Isolation of N-Terminal Protein Sequence Tags from Cyanogen Bromide Cleaved Proteins as a Novel Approach to Investigate Hydrophobic Proteins

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Abstract
A novel method for the isolation of protein sequence tags to identify proteins in a complex mixture of hydrophobic proteins is described. The PST (Protein Sequence Tag) technology deals with the isolation and MS/MS based identification of one N-terminal peptide from each polypeptide fragment generated by cyanogen bromide cleavage of a mixture of proteins. PST sampling takes place after sub-cellular fractionation of a complex protein mixture to give enrichment of mitochondrial proteins. The method presented here combines effective sample preparation with a novel peptide isolation protocol involving chemical and enzymatic cleavage of proteins coupled to chemical labeling and selective capture procedures. The overall process has been very successful for the analysis of complex mixtures of hydrophobic proteins, particularly membrane proteins. This method substantially reduces the complexity of a protein digest by “sampling” the peptides present in the digest. The sampled digest is amenable to analysis by liquid chromatography tandem mass spectrometry (LC−MS/MS). Methods of “sampling” protein digests have great value1 if they can provide sufficient information to identify substantially all of the proteins in the sample while reducing the complexity of the sample to maximize the efficient usage of LC−MS/MS capacity. The validity of the process is demonstrated for mitochondrial samples from S.cerevisiae. The proteins identified by the PST technology are compared to the proteins identified by the conventional technology 2-D gel electrophoresis as a control. Keywords: PST technology • protein sequence tag • N-terminal fragment • hydrophobic proteins • mass spectrometry • yeast mitochondrion • proteomics.