Protein Protocols Protein Protocols

Nanoelectrospray MS/MS for Peptide Sequencing 693
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Peptide Sequencing by Nanoelectrospray Tandem Mass
Spectrometry
Ole Nørregaard Jensen and Matthias Wilm
1. Introduction
Electrospray ionization mass spectrometry (ESI-MS) (1) has had a profound influence
on biological research over the last decade. With this technique it is possible to
generate and characterize gas-phase analyte ions from aqueous solutions of proteins,
peptides, and other classes of biomolecules. ESI is performed at atmospheric pressure,
which simplifies sample preparation and handling and allows on-line coupling of
chromatography, such as capillary high-performance liquid chromatography (HPLC),
to mass spectrometers (LC-MS). The use of ESI in combination with tandem mass
spectrometry (MS/MS) provides the capability for amino acid sequencing of peptides.
The optimization and miniaturization of peptide sample preparation methods for ESI
as well as the development of highly sensitive tandem mass spectrometers, such as
triple quadrupoles, ion traps, and quadrupole-time-of-flight (Q-TOF) hybrid instruments,
makes it possible and almost routine to obtain amino acid sequences from
subpicomole levels of protein in many laboratories. Peptide sequencing is typically
performed by nanoelectrospray MS/MS analysis of crude, concentrated peptide
mixtures or by hyphenated techniques, such as capillary HPLC coupled to micro/
nanoelectrospray-MS/MS. The sets of peptide tandem mass spectra generated in such
experiments are used to query biological sequence databases with the aim to identify
all protein components present in the sample. In this chapter we describe practical
aspects of nanoelectrospray mass spectrometry aimed at amino acid sequencing of
peptides at subpicomole levels. We do not consider LC-MS/MS, although many of the
features of the two analytical approaches are very similar.
1.1. The Nanoelectrospray Ion Source
The main characteristics of the nanoelectrospray ion source are low flow rate, high
ionization efficiency, and extended measurement time with concomitant improvements
in absolute sensitivity (2,3). These features are crucial for peptide sequencing by tandem
mass spectrometry. Nanoelectrospray tandem mass spectrometry is a reliable and
robust technique for identification or sequencing of gel-isolated proteins available in
sub-picomole amounts (4,5). The very low flow rate of 10–25 nL/min of the
From: The Protein Protocols Handbook, 2nd Edition
Edited by: J. M. Walker © Humana Press Inc., Totowa, NJ
693