Collision Induced Unfolding Classifies Ligands Bound to the Integral Membrane Translocator Protein

Abstract

Membrane proteins represent most current therapeutic targets, yet remain understudied due to their insolubility in aqueous solvents and generally low yields during purification and expression. Ion mobility-mass spectrometry and collision induced unfolding experiments have recently garnered attention as methods capable of directly detecting and quantifying ligand binding within a wide range of membrane protein systems. Despite prior success, ionized surfactant often creates chemical noise patterns resulting in significant challenges surrounding the study of small membrane protein–ligand complexes. Here, we present a new data analysis workflow that overcomes such chemical noise and then utilize this approach to quantify and classify ligand binding associated with the 36 kDa dimer of translocator protein (TSPO). Following our denoising protocol, we detect separate gas-phase unfolding signatures for lipid and protoporphyrin TSPO binders, molecular classes that likely interact with separate regions of the protein surface. Further, a detailed classification analysis reveals that lipid alkyl chain saturation levels can be detected within our gas-phase protein unfolding data. We combine these data and classification schemes with mass spectra acquired directly from liquid–liquid extracts to propose an identity for a previously unknown endogenous TSPO ligand.

Trapped Ion Mobility Spectrometry and Parallel Accumulation–Serial Fragmentation in Proteomics

“…In the study of protein unfolding, ion heating can be intentional, for example, in the case of collision-induced unfolding ( 51 , 53 ). Collision-induced unfolding as a technique is still relatively new; however, early examples show it may provide insight, for example, in the study of protein–ligand binding ( 54 ). Unintentional ion heating can occur while injecting ions into, or while holding ions in, the TIMS analyzer ( 55 , 56 , 57 ).…”

Toward Automation of Collision-Induced Unfolding Experiments through Online Size Exclusion Chromatography Coupled to Native Mass Spectrometry

“…Following the commercialization of quadrupole/TWIMS/time-of-flight mass spectrometers 4 , modern technological developments have further extended the range of CIU examples. Current applications of CIU encompass probing protein-protein complexes 12,13 , protein-ligand interactions 9,[14][15][16][17][18] , and binding of lipids to membrane proteins 19,20 . CIU also plays a key role to investigate therapeutically-relevant proteins, allowing more conformational insights into monoclonal antibodies (mAbs) [21][22][23][24] , biosimilars 25 and antibody-drugconjugates 26,27 .…”

Ion mobility–mass spectrometry reveals the role of peripheral myelin protein dimers in peripheral neuropathy

“…While chemical modification has been used to specifically study WT PMP22 dimers (50), many reports convolute the various oligomeric states of PMP22. The gas-phase unfolding of membrane proteins has been previously reported to inform on the conformational dynamics and stability of protein structures (43,45), and it allows for independent analysis of discrete protein forms that are separated in m/z. Advances in CIU analysis software have also enabled the automated identification of features and the fitting of sigmoidal transitions between such identified features (56).…”

“…The feature observed at the lowest drift time and collision voltage in CIU plots represents the most folded and native-like conformational family observed for the PMP22 monomer in our experiments, while features appearing at longer IM arrival times correspond to a more unfolded PMP22 conformations. Sigmoidal curves fit to the transitions between CIU features allow us to compute inflection points, or CIU50 values, that we use to compare the relative stabilities of PMP22 variants (45,56).…”

“…In CIU, the energies experienced by gas-phase protein ions are increased in a stepwise fashion causing gas-phase protein unfolding to occur. These dynamic measurements have been shown to be sensitive to ligand binding (38,39), glycosylation (40,41), and disulfide bonding (40) in soluble proteins, as well as selective lipid and small molecule binding in membrane proteins (42)(43)(44)(45). While CIU can clearly capture subtle structural changes in membrane proteins (43,45,46) and soluble mutant protein variants (47,48) its ability to characterize membrane protein variants is only beginning to be explored.…”