Dataset Information

A double-barrel LC-MS/MS system to quantify 96 interactomes per day

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 sub-proteomes 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 very high sequencing speed of the Q Exactive HF dramatically reduces required measurement time. We apply the pipeline to the yeast chromatin remodeling landscape, and demonstrate quantification of 96 pull-downs of chromatin complexes in about one day. This is achieved with only 500 µg 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 several-fold compared to established workflows. The pipeline can be extended to different types of interaction studies and to other medium complexity proteomes.

INSTRUMENT(S): LTQ Orbitrap, Q Exactive

ORGANISM(S): Saccharomyces Cerevisiae (baker's Yeast)

SUBMITTER: Mario Oroshi

LAB HEAD: Matthias Mann

PROVIDER: PXD001695 | Pride | 2015-05-07

REPOSITORIES: pride

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Dataset's files

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			Action	DRS
	20140602_QEp4_FaHo_SA_ADA2_01.raw	Raw
	20140602_QEp4_FaHo_SA_ADA2_02.raw	Raw
	20140602_QEp4_FaHo_SA_ADA2_03.raw	Raw
	20140602_QEp4_FaHo_SA_BDF1_01.raw	Raw
	20140602_QEp4_FaHo_SA_BDF1_02.raw	Raw

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Publications

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

Hosp Fabian F Scheltema Richard A RA Eberl H Christian HC Kulak Nils A NA Keilhauer Eva C EC Mayr Korbinian K Mann Matthias M

Molecular & cellular proteomics : MCP 20150417 7

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-t ...[more]

PMID: 25887394

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