Tài liệu Báo cáo khoa học: Top-down MS, a powerful complement to the high capabilities of

MINIREVIEW

Top-down MS, a powerful complement to the high

capabilities of proteolysis proteomics

Fred W. McLafferty1

, Kathrin Breuker2

, Mi Jin1

, Xuemei Han1

, Giuseppe Infusini1

, Honghai Jiang1

,

Xianglei Kong1 and Tadhg P. Begley1

1 Department of Chemistry and Chemical Biology, Baker Laboratory, Cornell University, Ithaca, NY, USA

2 Institute of Organic Chemistry and Center for Molecular Biosciences Innsbruck (CMBI), University of Innsbruck, Austria

Introduction

The MS techniques of ESI [1] and MALDI [2] have

been available for only two decades, but they have rev￾olutionized the introduction of large, nonvolatile mole￾cules such as proteins into the mass spectrometer [3,4].

Here we discuss two general types of such MS ‘proteo￾mics’ applications: (a) the identification of a protein

from among those predicted from the parent genome’s

DNA; and (b) the structural characterization of a pro￾tein, such as identifying and locating post-translational

modifications (PTMs) or errors in the predicted

sequence. Currently, by far the most common method￾ology for these in useful applications involves initial

protein proteolysis, an approach that we have termed

‘bottom-up’ [5]. The ‘top-down’ [5] approach described

Keywords

electron capture dissociation; MS; protein

characterization; protein identification;

post-translational modifications; top-down

proteomics

Correspondence

F. W. McLafferty, Baker Chemistry

Laboratory, Cornell University, Ithaca,

NY 14853, USA

Fax: +607 255 4137

E-mail: [email protected]

(Received 30 May 2007, revised 12 October

2007, accepted 17 October 2007)

doi:10.1111/j.1742-4658.2007.06147.x

For the characterization of protein sequences and post-translational modifi￾cations by MS, the ‘top-down’ proteomics approach utilizes molecular and

fragment ion mass data obtained by ionizing and dissociating a protein in

the mass spectrometer. This requires more complex instrumentation and

methodology than the far more widely used ‘bottom-up’ approach, which

instead uses such data of peptides from the protein’s digestion, but the top￾down data are far more specific. The ESI MS spectrum of a 14 protein

mixture provides full separation of its molecular ions for MS ⁄MS dissocia￾tion of the individual components. False-positive rates for the identification

of proteins are far lower with the top-down approach, and quantitation of

multiply modified isomers is more efficient. Bottom-up proteolysis destroys

the information on the size of the protein and the connectivities of the pep￾tide fragments, but it has no size limit for protein digestion. In contrast,

the top-down approach has a 500 residue, 50 kDa limitation for the

extensive molecular ion dissociation required. Basic studies indicate that

this molecular ion intractability arises from greatly strengthened electro￾static interactions, such as hydrogen bonding, in the gas-phase molecular

ions. This limit is now greatly extended by variable thermal and collisional

activation just after electrospray (‘prefolding dissociation’). This process

can cleave 287 inter-residue bonds in the termini of a 1314 residue

(144 kDa) protein, specify previously unidentified disulfide bonds between

eight of 27 cysteines in a 1714 residue (200 kDa) protein, and correct

sequence predictions in two proteins, one of 2153 residues (229 kDa).

Abbreviations

BCA, bovine carbonic anhydrase; CAD, collisionally-activated dissociation; ECD, electron-capture dissociation; HAD, 3-hydroxyanthranilate￾3,4-dioxygenase; IRMPD, infrared multiphoton dissociation; PFD, prefolding dissociation; PTM, post-translational modification;

PurL, formylglycinamide ribonucleotide amidotransferase.

6256 FEBS Journal 274 (2007) 6256–6268 ª 2007 The Authors Journal compilation ª 2007 FEBS