Accepted: Engen Deuterated Peptides

Introduction

A major bottleneck for the analysis of peptides originating from protein digests is the requirement for long chromatographic separation times. The introduction of hybrid ion mobility-TOF mass spectrometers has proven advantageous in retaining peak capacity when chromatographic times are shortened, due to the extra dimension of separation afforded by the ion mobility (IM) device. Recent advances in travelling-wave IM instrumentation have led to the development of cyclic IM (cIM), a technology that provides variable IM separation by simply increasing the number of passes, or cycles, before MS detection.  We investigated the utility of cIM for peptide separation under fast chromatographic conditions and by direct infusion. In addition, we monitored the retention of deuterium by exchanged peptides within the cIM device.

Methods

An enolase tryptic digest and peptide P1 (HHHHHHIIKIIK) were used as model systems in this study.  Experiments were performed on a cyclic ion mobility-enabled quadrupole time-of-flight (Q-cIM-oa 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 higher path length and hence resolution over a reduced (selected) mobility range. Data were acquired in both infusion and LC-MS modes. Increasing numbers of passes of the cyclic IM device were performed on the same sample, resulting in a resolution range of ~65 to 140 CCS/ΔCCS (between 1 and 5 passes).  Totally deuterated peptides were prepared by incubation in 100% D2O at pD 2 for 15 minutes.

Preliminary Data

Separation of peptides with chromatography.   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.
 
Direct analysis of peptides.  The same enolase 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 observed with chromatography were observed by direct infusion with distinct m/z and arrival times. Choosing groups of similar mass peptides, 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 did not overlap in the m/z scale. 
 
Deuterated peptides. 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.  The time of each pass was approximately 15 ms, and after 5 passes (approx. 75 msec), it is unsurprising that some side chain deuterium could have back-exchanged to hydrogen. Further data will be presented on deuterated peptides, including those generated using a hyphenated HDX system.

Novel Aspect

Investigation of a cyclic ion mobility device as a means to increase peak capacity of peptide/ deuterated peptide separations

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Poster:
Ion Mobility: Applications

Submitting Author:
Martin Palmer
Waters MS Technologies
Wilmslow,  
martin_palmer@waters.com