title: Exploring key parameters to detect subtle ligand-induced protein conformational changes using traveling wave ion mobility mass spectrometry.
abstract: Evidencing subtle conformational transitions in proteins occurring upon small modulator binding usually requires atomic resolution techniques (X-ray crystallography or NMR). Recently, hyphenation of ion mobility and mass spectrometry (IM-MS) has greatly enlarged the potentials for biomolecular assembly structural characterization. Using the well 3D-characterized Bcl-xL/ABT-737 protein model, we explored in the present report whether IM-MS can be used to differentiate close conformers and monitor collision cross section (CCS) differences correlating with ligand-induced conformational changes. Because comparing CCS derived from IM-MS data with 3D-computed CCS is critical for thorough data interpretation, discussing pitfalls related to protein construct similarity and missing sequence sections in PDB files was of primary importance to avoid misinterpretation. The methodic exploration of instrument parameters showed enhanced IM separation of Bcl-xL conformers by combining high wave heights and velocities with low helium and nitrogen flow rates while keeping a high He/N(2) flow rate ratio (>3). The robustness of CCS measurements was eventually improved with a modified IM calibration method providing constant CCS values regardless of instrument settings. Altogether, optimized IM-MS settings allowed a 0.4 nm(2) increase (i.e., 2%) of Bcl-xL CCS to be evidenced upon ABT-737 binding.
authors.affiliation: NovAliX, Structural Biology, Parc d'Innovation Bioparc, Boulevard Sébastien Brant, 67400 Illkirch, France.
authors.forename: Cédric
authors.initials: C
authors.lastname: Atmanene
authors.forename: Stéphanie
authors.initials: S
authors.lastname: Petiot-Bécard
authors.forename: Denis
authors.initials: D
authors.lastname: Zeyer
authors.forename: Alain
authors.initials: A
authors.lastname: Van Dorsselaer
authors.forename: Valérie
authors.initials: V
authors.lastname: Vivat Hannah
authors.forename: Sarah
authors.initials: S
authors.lastname: Sanglier-Cianférani
mesh_terms: D000595:Amino Acid Sequence; D001713:Biphenyl Compounds / Q000737:chemistry; D018360:Crystallography, X-Ray; D006371:Helium; D006801:Humans; D007477:Ions / Q000032:analysis*; D008024:Ligands; D013058:Mass Spectrometry / Q000295:instrumentation / Q000379:methods*; D008969:Molecular Sequence Data; D009584:Nitrogen; D009596:Nitrophenols / Q000737:chemistry; D010879:Piperazines / Q000737:chemistry; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000737:chemistry; D012680:Sensitivity and Specificity; D016415:Sequence Alignment; D013449:Sulfonamides / Q000737:chemistry; D051020:bcl-X Protein / Q000032:analysis* / Q000737:chemistry
chemical_list: C501332:ABT-737; C494811:BCL2L1 protein, human; D001713:Biphenyl Compounds; D007477:Ions; D008024:Ligands; D009596:Nitrophenols; D010879:Piperazines; D011993:Recombinant Fusion Proteins; D013449:Sulfonamides; D051020:bcl-X Protein; D006371:Helium; D009584:Nitrogen