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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,
Bruderer Roland,
Uhlmann Thomas,
Rezwan Mandana
Publication year - 2012
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.26.1_supplement.978.12
Subject(s) - computational biology , workflow , robustness (evolution) , protein–protein interaction , proteomics , computer science , chemistry , fibrodysplasia ossificans progressiva , orbitrap , microbiology and biotechnology , heterotopic ossification , mass spectrometry , biology , biochemistry , gene , database , anatomy , chromatography
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 use this pipeline to study interactions associated with BMP signaling systems. In particular we focus on ACTR‐1/Alk2 and FKBP12; two proteins intimately linked to Fibrodysplasia Ossificans Progressiva (FOP) a catastrophic human disorder leading to heterotopic ossification throughout the body. 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.