A Protocol for Top-Down Proteomics Using HPLC and ETD/PTR-MS - LC-MS/MS in Proteomics: Methods and Applications - page 336

Chapter 21

A Protocol for Top-Down Proteomics Using HPLC
and ETD/PTR-MS

Sarah R. Hart

Abstract

Analysis of intact proteins by tandem mass spectrometry has mostly been confined to high-end mass
spectrometry platforms. This protocol describes the application of routine HPLC to separate proteins,
MALDI-ToF mass spectrometry to interrogate intact protein species and electron transfer dissocia-
tion/proton transfer reaction within a quadrupole ion trap to perform tandem mass spectrometry.

Key words: Mass spectrometry, intact protein, electron transfer dissociation, proton transfer
reaction high-performance liquid chromatography.

1. Introduction

Mass spectrometric characterisation of proteomic samples has
matured over the last decade to the point where identification of
protein components within biological samples of moderate com-
plexity is now routine ( 1 ) . The overwhelming majority of pro-
teomic studies rely on a few basic principles, reviewed exten-
sively elsewhere ( 2 - 4 ) . Briefly, protein-containing samples are
subjected to either one- or two-dimensional polyacrylamide gel
electrophoresis and stained proteins excised and proteolysed ( 5 ) .
Alternatively in 'shotgun' proteomics, crude extracts are prote-
olysed and the peptides separated, often by ion exchange chro-
matography (IEX) ( 6 ) . Both gel and shotgun approaches con-
verge upon reversed-phase liquid chromatography of peptides,