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Data-Dependent Middle-Down Nano-Liquid Chromatography–Electron Capture Dissociation-Tandem Mass Spectrometry: An Application for the Analysis of Unfractionated Histones

Kalli, Anastasia and Sweredoski, Michael J. and Hess, Sonja (2013) Data-Dependent Middle-Down Nano-Liquid Chromatography–Electron Capture Dissociation-Tandem Mass Spectrometry: An Application for the Analysis of Unfractionated Histones. Analytical Chemistry, 85 (7). pp. 3501-3507. ISSN 0003-2700. doi:10.1021/ac303103b. https://resolver.caltech.edu/CaltechAUTHORS:20130522-104357653

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Abstract

Middle-down mass spectrometry (MS) combined with electron capture dissociation (ECD) represents an attractive method for characterization of proteins and their post-translational modifications (PTMs). Coupling online chromatographic separation with tandem mass spectrometry enables a high-throughput analysis, while improving sensitivity of the electrosprayed peptides and reducing sample amount requirements. However, middle-down ECD has not been thus far coupled with online chromatographic separation. In this work, we examine the feasibility of coupling middle-down ECD with online nanoflow-liqiud chromatography (nano-LC) for the analysis of large, >3 kDa, and highly modified polypeptides in a data-dependent acquisition mode. We evaluate the effectiveness of the method by analyzing peptides derived from Asp-N and Glu-C digestions of unfractionated histones from calf thymus and acid-extracted histones from HeLa, MCF-7, and Jurkat cells. Our results demonstrate that middle-down ECD is compatible with online chromatographic separation, providing high peptide and protein sequence coverage while allowing precise mapping of PTM sites. The high mass accuracy, obtained by the ICR mass analyzer, for both the precursor and product ions greatly increases confidence in peptide identification, particularly for modified peptides. Overall, for all samples examined, several histone variants were identified and modification sites were successfully localized, including single, multiple, and positional isomeric PTM sites. The vast majority of the identified peptides were in the mass range from 3 to 9 kDa. The data presented here highlight the feasibility and utility of nano-LC–ECD-MS/MS for high-throughput middle-down analysis.


Item Type:Article
Related URLs:
URLURL TypeDescription
http://pubs.acs.org/doi/abs/10.1021/ac303103bPublisherArticle
http://dx.doi.org/10.1021/ac303103bDOIArticle
ORCID:
AuthorORCID
Sweredoski, Michael J.0000-0003-0878-3831
Hess, Sonja0000-0002-5904-9816
Additional Information:© 2013 American Chemical Society. Received October 23, 2012; Accepted February 28, 2013; Published February 28, 2013. This work was supported by the Betty and Gordon Moore Foundation and the Beckman Institute. We thank Tsui-Fen Chou and Raymond J. Deshaies for providing the HeLa, Jurkat, and MCF-7 cells.
Funders:
Funding AgencyGrant Number
Gordon and Betty Moore FoundationUNSPECIFIED
Caltech Beckman InstituteUNSPECIFIED
Issue or Number:7
DOI:10.1021/ac303103b
Record Number:CaltechAUTHORS:20130522-104357653
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20130522-104357653
Official Citation:Data-Dependent Middle-Down Nano-Liquid Chromatography–Electron Capture Dissociation-Tandem Mass Spectrometry: An Application for the Analysis of Unfractionated Histones. Anastasia Kalli, Michael J. Sweredoski, and Sonja Hess. Analytical Chemistry 2013 85 (7), 3501-3507
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:38632
Collection:CaltechAUTHORS
Deposited By: John Wade
Deposited On:22 May 2013 18:41
Last Modified:09 Nov 2021 23:38

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