Disordered Motifs and Domains in Cell Control - October 11-15, 2014

Disordered Motifs and Domains in Cell Control

Poster Session II

36-POS Board 12 A Mass Spectrometry-Based Framework to Identify (Non)-Structural Order in p27 Rebecca Beveridge 1 , Yongqi Huang 2 , Rohit V. Pappu 3 , Richard Kriwacki 2 , Perdita E. Barran 1 . 1 University of Manchester, Manchester, United Kingdom, 2 St. Jude Children's Research Hospital, Memphis, TN, USA, 3 Washington University, St. Loius, MO, USA. In the last ten years mass spectrometry (MS) coupled with electrospray ionisation (ESI) has been extensively applied to identify proteins and elucidate stoichiometry of protein complexes, often without the need for labels. Because desolvated species are affected by solvent conditions such as pH, buffer strength and concentration, ESI-MS is an appropriate method by which to consider the range of conformational states that proteins may occupy including natively folded, disordered, denatured and amyloid. Rotationally averaged collision cross sections of the ionised forms of proteins, provided by the combination of mass spectrometry and ion mobility (IM-MS), are also instructive in exploring conformational landscapes in the absence of solvent. We compare these experimental parameters to a simple model which allows the prediction of the theoretical smallest and largest possible collision cross sections based on the volume of the amino acids in the sequences. Consideration of the occupancy of conformational states (based on the intensities of ions in the mass spectra) allows us to qualitatively predict the potential energy landscape of each protein. This empirical approach to assess order or disorder has more accuracy than theoretical methods based on the amino acid sequences for the chosen systems, and could provide an initial route to characterisation. Here we apply IM-MS analysis to the intrinsically disordered protein p27. We compare the range of rotationally averaged collision cross sections of the wildtype protein with various permutants, to investigate the effects of different distributions of charge. We also monitor conformations of the different permutants when they are sprayed from buffers of different ionic strengths and relate this back to the charge distribution.

- 88 -