CHAPTER 1
Strategies and Methods for Proteome Analysis
D. R. GOODLETTl A. TIMPERMAN l , S. P. GYGIl, J. WATTSl, G. CORTHALSI,
D. FIGEYS l and R. AEBERSOLD l
A proteome has been defined as the protein complement expressed by the
genome of an organism (Wilkins, et al. 1996). In multicellular organisms the proteome is the protein complement expressed by a tissue or differentiated cell. The
most common approach to proteome analysis involves separation of proteins by
one- or two-dimensional gel electrophoresis (IEF/SDS-PAGE), enzymatic cleavage of selected proteins, tandem mass spectrometry (MS/MS) of peptides, and
finally data interpretation by computer routines which also search databases
(Fig. 1.1). In most cases the availability of protein and DNA sequences in public
databases eliminates the need for complete protein sequence analysis. Protein
sequences are more rapidly identified by partial sequence analysis using tandem
mass spectrometry which allows rapid, complete gene identification. As reliable
as protein identification by mass spectrometry has become there are still many
obstacles that prevent proteome analysis from becoming as automated and routine as genome analysis.
Fig. 1.1. Schematic of Proteomic Analytical
Process. Proteins are 1) separated by 2DE
and visualized by silver staining, 2) spots of
interest are excised and digested with trypsin, 3) peptides are analyzed by automated
~LC-MS/MS and 4) data screened for hits
against protein or DNA databases using the
database search routine SEQUEST
•
,e,
. . . . " ..
~ : ',' ' I
j'
1) 2D E
2) Excise/Digest
/3) LC-MSIMS
rvlodeled CI D \IS Observed CI D
111111111111 II ,JL,dlll" I
4) SEOUEST
1 Department of Molecular Biotechnology, University of Washington, Seattle, Washington 98195 USA.
E-mail: ruedi@u.washington.edu.
Strategies and Methods for Proteome Analysis
D. R. GOODLETTl A. TIMPERMAN l , S. P. GYGIl, J. WATTSl, G. CORTHALSI,
D. FIGEYS l and R. AEBERSOLD l
A proteome has been defined as the protein complement expressed by the
genome of an organism (Wilkins, et al. 1996). In multicellular organisms the proteome is the protein complement expressed by a tissue or differentiated cell. The
most common approach to proteome analysis involves separation of proteins by
one- or two-dimensional gel electrophoresis (IEF/SDS-PAGE), enzymatic cleavage of selected proteins, tandem mass spectrometry (MS/MS) of peptides, and
finally data interpretation by computer routines which also search databases
(Fig. 1.1). In most cases the availability of protein and DNA sequences in public
databases eliminates the need for complete protein sequence analysis. Protein
sequences are more rapidly identified by partial sequence analysis using tandem
mass spectrometry which allows rapid, complete gene identification. As reliable
as protein identification by mass spectrometry has become there are still many
obstacles that prevent proteome analysis from becoming as automated and routine as genome analysis.
Fig. 1.1. Schematic of Proteomic Analytical
Process. Proteins are 1) separated by 2DE
and visualized by silver staining, 2) spots of
interest are excised and digested with trypsin, 3) peptides are analyzed by automated
~LC-MS/MS and 4) data screened for hits
against protein or DNA databases using the
database search routine SEQUEST
•
,e,
. . . . " ..
~ : ',' ' I
j'
1) 2D E
2) Excise/Digest
/3) LC-MSIMS
rvlodeled CI D \IS Observed CI D
111111111111 II ,JL,dlll" I
4) SEOUEST
1 Department of Molecular Biotechnology, University of Washington, Seattle, Washington 98195 USA.
E-mail: ruedi@u.washington.edu.