Techniques for sample preparation including methods for concentrating peptide samples.
In the current era of proteomics two main analytical techniques are employed for protein identification. By far the fastest and most sensitive procedure for protein identification employs biological mass spectrometry, while de novo sequence analysis by classical Edman degradation is currently diminishing. In order to achieve the highest sensitivity for both techniques, great demands need to be put on sample preparation. In this paper we review three different aspects of protein sample preparation. Firstly, we discuss the use of polyacrylamide or agarose gel systems in which, during electrophoresis, proteins present in multiple primary gel pieces are eluted and simultaneously concentrated in a small secondary gel volume, whereby the overall sensitivity of Edman sequencing can be greatly increased. In a second chapter we review automation strategies occurring in the protein field which allow the automatic handling of multiple protein spots at the same time. In this context, we describe the use of auto-sampling techniques for further mass spectrometric studies and protein digestion robots allowing the simultaneous preparation of tens of gel-separated proteins. Finally we discuss various strategies for the preparation of biological peptide samples such as protein digests for both matrix-assisted laser desorption ionisation and electrospray ionisation mass spectrometry.
Gevaert
K
K
Flanders Interuniversity Institute for Biotechnology, Department of Biochemistry, Universiteit Ghent, Belgium.
Houthaeve
T
T
Vandekerckhove
J
J
D001331:Automation; D004587:Electrophoresis, Agar Gel / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D013058:Mass Spectrometry / Q000295:instrumentation / Q000379:methods; D010455:Peptides / Q000737:chemistry* / Q000302:isolation & purification; D011506:Proteins / Q000737:chemistry* / Q000302:isolation & purification
D010455:Peptides; D011506:Proteins