Informace o publikaci

Utilization of HDX Workbench software for evaluation of H/D data obtained from Orbitrap Elitemass spectrometer



Rok publikování 2014
Druh Konferenční abstrakty
Fakulta / Pracoviště MU

Přírodovědecká fakulta

Popis Hydrogen deuterium exchange combined with high resolution mass spectrometry is a unique method for analyzing of protein conformation, dynamics and protein-protein interactions. In this study, the Translocase of outer mitochondrial membrane 34 (Tomm34) protein either free or in complex with Cterminal peptides Hsp70 or Hsp90 was investigated. The Tomm34 consists of two tetratricopeptide repeat (TPR) domains and has recently been discovered as a co-chaperone of molecular chaperones Hsp70 and Hsp90. These chaperones play role in folding and unfolding of other proteins and their function has been influenced by interactions with co-chaperones. The deuterium exchange reaction was initiated by dilution of samples in buffer with D2O and quenched at 60s, 30min and 1h by 1M Glycin. Each sample was digested by pepsin and obtained peptides were desalted and separated. The peptic peptides were measured on the Orbitrap Elite mass spectrometer (Thermo Fisher Scientific). Sample for peptide mapping was measured in LC-MS/MS mode and spectra were searched using SequestHT against cRAP database ( containing sequence of the Tomm34 protein. Deuterated samples were measured in LC-MS mode. Obtained data were analysed by HDX WorkBench software (1). Protein was defined by submitted resulting list of peptides from SequestHT and amino-acid sequence of Tomm34. All MS data of undeuterated and deuterated samples were loaded through experiment wizard. In the initial detection only MS spectra of undeuterated samples (T0) was searched for submitted peptides. Founded peptides in T0 were used for detection of corresponding peptides in MS spectra of deuterated samples. Percentage of deuteration was calculated by comparison of isotopic envelop of undeuterated and deuterated peptides. Results were manually verified and level of deuteration was presented in graph separately for each peptide. This study focused on finding sites of interactions between the Tomm34 and chaperone Hsp90. Our results suggest that interaction between Tomm34 and C-terminal peptide Hsp90 is preferentially mediated by TPR2 domain. 1) Pascal BD1, Willis S, Lauer JL, Landgraf RR, West GM, Marciano D, Novick S, Goswami D, Chalmers MJ, Griffin PR.: HDX workbench: software for the analysis of H/D exchange MS data. J Am Soc Mass Spectrom. 2012 Sep;23(9):1512-21.