Simple Protein Complex Purification and Identification Method Suitable for High- throughput Mapping of Protein Interaction Networks

Journal Article · Mon Apr 11 00:00:00 EDT 2005 · Journal of Proteome Research, 4(2):268-274

DOI:https://doi.org/10.1021/pr049847a· OSTI ID:15015959

Markillie, Lye Meng; Lin, Chiann Tso; Adkins, Joshua N; Auberry, Deanna L; Hill, Eric A; Hooker, Brian S; Moore, Priscilla A; Moore, Ronald J; Shi, Liang; Wiley, H S; Kery, Vladimir

Most of the current methods for purification and identification of protein complexes use endogenous expression of affinity tagged bait, tandem affinity tag purification of protein complexes followed by specific elution of complexes from beads, gel separation, in-gel digestion and mass spectrometric analysis of protein interactors. We propose a single affinity tag in vitro pulldown assay with denaturing elution, trypsin digestion in organic solvent and LC ESI MS/MS protein identification using SEQUEST analysis. Our method is simple, easy to scale up and automate thus suitable for high throughput mapping of protein interaction networks and functional proteomics.

• Research Organization: Pacific Northwest National Lab. (PNNL), Richland, WA (United States)
• Sponsoring Organization: USDOE
• DOE Contract Number: AC05-76RL01830
• OSTI ID: 15015959
• Report Number(s): PNNL-SA-42494; KP1501021; TRN: US200509%%518
• Journal Information: Journal of Proteome Research, 4(2):268-274, Vol. 4, Issue 2
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
AFFINITY
DIGESTION
FUNCTIONALS
IN VITRO
ORGANIC SOLVENTS
PROTEINS
PURIFICATION
TRYPSIN
Protein complex purification
identification
mass spectrometry
mapping protein interaction networks