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Fast Fix, Fish and Filter, 4Facts strategy to identify real time protein‐protein interactions in situ with few false positives
Author(s) -
Zhu Lisi,
Li Menglin,
Jia Lulu,
Guo Zhengguang,
Wei Lilong,
Gao Youhe
Publication year - 2013
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.27.1_supplement.810.2
Subject(s) - chemistry , transferrin , false positive paradox , covalent bond , albumin , target protein , biochemistry , combinatorial chemistry , gene , computer science , organic chemistry , machine learning
To study dynamic protein‐protein interactions in physiological state, F ast F ix is essential. Formaldehyde cross‐linking can fix the transient and weak protein interactions, but produces great complexity. To reduce the complexity, immunoaffinity purification can F ish complexes of the target protein, but co‐purification by affinity has limited ability in eliminating the nonspecific interactions of beads and antibodies. To F ilter the complexes, SDS‐PAGE was used to disrupt non‐covalent bonds thereby eliminated uncross‐linked complexes and at the same time provided molecular weight information for quality control of identified complexes. Only proteins appeared in ranges with molecular weight higher than the sum of their theoretic molecular weights were considered ligands in situ. This strategy tried to combine the advantages of above methods. Proteins which have post‐translational modification can also show up in gels at much higher than that of potential complexes. So the corresponding control was used to eliminate this possibility. In the present study, human blood was added to10% formaldehyde to cross‐link with ligands for five seconds, complexes of albumin and transferrin were purified using their antibodies respectively, then separated in SDS‐PAGE and analyzed by LC‐MS/MS. Proteins identified were further filtered by molecular weight information. Application of this strategy in human blood generated 33 and 24 ligands for albumin and transferrin respectively. Comparing with three previous major albuminome studies, 30% of 33 ligands in our study were also identified in other studies. But only around 15% of their ligands in each of three studies were identified in our study. This 4facts strategy can be used to reveal real time transient and weak protein interactions in situ with less false positives.