Interlaboratory reproducibility of large-scale human protein-complex analysis by standardized AP-MS

Abstract

The characterization of all protein complexes of human cells under defined physiological conditions using affinity purification-mass spectrometry (AP-MS) is a highly desirable step in the quest to understand the phenotypic effects of genomic information. However, such a challenging goal has not yet been achieved, as it requires reproducibility of the experimental workflow and high data consistency across different studies and laboratories. We systematically investigated the reproducibility of a standardized AP-MS workflow by performing a rigorous interlaboratory comparative analysis of the interactomes of 32 human kinases. We show that it is possible to achieve high interlaboratory reproducibility of this standardized workflow despite differences in mass spectrometry configurations and subtle sample preparation-related variations and that combination of independent data sets improves the approach sensitivity, resulting in even more-detailed networks. Our analysis demonstrates the feasibility of obtaining a high-quality map of the human protein interactome with a multilaboratory project.

VRK1 interacts with p53 forming a basal complex that is activated by UV‐induced DNA damage

“…VRK1, the most abundant Ser-Thr kinase in nuclei [23], is a nucleosomal/chromatin kinase that can form complexes and phosphorylate several transcription factors [24][25][26], histones [18,[27][28][29] and other chromatin proteins [30,31]. Moreover, VRK1 kinase activity can also be regulated by these proteins interactions, including those with histones [28][29][30].…”

Isolation, characterization and complete genome sequence of PhaxI: a phage of Escherichia coli O157 : H7

“…Tryptic peptide digests were quenched with 10 % Trifluoroacetic acid (TFA), concentrated and purified by reversed-phase chromatography columns (C18 material, eluted with 90 % CH 3 CN, 0.1 % TFA) (Varjosalo et al, 2013). The dried peptides were reconstituted (2 % CH 3 CN, 0.1 % TFA) and the MS analysis was performed on an Orbitrap Elite ETD mass spectrometer (Thermo Scientific), using Xcalibur version 2.7.1, coupled to an Thermo Scientific nLCII nanoflow HPLC system.…”

Studying post-translational modifications with protein interaction networks

“…Much of the work to improve data quality focused on determining and improving the specificity of large scale PPI approaches [4][5][6]. Given high specificity, it is relatively low coverage large unbiased data sets suffer from.…”

“…Providing a more global context for kinase function, two recent studies by Varjosalo et al presented proteomic analysis of complexes of a large number of human CMGC kinases [35 ] and of selected kinases from other major families [6]. 481 and 345 co-complex members were identified for 57 and 32 kinases (512 and 488 PPIs) via LC-MS/MS, in the two studies respectively.…”