TY - JOUR

T1 - A double-barrel LC-MS/MS system to quantify 96 interactomes per day

AU - Hosp, Fabian

AU - Scheltema, Richard A

AU - Eberl, Christian

AU - Kulak, Nils A

AU - Keilhauer, Eva C

AU - Mayr, Korbinian

AU - Mann, Matthias

N1 - Copyright © 2015, The American Society for Biochemistry and Molecular Biology.

PY - 2015/4/17

Y1 - 2015/4/17

N2 - 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 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 pull-downs of chromatin complexes in about one day. This is achieved with only 500 ug 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.

AB - 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 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 pull-downs of chromatin complexes in about one day. This is achieved with only 500 ug 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.

U2 - 10.1074/mcp.O115.049460

DO - 10.1074/mcp.O115.049460

M3 - Journal article

C2 - 25887394

VL - 14

SP - 2030

EP - 2041

JO - Molecular and Cellular Proteomics

JF - Molecular and Cellular Proteomics

SN - 1535-9476

IS - 7

ER -