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AN INTEGRATED EXPERIMENTAL WORKFLOW TO INCREASE THROUGHPUT AND DATA ROBUSTNESS FOR ANALYSIS OF MAMMALIAN PROTEIN INTERACTION NETWORKS
Author(s) -
Auerbach Daniel,
Lentze Nicolas,
Lampart Thomas,
Gstaiger Matthias,
Aebersold Ruedi,
Glatter Timo
Publication year - 2010
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.24.1_supplement.lb165
Subject(s) - robustness (evolution) , protein–protein interaction , computational biology , workflow , systems biology , chemistry , microbiology and biotechnology , computer science , biology , biochemistry , gene , database
To understand the organization of protein complexes and how biological information is propagated via protein‐protein interaction is becoming increasingly important to get new functional insights on cellular processes. Affinity purification coupled to mass spectrometry (AP‐MS) has been successfully used in the past to characterize protein complexes. However, mainly due to significant experimental limitations the success and power of the AP‐MS strategy has been underrated to date. Here, we describe an integrated workflow for characterization of mammalian protein complexes. The experimental pipeline comprises rapid generation of isogenic mammalian cell lines, protein complex analysis using an efficient double affinity purification strategy followed by state‐of‐the‐art nano‐LC‐Orbitrap‐MS analysis. We have used the system to study interactions associated with the human protein phosphatase 2A and NF‐kB systems. Analysis of these systems revealed new components of protein network structures that are linked to a number of important biological processes such as transcription, the cell proliferation, apoptosis and inflammation. The high performance of our strategy is well suited to identifying protein interactions partners that can subsequently be used as (1) entry points for focused research projects and (2) to more globally map out whole signaling systems linked to important biological processes.