A Structured Workflow for Mapping Human Sin3 Histone Deacetylase Complex Interactions Using Halo-MudPIT Affinity-Purification Mass Spectrometry

Abstract

Although a variety of affinity purification mass spectrometry (AP-MS) strategies have been used to investigate complex interactions, many of these are susceptible to artifacts because of substantial overexpression of the exogenously expressed bait protein. Here we present a logical and systematic workflow that uses the multifunctional Halo tag to assess the correct localization and behavior of tagged subunits of the Sin3 histone deacetylase complex prior to further AP-MS analysis. Using this workflow, we modified our tagging/expression strategy with 21.7% of the tagged bait proteins that we constructed, allowing us to quickly develop validated reagents. Specifically, we apply the workflow to map interactions between stably expressed versions of the Sin3 subunits SUDS3, SAP30, or SAP30L and other cellular proteins. Here we show that the SAP30 and SAP30L paralogues strongly associate with the core Sin3 complex, but SAP30L has unique associations with the proteasome and the myelin sheath. Next, we demonstrate an advancement of the complex NSAF (cNSAF) approach, in which normalization to the scaffold protein SIN3A accounts for variations in the proportion of each bait capturing Sin3 complexes and allows a comparison among different baits capturing the same protein complex. This analysis reveals that although the Sin3 subunit SUDS3 appears to be used in both SIN3A and SIN3B based complexes, the SAP30 subunit is not used in SIN3B based complexes. Intriguingly, we do not detect the Sin3 subunits SAP18 and SAP25 among the 128 high-confidence interactions identified, suggesting that these subunits may not be common to all versions of the Sin3 complex in human cells. This workflow provides the framework for building validated reagents to assemble quantitative interaction networks for chromatin remodeling complexes and provides novel insights into focused protein interaction networks.

Differential complex formation via paralogs in the human Sin3 protein interaction network

“…Recombinant proteins were affinity-purified and analyzed by MudPIT (Tables S1, S2A-C) as previously described (15). Statistically-significant protein enrichment over negative controls was assessed with QSPEC v 1.3.…”

Integrative Modeling of a Sin3/HDAC Complex Sub-structure

“…Relative abundance of Sin3 complex subunits copurifying with the Halo-SAP30L subunit determined by AP-MS. Values for each subunit are 1000 x mean dNSAF (data and experimental details previously published inBanks et al 2018). purified complexes treated with or without DSSO cross-linking.…”

Tandem Affinity Purification and Mass Spectrometry (TAP‐MS) for the Analysis of Protein Complexes

“…Single‐step affinity purification followed by mass spectrometry analysis (AP‐MS) (Banks et al., ; Dunham, Mullin, & Gingras, ; Giambruno et al., ; Greco, Guise, & Cristea, ; Kwan & Emili, ; Morris et al., ) has recently been used to build protein interaction networks which cover entire or defined subsets of proteomes for model organisms (Butland et al., ; Krogan et al., ) or homo sapiens (Hein et al., ; Huttlin et al., ). Although these data have a broad scope, they are limited in context (Washburn, ) in that disease‐specific proteoforms, gene variants, as well as infectious pathogens or other exogenous factors, may drastically alter the landscape of cellular protein interactions (Koleva et al., ; Rozenblatt‐Rosen et al., ).…”