I-DIRT, A General Method for Distinguishing between Specific and Nonspecific Protein Interactions

Abstract

Isolation of protein complexes via affinity-tagged proteins provides a powerful tool for studying biological systems, but the technique is often compromised by co-enrichment of nonspecifically interacting proteins. We describe a new technique (I-DIRT) that distinguishes contaminants from bona fide interactors in immunopurifications, overcoming this most challenging problem in defining protein complexes. I-DIRT will be of broad value for studying protein complexes in biological systems that can be metabolically labeled.

Ringo/Cyclin-dependent Kinase and Mitogen-activated Protein Kinase Signaling Pathways Regulate the Activity of the Cell Fate Determinant Musashi to Promote Cell Cycle Re-entry in Xenopus Oocytes

“…Under these conditions, the GST-Musashi1 protein appeared to undergo a progesterone-dependent mobility shift. Control and treated GST-Musashi1 protein bands were excised, in-gel-digested with 100 ng of GluC, and prepared for MALDI mass spectrometric analysis (21,22). MALDI mass spectra were collected with a PerkinElmerSciex prOTOF, whereas MS 2 and MS 3 spectra were collected with a Thermo vMALDI-LTQ.…”

Identification of the Major Phosphorylation Site in Bcl-xL Induced by Microtubule Inhibitors and Analysis of Its Functional Significance

“…Each gel band was divided into two portions of which half was digested with 75 ng of trypsin and the other half with 100 ng of endoproteinase AspN (Roche Applied Science). The resulting peptides were spotted in 2,5-dihydroxybenzoic acid for MALDI-MS and MALDI-MS 2 analysis (20,21). A high accuracy (ϳ5 ppm) mass spectrum (MALDI-MS) was taken of each sample with a PerkinElmer Life Sciences prOTOF mass spectrometer.…”

“…Utilizing a custom engineered sample stage, the peptides observed from the spectra collected with the prOTOF mass spectrometer were subjected to tandem mass spectrometric analysis (MALDI-MS 2 ) with a Thermo vMALDI-LTQ ion trap mass spectrometer. A single data file containing the high accuracy peptide measurements and fragmentation information was used to identify Bcl-xL via data base searching with the program XProteo (21). The combination of peptides resulting from trypsin and AspN digestion provided for 64% sequence coverage of Bcl-xL.…”

Protein Complex Affinity Capture from Cryomilled Mammalian Cells

“…As a result, significant effort must be devoted to optimizing the capture process. The detection of FPs remains a central challenge for which a large number of different tools have been developed, including experimental [38][39][40] and computational approaches [41][42][43] , each with different pros and cons. In our own experiments we previously noted a pattern of FP protein binding, obtained after incubation of α-FLAG magnetic medium with control cell extracts, that was distinct from the pattern obtained in the presence of 3xFLAG-tagged proteins.…”