Sample handling for proteome analysis.
The main factor limiting the sensitivity range for the identification of proteins isolated by two-dimensional (2-D) electrophoresis is sample handling: protein detection limits on the gel, losses during extraction and digestion, as well as interference of gel contaminants and detergents with the mass spectrometry (MS) detection increasing background noise. At the one hundred picomole level, losses are fairly negligible but when the amounts drop below 1 picomole (and subfemtomole peptide detection limits have been reported recently by MS), the losses become a critical point. In order to extend proteome analysis to include very low copy number proteins, methods must be developed to minimize losses and handling steps, maximize digestion and extraction yields, as well as to lower chemical noise. We present several methods that we have developed in our laboratory to: (i) increase the amount of material available in a sodium dodecyl sulfate (SDS)-free form which does not require staining, (ii) increase protein extraction and digestion yields and lower the contamination by autoproteolytic products, and (iii) allow direct modification of the peptide mixture to generate sequence tags.
Staudenmann
W
W
Protein Chemistry Laboratory, Swiss Federal Institute of Technology, ETH-Zentrum, Zürich.
Hatt
P D
PD
Hoving
S
S
Lehmann
A
A
Kertesz
M
M
James
P
P
D001426:Bacterial Proteins / Q000032:analysis*; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D004926:Escherichia coli / Q000737:chemistry; D005782:Gels; D010449:Peptide Mapping / Q000295:instrumentation / Q000379:methods
D001426:Bacterial Proteins; D005782:Gels