Gallagher, K., Palasser, M., Hughes, S., Mackay, C.L., Kilgour, D. ORCID: 0000-0002-3860-7532 and Clarke, D.J., 2020. Isotope Depletion Mass Spectrometry (ID-MS) for accurate mass determination and improved top-down sequence coverage of intact proteins. Journal of The American Society for Mass Spectrometry, 31 (3), pp. 700-710. ISSN 1044-0305
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Abstract
Top-down mass spectrometry (MS) is an increasingly important technique for protein characterization. However, in many biological MS experiments, the practicality of applying top-down methodologies is still limited at higher molecular mass. In large part, this is due to the detrimental effect resulting from the partitioning of the mass spectral signal into an increasing number of isotopic peaks as molecular mass increases. Reducing the isotopologue distribution of proteins via depletion of heavy stable isotopes was first reported over 20 years ago (Marshall, A. G.; Senko, M. W.; Li, W.; Li, M.; Dillon, S., Guan, S.; Logan, T. M.. Protein Molecular Mass to 1 Da by 13C, 15N Double-Depletion and FT-ICR Mass Spectrometry. J. Am. Chem. Soc. 1997, 119, 433−434.) and has been demonstrated for several small proteins. Here we extend this approach, introducing a new highly efficient method for the production of recombinant proteins depleted in 13C and 15N and demonstrating its advantages for top-down analysis of larger proteins (up to ∼50 kDa). FT-ICR MS of isotopically depleted proteins reveals dramatically reduced isotope distributions with monoisotopic signal observed up to 50 kDa. In top-down fragmentation experiments, the reduced spectral complexity alleviates fragment-ion signal overlap, the presence of monoisotopic signals allows assignment with higher mass accuracy, and the dramatic increase in signal-to-noise ratio (up to 7-fold) permits vastly reduced acquisition times. These compounding benefits allow the assignment of ∼3-fold more fragment ions than comparable analyses of proteins with natural isotopic abundances. Finally, we demonstrate greatly increased sequence coverage in time-limited top-down experiments—highlighting advantages for top-down LC–MS/MS workflows and top-down proteomics.
Item Type: | Journal article | ||||||
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Alternative Title: | Top-down isotope depletion mass spectrometry [running title] | ||||||
Publication Title: | Journal of The American Society for Mass Spectrometry | ||||||
Creators: | Gallagher, K., Palasser, M., Hughes, S., Mackay, C.L., Kilgour, D. and Clarke, D.J. | ||||||
Publisher: | Springer Verlag | ||||||
Date: | 2020 | ||||||
Volume: | 31 | ||||||
Number: | 3 | ||||||
ISSN: | 1044-0305 | ||||||
Identifiers: |
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Divisions: | Schools > School of Science and Technology | ||||||
Record created by: | Linda Sullivan | ||||||
Date Added: | 04 Feb 2020 12:56 | ||||||
Last Modified: | 31 May 2021 15:07 | ||||||
URI: | https://irep.ntu.ac.uk/id/eprint/39165 |
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