Accepted: Scrivens hemoglobin

Introduction

All hemoglobin molecules consist of two identical copies of an α-like chain and a β-like chain each containing a prosthetic hemegroup located in a small pocket of the protein. In adult humans 96-98% of all hemoglobin is hemoglobin A (HbA) consisting of two α and two β-globin chains. Disorders of hemoglobin known collectively as hemoglobinopathies are some of the most common of all inherited disorders. Structural variants result from mutations to the genes that manifest as amino acid substitutions in the β-chain primary sequence. Structural variants of clinical significance include HbS, HbC, HbE, HbDPunjab and HbOArab. Here we measure the rotationally averaged cross sections and fragmentation pathways of hemoglobin variants to understand structure-property variations.

Methods

Studies were performed using a cyclic ion mobility-enabled quadrupole time-of-flight (Q-cIM-oaToF) mass spectrometer. The cIM device minimizes instrument footprint whilst providing a longer, higher mobility resolution separation path; a multi-pass capability provides significantly higher resolution over a reduced (selected) mobility range; the device can be enabled for mobility separation or by-passed if not required and, the multifunctional ion entry/exit array can select species within a range of mobilities, providing additional functionality. The cIM device consists of a 100 cm path length RF ion guide comprising over 600 electrodes around which T-Waves circulate to provide mobility separation. Native mass spectra were generated using ESI. Mobility separated species were subjected to CID and fragmentation maps generated.

Preliminary Data

Clinically relevant structural variants of hemoglobin exhibit only minor changes in amino acid sequence. Although the Glu-Val replacement in HbS introduces a molecular weight difference of 30 Da, the amino acid substitutions in HbC, HbD, HbE and HbO only result in one Dalton -changes. Accurate measurement of rotationally averaged cross sections requires careful native ionisation (in order to minimise cation attachment) and good mobility resolution. We have demonstrated that HbA, HBs and HbC intact hemoglobins have statistically different cross sections under native conditions and that the unfolding introduced by the addition of further protons populates different pathways. CID studies, at varying collision energies, of mobility selected intact hemoglobin species show variations in the product ion spectra obtained which can be correlated with variations in stability. The Q-cIM-oaToF mass spectrometer used in these investigations) introduces a number of potential experimental improvements including an improved mobility resolution of Ω/∆Ω of 65 for single pass operation (and the capability of improving this with multiple passes), higher TOF analyser resolution and an enhanced dynamic range. Results obtained with the cIM-enabled instrument were compared with those obtained using a Synapt G2 Q-TOF (Waters Corporation) and demonstrated that an increased quality of the experimental data could be obtained.

Novel Aspect

Use of cIM approach to characterise hemoglobin variants of clinical significance.

Options:

A post-doc is presenting author on this abstract? No
A graduate student is presenting author on this abstract? Yes
Research advisor's name Professor james scrivens
Advisor's email j.scrivens@tees.ac.uk
An undergraduate student is presenting author on this abstract? No

Poster:

Ion Mobility: Applications

Submitting Author:

James Scrivens
Teesside University
Middlesbrough,
jim.scrivens@gmail.com