Improved Sensitivity and Separations for Phosphopeptides using Online Liquid Chromotography Coupled with Structures for Lossless Ion Manipulations Ion Mobility–Mass Spectrometry

Journal Article · Fri Aug 17 04:00:00 EDT 2018 · Analytical Chemistry

DOI:https://doi.org/10.1021/acs.analchem.8b02397· OSTI ID:1503500

Chouinard, Christopher D. ^[1]; Nagy, Gabe ^[1]; Webb, Ian K. ^[1]; Shi, Tujin ^[1]; Baker, Erin S. ^[1]; Prost, Spencer A. ^[1]; Liu, Tao ^[1]; Ibrahim, Yehia M. ^[1]; Smith, Richard D. ^[1]

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1. Biological Sciences Division, Pacific Northwest National Laboratory, Richland, Washington 99352, United States

Phosphoproteomics greatly augments proteomics and holds tremendous potential for insights into the modulation of biological systems for various disease states, etc. However, numerous challenges hinder conventional methods in terms of measurement sensitivity, throughput, and capabilities for confident phosphopeptide and phosphosite identification. In this work, we report the first example of integrating structures for lossless ion manipulations ion mobility (SLIM) IM-MS with online reversed-phase liquid chromatography (LC) to evaluate its potential for addressing the aforementioned challenges. A mixture of 51 heavy-labeled phosphopeptides was analyzed with a SLIM IM module having integrated ion accumulation and long-path separation regions. The SLIM IM-MS provided improved sensitivity over an established LC-SRM method, with limits of detection as low as 50 pM (50 amol/µl) for several peptides. Conventionally problematic phosphopeptide isomers could be resolved following an 18 m SLIM IM separation. The 2-D peak capacity was estimated as ~9000 for 90 min LC separation coupled to an 18 m SLIM IM separation, considerably higher than LC alone and providing a basis for both improved identification and quantification, with additional gains projected with the future use of longer path SLIM IM separations. Thus, LC-SLIM IM-MS offers great potential for improving the sensitivity, separation, and throughput of phosphoproteomics analyses.

• Research Organization: Pacific Northwest National Laboratory (PNNL), Richland, WA (United States)
• Sponsoring Organization: USDOE
• DOE Contract Number: AC05-76RL01830
• OSTI ID: 1503500
• Report Number(s): PNNL-SA-135107
• Journal Information: Analytical Chemistry, Journal Name: Analytical Chemistry Journal Issue: 18 Vol. 90; ISSN 0003-2700
• Publisher: American Chemical Society (ACS)
• Country of Publication: United States
• Language: English