughput allowed probing with many bait proteins. Even alternative complex compositions were detectable in these very short gradients. Thus, sample throughput, sensitivity and LC/MS-MS duty cycle are improved severalfold compared with established workflows. The pipeline can be extended to different types of interaction studies and to other medium complexity proteomes.

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Language(s): eng - English
 Dates: 2015
 Publication Status: Published in print
 Pages: 12
 Publishing info: -
 Table of Contents: -
 Rev. Type: Peer
 Identifiers: ISI: 000357434400022
DOI: 10.1074/mcp.O115.049460
 Degree: -

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Title: MOLECULAR & CELLULAR PROTEOMICS
Source Genre: Journal
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Publ. Info: 9650 ROCKVILLE PIKE, BETHESDA, MD 20814-3996 USA : AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
Pages: - Volume / Issue: 14 (7) Sequence Number: - Start / End Page: 2030 - 2041 Identifier: ISSN: 1535-9476