Application of cyclic ion mobility coupled to mass spectrometry for high peak capacity analysis of native and deuterated peptide mixtures

Introduction

A major bottleneck for the analysis of protein digests is the need for long chromatographic separation times. The introduction of ion mobility (IM) has proven advantageous in retaining peak capacity when chromatographic times are shortened, due to the extra dimension of separation by ion mobility. We investigated the utility of cyclic IM for peptide separation under fast chromatography and infusion. In addition, we monitored the retention of deuterium by exchanged peptides within the cIM device.

Experimental and results

An enolase digest and synthetic peptide P1 were used as model systems in this study.  Experiments were performed on a cIM enabled Q-Tof mass spectrometer. The cIM device has a circular path that provides a longer, higher mobility resolution separation path (~1m) whilst a multi-pass capability provides significantly longer path length and hence resolution over a reduced (selected) mobility range. Data were acquired in both infusion and LC-MS modes.

The enolase digest was used as a model system for the separation of peptides by rapid LC-MS. 82 % sequence coverage was obtained with a gradient of less than 3 minutes, afforded by the enhanced mobility and TOF resolution. Partially and fully co-eluting species were separated by a single pass through the cIM device, even with this short chromatographic gradient, showing promise for data-independent LC-MS workflows. The same digest was used to assess separation in the absence of chromatographic separation. Strikingly, using a single pass of the cIM device 91 % of the peptides detected with chromatography were observed. Multipass cIM experiments were used to investigate peptide separation.

We used the model peptide P1 to also assess hydrogen/deuterium back exchange within the cIM device. The deuterium level in the peptide changed depending on the number of passes in the IM device: more passes resulted in decreased average deuterium, most likely the result of losses of deuterium from extremely labile side-chain positions. 

Preliminary Data

A high level of peptide sequence coverage was maintained when using cyclic ion mobility and rapid chromatographic and direct infusion methods. When direct infusion was employed the resolving power of the cIM device was compared with changes in the number of passes.  Increasing the number of passes resolved peptide species in drift time such that their isotope distributions no longer overlapped in the m/z scale.

The observed level of deuteration decreased with increasing number of passes of the cIM device, this was attributed to the loss of deuterium from extremely labile side-chains.