Journal Article

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

Varjosalo, Markku, Stukalov, Alexey, Colinge, Jacques, Sacco, Roberto, Gstaiger, Matthias, Planyavsky, Melanie, Aebersold, Ruedi, Hauri, Simon, Superti-Furga, Giulio, van Drogen, Audrey, Bennett, Keiryn L

2013

Overview

A systematic study of the intra- and interlaboratory reproducibility of a standardized affinity purification–mass spectrometry protocol demonstrates the high reproducibility of this technique and hints at the feasibility of a large-scale human interactome project through interlaboratory efforts. 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.

• Publisher: Nature Publishing Group US,Nature Publishing Group
• Subject: 631/114/1314 / 631/1647/2230/1378 / 631/1647/48 / 631/1647/527/296 / 631/45/475/2290 / 631/553 / analysis / Bioinformatics / Biological Microscopy / Biological Techniques / Biomedical Engineering/Biotechnology / Cell Line / Cellular biology / Chromatography, Affinity / Databases, Factual / Genomics / Humans / Integrins / Interactomes / Kinases / Laboratories - standards / Laboratory tests / Life Sciences / Mass spectrometry / Mass Spectrometry - methods / Mass Spectrometry - standards / Methods / Molecular biology / Multiprotein Complexes - chemistry / Phosphotransferases - chemistry / Phosphotransferases - genetics / Phosphotransferases - metabolism / Phylogeny / Physiology / Protein Interaction Maps / Proteins / Proteomics / Reproducibility of Results / Sample preparation