Identification of Biotinylation Sites on Proteins by Selective Retrieval of 2-Iminobiotinylated Peptides from Proteolytic Peptide Mixtures: Localization of the Accessible Lysine Residues on the Photosystem I Subunits PsaD and PsaE
✍ Scribed by Achim von Leoprechting; Patric Hörth; Wolfgang Haehnel; Emile Schilz; Ulrich Mühlenhoff
- Publisher
- Elsevier Science
- Year
- 1998
- Tongue
- English
- Weight
- 386 KB
- Volume
- 262
- Category
- Article
- ISSN
- 0003-2697
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✦ Synopsis
An affinity purification technique was established that allows the selective isolation of 2-iminobiotinylated peptides from proteolytic digest of proteins in order to identify surface-exposed protein domains. Serving as model systems, two photosystem I subunits, PsaD and PsaE from the cyanobacterium Synechococcus elongatus, were overexpressed in Escherichia coli, modified in vitrowith NHS-2-iminobiotin which incorporates 2-iminobiotin at exposed amino groups, and subjected to proteolytic digestion by Glu-C and Arg-C protease, respectively. 2-Iminobiotin-containing proteolytic peptides were subsequently extracted from the proteolytic digests using avidin agarose in a batch procedure and the extracted peptides were separated by HPLC chromatography. The analysis of the peptide maps by electrospray ionization mass spectrometry or N-terminal sequencing showed that avidin-extracted peptide fractions contain almost exclusively 2-iminobiotinylated proteolytic fragments of PsaE or PsaD. No unmodified peptides of PsaD or PsaE were detected. According to this analysis, PsaE is accessible to biotinylation at all of its 7 lysine residues and at its N-terminus. Similarly, all 11 lysine residues of PsaD can be biotinylated and only the N-terminus of PsaD is not accessible.