Guy Bauw

Patatins, Kunitz protease inhibitors and other major proteins in tuber of potato cv. Kuras

The major potato tuber proteins of the Kuras cultivar, which is the dominant cultivar used in Northern Europe for industrial starch production, were analysed using 1D and 2D gel electrophoresis. The electrophoretic patterns varied significantly depending on the method of preparation and the potato variant (Solanumu2003tuberosum). Proteins were characterized using MS and scored against potato protein databases, derived from both ‘Kuras only’ and ‘all potato’ expressed sequence tags (EST) and full‐length cDNAs. Despite the existence of ∼u2003180u2003000 ESTs, the currently available potato sequence data showed a severe under‐representation of genes or long transcripts encoding proteins >u200a50u2003kDa (3.5% of all) compared with the complete proteome of Arabidopsisu2003thaliana (33% of all). We found that patatin and Kunitz protease inhibitor (KPI) variants are extraordinarily dominant in Kuras tuber and, most significantly, that their amino acid sequences are specific to Kuras. Other proteins identified include annexin, glyoxalaseu2003I, enolase and two lipoxygenases, the sequences of which are highly conserved among potato variants. Known S.u2003tuberosum patatins cluster into three clades all represented in Kuras. S.u2003tuberosum KPIs cluster into more diverse clades of which five were found in Kuras tuber, including a novel clade, KPIu2003K, found to date only in Kuras. Furthermore, protein abundance was contrasted with the levels of corresponding gene transcripts found in our previous EST and LongSAGE studies of Kuras tuber.

The amyloplast proteome of potato tuber

Potato (Solanumu2003tuberosum) is the fourth largest crop worldwide in yield, and cv. Kuras is the major starch potato of northern Europe. Storage starch is packed densely in tuber amyloplasts, which become starch granules. Amyloplasts of soil‐grown mini‐tubers and agar‐grown micro‐tubers of cv. Kuras were purified. The mini‐tuber amyloplast preparation was enriched 10–20‐fold and the micro‐tuber amyloplast approximately fivefold over comparative total protein extracts. Proteins separated by SDS–PAGE were digested with trypsin, analysed by mass spectrometry and identified by mascot software searches against an in‐house potato protein database and the NCBI non‐redundant plant database. The differential growth conditions for mini‐ and micro‐tubers gave rise to rather different protein profiles, but the major starch granule‐bound proteins were identical for both and dominated by granule‐bound starch synthaseu2003I, starch synthaseu2003II and α‐glucan water dikinase. Soluble proteins were dominated by starch phosphorylase L‐1, other large proteins of the classes ‘starch and sucrose metabolism’, ‘pentose phosphate pathway’, ‘glycolysis’, ‘amino acid metabolism’, and other proteins such as plastid chaperonins. The majority of the identified proteins had a predicted plastid transit peptide, supporting their presence in the amyloplast. However, several highly expressed proteins had no transit peptide, such as starch phosphorylaseu2003H, or had a predicted mitochondrial location. Intriguingly, all polyphenol oxidases, a family of enolases, one transketolase, sulfite reductase, deoxynucloside kinase‐like and dihydroxy‐acid dehydrase had twin‐arginine translocation motifs, and a homologue to dihydrolipoamide dehydrogenase had a Sec (secretory) motif; these motifs usually target thylakoid‐like structures.

Interpreting peptide mass spectra by VEMS

Most existing Mass Spectra (MS) analysis programs are automatic and provide limited opportunity for editing during the interpretation. Furthermore, they rely entirely on publicly available databases for interpretation. VEMS (Virtual Expert Mass Spectrometrist) is a program for interactive analysis of peptide MS/MS spectra imported in text file format. Peaks are annotated, the monoisotopic peaks retained, and the b-and y-ion series identified in an interactive manner. The called peptide sequence is searched against a local protein database for sequence identity and peptide mass. The report compares the calculated and the experimental mass spectrum of the called peptide. The program package includes four accessory programs. VEMStrans creates protein databases in FASTA format from EST or cDNA sequence files. VEMSdata creates a virtual peptide database from FASTA files. VEMSdist displays the distribution of masses up to 5000 Da. VEMSmaldi searches singly charged peptide masses against the local database.