Nature Biotechnology21, 566 - 569 (2003)
Published online: 31 March 2003; | doi:10.1038/nbt810
Exploring proteomes and analyzing protein processing by mass
spectrometric identification of sorted N-terminal peptides
Kris Gevaert, Marc Goethals, Lennart Martens, Jozef Van Damme, An Staes, Grégoire R. Thomas
& Joël Vandekerckhove
Department of Medical Protein Research, Flanders
Interuniversity Institute for Biotechnology, Department of Biochemistry, Ghent
University, A. Baertsoenkaai 3, B-9000
Ghent, Belgium.
Current non-gel techniques for analyzing proteomes rely heavily on
mass spectrometric analysis of enzymatically digested protein mixtures. Prior
to analysis, a highly complex peptide mixture is either separated on a
multidimensional chromatographic system1,
2 or it is first
reduced in complexity by isolating sets of representative peptides3,
4,
5,
6,
7,
8. Recently, we developed a peptide isolation procedure
based on diagonal electrophoresis9 and diagonal
chromatography10. We call it combined fractional diagonal
chromatography (COFRADIC). In previous experiments, we used COFRADIC to
identify more than 800 Escherichia coli proteins by tandem mass
spectrometric (MS/MS) analysis of isolated methionine-containing peptides11. Here, we describe a diagonal method to isolate N-terminal peptides.
This reduces the complexity of the peptide sample, because each protein has one
N terminus and is thus represented by only one peptide. In this new procedure,
free amino groups in proteins are first blocked by acetylation12
and then digested with trypsin. After reverse-phase (RP) chromatographic
fractionation of the generated peptide mixture, internal peptides are blocked
using 2,4,6-trinitrobenzenesulfonic acid (TNBS)13,
14; they
display a strong hydrophobic shift and therefore segregate from the unaltered
N-terminal peptides during a second identical separation step. N-terminal
peptides can thereby be specifically collected for further liquid
chromatography (LC)-MS/MS analysis. Omitting the acetylation step results in
the isolation of non-lysine-containing N-terminal peptides from in vivo
blocked proteins.
