title: A Double-Barrel Liquid Chromatography-Tandem Mass Spectrometry (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 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 pull-downs of chromatin complexes in about 1 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 severalfold compared with established workflows. The pipeline can be extended to different types of interaction studies and to other medium complexity proteomes.
authors.affiliation: From the ‡Department of Proteomics and Signal Transduction, Max-Planck Institute of Biochemistry, Am Klopferspitz 18, D-82152 Martinsried, Germany.
authors.forename: Fabian
authors.initials: F
authors.lastname: Hosp
authors.affiliation: From the ‡Department of Proteomics and Signal Transduction, Max-Planck Institute of Biochemistry, Am Klopferspitz 18, D-82152 Martinsried, Germany.
authors.forename: Richard A
authors.initials: RA
authors.lastname: Scheltema
authors.affiliation: From the ‡Department of Proteomics and Signal Transduction, Max-Planck Institute of Biochemistry, Am Klopferspitz 18, D-82152 Martinsried, Germany.
authors.forename: H Christian
authors.initials: HC
authors.lastname: Eberl
authors.affiliation: From the ‡Department of Proteomics and Signal Transduction, Max-Planck Institute of Biochemistry, Am Klopferspitz 18, D-82152 Martinsried, Germany.
authors.forename: Nils A
authors.initials: NA
authors.lastname: Kulak
authors.affiliation: From the ‡Department of Proteomics and Signal Transduction, Max-Planck Institute of Biochemistry, Am Klopferspitz 18, D-82152 Martinsried, Germany.
authors.forename: Eva C
authors.initials: EC
authors.lastname: Keilhauer
authors.affiliation: From the ‡Department of Proteomics and Signal Transduction, Max-Planck Institute of Biochemistry, Am Klopferspitz 18, D-82152 Martinsried, Germany.
authors.forename: Korbinian
authors.initials: K
authors.lastname: Mayr
authors.affiliation: From the ‡Department of Proteomics and Signal Transduction, Max-Planck Institute of Biochemistry, Am Klopferspitz 18, D-82152 Martinsried, Germany mmann@biochem.mpg.de.
authors.forename: Matthias
authors.initials: M
authors.lastname: Mann
mesh_terms: D042002:Chromatin Assembly and Disassembly; D002851:Chromatography, High Pressure Liquid; D002853:Chromatography, Liquid / Q000379:methods*; D025941:Protein Interaction Mapping / Q000379:methods*; D015203:Reproducibility of Results; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism; D053719:Tandem Mass Spectrometry / Q000379:methods*; D013997:Time Factors
chemical_list: D029701:Saccharomyces cerevisiae Proteins