Tài liệu Báo cáo khoa học: Analysis of proteins and peptides on a chromatographic timescale by

MINIREVIEW

Analysis of proteins and peptides on a chromatographic

timescale by electron-transfer dissociation MS

Namrata D. Udeshi1

, Jeffrey Shabanowitz1

, Donald F. Hunt1,2 and Kristie L. Rose1

1 Department of Chemistry, University of Virginia, Charlottesville, VA, USA

2 Department of Pathology, University of Virginia, Charlottesville, VA, USA

Introduction

The traditional method of identifying proteins in com￾plex mixtures by tandem MS involves the following

steps: (a) enzymatic digestion with trypsin; (b) fraction￾ation of the resulting tryptic peptides (usually

10–30 residues in length) by nanoflow HPLC inter￾faced to a mass spectrometer equipped for ESI; (c)

fragmentation of individual peptides by collision-acti￾vated dissociation (CAD); and (d) a search of the

resulting tandem mass spectra against a database of

spectra predicted for tryptic peptides of all known pro￾teins. Thousands of proteins in cultured cells, tissues

and biological fluids have been identified by this

approach. Unfortunately, the CAD step in the above

protocol often promotes the loss of labile post-transla￾tional modifications (PTMs) (i.e. phosphate [1–3] and

carbohydrate [4] modifications) and provides only

limited sequence information from large peptides and

intact proteins.

Electron-capture dissociation (ECD), a technique

introduced by the McLafferty laboratory in 1998,

overcomes the above limitations [5]. In this method,

multiply protonated peptides or proteins are confined

in the Penning trap of an FT ion cyclotron resonance

mass spectrometer and allowed to interact with a beam

of electrons having thermal or near- thermal energies.

Capture of a thermal electron by a protonated peptide

or protein is exothermic by 6 eV and causes the pep￾tide backbone to fragment by a nonergodic process,

Keywords

cell migration; chromatin; HIV regulator of

expression of virion products; mass

spectrometry; O-GlcNAcylation;

phosphorylation; post-translational

modifications; protein identification

Correspondence

K. L. Rose, Department of Chemistry,

University of Virginia, Charlottesville,

VA 22904, USA

Fax: +434 982 2781

Tel: +434 924 7994

E-mail: [email protected]

(Received 10 July 2007, revised 13 August

2007, accepted 17 October 2007)

doi:10.1111/j.1742-4658.2007.06148.x

Peptide and protein sequence analysis using a combination of gas-phase

ion–ion chemistry and tandem MS is described. Samples are converted to

multiply charged ions by ESI and then allowed to react with fluoranthene

radical anions in a quadrupole linear ion trap mass spectrometer. Electron

transfer from the radical anion to the multiply charged peptide or protein

promotes random fragmentation along the amide backbone that is inde￾pendent of peptide or protein size, sequence, or the presence of post-trans￾lational modifications. Examples are provided that demonstrate the utility

of electron-transfer dissociation for characterizing post-translational modi￾fications and for identifying proteins in mixtures on a chromatographic

timescale (500 ms⁄ protein).

Abbreviations

CAD, collision-activated dissociation; ECD, electron-capture dissociation; ESI, electrospray ionization; ETD, electron-transfer dissociation;

FT, Fourier transform; O-GlcNAc, N-acetylglucosamine; PTM, post-translational modification; PTR, proton transfer charge reduction; QLT,

quadrupole linear ion trap; Rev, regulator of expression of virion products.

FEBS Journal 274 (2007) 6269–6276 ª 2007 The Authors Journal compilation ª 2007 FEBS 6269