U.S. Department of Energy

Pacific Northwest National Laboratory

CZE-nanoESI-SLIM-IMS-MS Platforms for Comprehensive, Ultrasensitive Proteome Analyses

Introduction 

Progress towards comprehensive proteome characterization relies on the development of high resolution front end separation techniques, efficient ionization and improved ion transmission and overall utilization in conjunction with mass spectrometric analysis.  Here we explore the coupling of capillary zone electrophoresis and nano-electrospray ionization (CZE-nanoESI) with ion mobility separations conducted in a Structures for Lossless Ion Manipulation device (IMS-SLIM) and mass spectrometry. One aim of these efforts is to enable CZE-nano-ESI-MS proteomic analyses with sensitivity extending to the single cell level.  In addition we aim to enable protein analyses amenable to both denatured and native states, as well as preservation of non-covalent complexes and utilization of sheath flow chemistry, such as H/D exchange. 
Methods  
CZE separations were performed with an in-house built instrument, consisting of a CE injection block and two high voltage power supplies. The CZE-nano-electrospray interface was constructed as previously described (Wojcik, RCMS 24:2554–2560).  CZE separations were performed using protein standards (including Myoglobin, Cytochrome C and Carbonic Anhydrase), standard protein digests and post-translationally modified peptide standards. Deuterated and non-deuterated sheath liquids were employed.  CZE separation buffers included acetic acid and ammonium acetate at various pH conditions. The IMS-SLIM setup coupled to an Agilent TOF instrument consisted of the ion transfer capillary, followed by an ion funnel trap, a multi-path length SLIM-IMS board, and a rear ion funnel. An in-house built acquisition program controlled and synchronized CZE and ion mobility separations. 

Preliminary data 
The advantage of coupling CZE-nanoESI is the capability to retain a sample plug inside the separation capillary, without incurring sample losses, and allowing use in conjunction with long path, high resolution ion mobility separations. Owing to the ability to decouple the CE separation voltage from the ESI voltage in the CZE-nano-ESI interface, the electrospray can be continuously maintained while the CE voltage can also be turned off to ‘park’ or pause the separation without any significant loss of performance. The flexibility of CE capillary lengths and IMS path lengths as well as acquisition automation and synchronization of CZE-IMS-SLIM separation cycles allow for optimization of the multidimensional separations to counteract the diffusional band broadening in both separation dimensions. Hence, the flexible setup is expected to increase the overall peak capacity of separated proteins and peptides for either targeted or comprehensive analysis.  Flexibility of the composition and pH of the CZE separation buffers and the composition of sheath liquid used to close the CE electrical circuit will be exploited for signal optimization, to allow manipulation of charge states of separated proteins and preservation of non-covalent complexes. The coupling with SLIM-IMS technology aims to achieve lossless ion transmission and enables short and long, high resolution IMS separations with high ion utilization efficiency. We anticipate, that CZE-nanoESI-SLIM-IMS-MS can become a universal tool for comprehensive top-down and bottom up proteomic analyses.

Novel aspect 
A powerful new CZE-IMS-MS platform enabling high sensitivity characterization of very small samples. 

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