Methods for analyzing peptides and proteins on a chromatographic timescale by electron-transfer dissociation mass spectrometry

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Abstract

Advancement in proteomics research relies on the development of new, innovative tools for identifying and characterizing proteins. Here, we describe a protocol for analyzing peptides and proteins on a chromatographic timescale by coupling nanoflow reverse-phase (RP) liquid chromatography (LC) to electron-transfer dissociation (ETD) mass spectrometry. For this protocol, proteins can be proteolytically digested before ETD analysis, although digestion is not necessary for all applications. Proteins ≤30 kDa can be analyzed intact, particularly if the objective is protein identification. Peptides or proteins are loaded onto a RP column and are gradient-eluted into an ETD-enabled mass spectrometer. ETD tandem mass spectrometry (MS/MS) provides extensive sequence information required for the unambiguous identification of peptides and proteins and for characterization of posttranslational modifications. ETD is a powerful MS/MS technique and does not compromise the sensitivity and speed necessary for online chromatographic separations. The described procedure for sample preparation, column packing, sample loading and ETD analysis can be implemented in 5–15 h.

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Figure 1: Fragmentation scheme for production of ions of type c′ and z′ resulting from the reaction of a fluoranthene radical anion and a multiply protonated peptide6,8.
Figure 2: ETD mass spectrum of the [M + 3H]+3 ion (m/z 519.8) of a phosphorylated peptide derived from the HIV-1 Rev protein.
Figure 3: Diagram of a capillary column utilized for online nanoflow LC analyses.
Figure 4: ETD mass spectra recorded, with and without supplemental activation, on [M + 3H]+3 ions (m/z 823) from residues 18–39 of Adrenocorticotropic Hormone (ACTH 18–39).
Figure 5: ETD/PTR mass spectrum of a 90-residue peptide derived from the Sam68 protein.
Figure 6: ETD/PTR mass spectrum of intact 21-kDa protein, p21.

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Acknowledgements

The authors thank Marie-Louise Hammarskjold, David Rekosh, Yukiko Misawa and Emily Sloan for providing the Sam68 and Rev protein samples and Dorothy Schafer and Tatyana Kotova for providing the Arp2/3 protein sample. This work was supported by grants from the National Institutes of Health (GM37537 to D.F. Hunt).

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Correspondence to Donald F Hunt or Kristie L Rose.

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