A method to resolve the composition of heterogeneous affinity-purified protein complexes assembled around a common protein by chemical cross-linking, gel electrophoresis and mass spectrometry

Abstract

Protein complexes form, dissociate and re-form in order to perform specific cellular functions. In this two-pronged protocol, noncovalent protein complexes are initially isolated by affinity purification for subsequent identification of the components by liquid chromatography high-resolution mass spectrometry (LC-MS) on a hybrid LTQ Orbitrap Velos. In the second prong of the approach, the affinity-purification strategy includes a chemical cross-linking step to 'freeze' a series of concurrently formed, heterogeneous protein subcomplex species that are visualized by gel electrophoresis. This branch of the methodology amalgamates standard and well-practiced laboratory methods to reveal compositional changes that occur in protein complex architecture. By using mouse N-terminally tagged streptavidin-binding peptide-hemagglutinin-TANK-binding kinase 1 (SH-TBK1), we chemically cross-linked the affinity-purified complex of SH-TBK1 with the homobifunctional lysine-specific reagent bis(sulfosuccinimidyl) suberate (BS(3)), and we separated the resultant protein complexes by denaturation and by silver-stained one- and two-dimensional SDS-PAGE. We observed a range of cross-linked TBK1 complexes of variable pI and M(r) and confirmed them by immunoblotting. LC-MS analysis of in situ-digested cross-linked proteins shows differences in the composition of the TBK1 subcomplexes. The protocol is inherently simple and can be readily extended to the investigation of a range of protein complexes. From cell lysis to data generation by LC-MS, the protocol takes approximately 2.5 to 5.5 d to perform.

JAGN1 deficiency causes aberrant myeloid cell homeostasis and congenital neutropenia

“…The bound material was eluted with 100 mM formic acid (Suprapur, Merck Millipore, Darmstadt, Germany) and neutralized with triethylammonium bicarbonate (TEAB) (Sigma-Aldrich). The samples were digested with trypsin (Promega) and 2 × 5% v/v of the resultant peptides were analyzed by LC–MSMS on a linear trap quadrupole (LTQ) Orbitrap Velos mass spectrometer (Thermo Fisher Scientific) coupled to an 1200 series high-performance liquid chromatography system (Agilent Technologies, Santa Clara, CA, USA) 24 .…”

“…N- and C-terminal JAGN1 streptavidin binding peptide haemagluttinin (STREP-HA) constructs were generated in HEK293T cells and tandem affinity purifications performed as previously described 24 . Three members of the Coat Protein I (COPI) complex (COPA, COPB2, and COPG2) were identified (Figure 3c and Supplementary Data).…”

Deregulated expression of TANK in glioblastomas triggers pro-tumorigenic ERK1/2 and AKT signaling pathways

“…Detailed biochemical studies showed mutual exclusive interaction of the adapters with their kinases, thus revealing the existence of distinct alternative protein complexes. 18, 19 All four adapters compete for binding to a C-terminal coiled-coil region of TBK1, an interaction that is required for TBK1 function. 20, 21 …”

Affinity Purification Strategies for Proteomic Analysis of Transcription Factor Complexes

“…Standardized two-step STREP-HA purifications 17 from 15 mg of pretreated nuclear extracts were conducted on C/EBPalpha-overexpressing FDCP-1 cells and control mock-infected cells that do not express any STREP-HA-tagged protein. Eluates were analyzed by 1D-gel-LC–MS and 1D-LC–MS, and the identified proteins were filtered by subtracting all proteins detected in the purifications from the control cell line.…”