Fast fixing and comprehensive identification to help improve real-time ligands discovery based on formaldehyde crosslinking, immunoprecipitation and SDS-PAGE separation.
BACKGROUND
Fast Fixation is necessary to study real-time protein-protein interactions under physiological conditions. Fast formaldehyde cross-linking can fix transient and weak protein interactions, thereby reducing the number of false negatives but producing great complexity. To reduce this complexity, immunoaffinity purification can Fish out complexes that include particular target proteins, but affinity-based co-purification has a limited capacity to eliminate nonspecific binding to beads and/or antibodies. To Filter out these complexes, SDS-PAGE is used to disrupt non-covalent bonds, thereby eliminating uncross-linked complexes and simultaneously providing molecular weight information for identification.


RESULTS
We described a 4 F strategy to help improve real-time ligands discovery based on formaldehyde crosslinking, immunoprecipitation and SDS-PAGE separation: Fast Fix, Fish, and Filter, using albumin interactome as an example. The use of gel excision without staining makes this strategy comprehensive and sensitive. The target protein must be identified in the same slice as its ligands. The ligands must be identified in slices for the experimental group but not in the corresponding control slices. Only proteins that appear in the range of molecular weights equal to or greater than the sum of the proteins' theoretical molecular weights, together with the target, are considered ligands. In this study, 5 s of cross-linking with 10% formaldehyde was achieved in human blood. The use of this strategy identified 35 ligands for albumin. Comparison with four major previous studies of the albuminome revealed that 68.57% of the 35 ligands identified in our study were identified in these other studies.


CONCLUSIONS
Fast cross-linking was achieved. The 4 F strategy can be used to identify real-time in situ interactions without prior intervention and to comprehensively identify ligands of particular target proteins with fewer false positives.
Lisi
L
Zhu
Menglin
M
Li
Lilong
L
Wei
Xuejiao
X
Liu
Jianrui
J
Yin
Department of Physiology and Pathophysiology, National Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing 100005, China. gaoyouhe@pumc.edu.cn.
Youhe
Y
Gao