Journal
ANALYTICAL CHEMISTRY
Volume 90, Issue 4, Pages 2918-2925Publisher
AMER CHEMICAL SOC
DOI: 10.1021/acs.analchem.7b05224
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Funding
- NIH COBRE [P30 GM110761, P20 RR17708]
- NSF [CHE-1552640, CHE-1654274, R21DA041287]
- VILLUM Foundation
- Lundbeck Foundation
- HRSA [C76HF16266]
- NATIONAL CENTER FOR RESEARCH RESOURCES [P20RR017708] Funding Source: NIH RePORTER
- NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES [P30GM110761] Funding Source: NIH RePORTER
- NATIONAL INSTITUTE ON DRUG ABUSE [R21DA041287] Funding Source: NIH RePORTER
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Comprehensive characterization of proteomes comprising the same proteins with distinct post-translational modifications (PTMs) is a staggering challenge. Many such proteoforms are isomers (localization variants) that require separation followed by top-down or middle-down mass spectrometric analyses, but condensed-phase separations are ineffective in those size ranges. The variants for middle-down peptides were resolved by differential ion mobility spectrometry (FAIMS), relying on the mobility increment at high electric fields, but not previously by linear IMS on the basis of absolute mobility. We now use complete histone tails with diverse PTMs on alternative sites to demonstrate that high resolution linear IMS, here trapped IMS (TIMS), broadly resolves the variants of 50 residues in full or into binary mixtures quantifiable by tandem MS, largely thanks to orthogonal separations across charge states. Separations using traveling-wave (TWIMS) and/or involving various time scales and electrospray ionization source conditions are similar (with lower resolution for TWIMS), showing the transferability of results across linear IMS instruments. The linear IMS and FAIMS dimensions are substantially orthogonal, suggesting FAIMS/IMS/MS as a powerful platform for proteoform analyses.
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