Identification of a novel Plasmopara halstedii elicitor protein combining de novo peptide sequencing algorithms and RACE-PCR

Abstract

Background: Often high-quality MS/MS spectra of tryptic peptides do not match to any database entry because of only partially sequenced genomes and therefore, protein identification requires de novo peptide sequencing. To achieve protein identification of the economically important but still unsequenced plant pathogenic oomycete Plasmopara halstedii, we first evaluated the performance of three different de novo peptide sequencing algorithms applied to a protein digests of standard proteins using a quadrupole TOF (QStar Pulsar i).

Results: The performance order of the algorithms was PEAKS online > PepNovo > CompNovo. In summary, PEAKS online correctly predicted 45% of measured peptides for a protein test data set.All three de novo peptide sequencing algorithms were used to identify MS/MS spectra of tryptic peptides of an unknown 57 kDa protein of P. halstedii. We found ten de novo sequenced peptides that showed homology to a Phytophthora infestans protein, a closely related organism of P. halstedii. Employing a second complementary approach, verification of peptide prediction and protein identification was performed by creation of degenerate primers for RACE-PCR and led to an ORF of 1,589 bp for a hypothetical phosphoenolpyruvate carboxykinase.

Conclusions: Our study demonstrated that identification of proteins within minute amounts of sample material improved significantly by combining sensitive LC-MS methods with different de novo peptide sequencing algorithms. In addition, this is the first study that verified protein prediction from MS data by also employing a second complementary approach, in which RACE-PCR led to identification of a novel elicitor protein in P. halstedii.

Figure 1

Prediction accuracy of QSTAR data. Number of correct peptides in percent with up to three false amino acid assignments allowed and no identification are shown.

Figure 2

Elicitor activity of identified P. halstedii 57 kDa protein. Figure (A) shows a 13% SDS-PAGE of the P. halstedii protein purification. Lane 2 (P) displays the ammonium sulphate precipitate, lane 3 (FT) shows the purified proteins of the flow-through, lane 4 (57 kDa) displays the gel extraction section from lane 3 at M_r 50-70 kDa and lane 5 (<35 kDa) shows the gel extraction section from lane 3 at M_r below 35 kDa. The diagram (B) shows the effect of the extracted proteins of the flow-through to the ethylene induction of sunflower leave-disks 3 h after infiltration. The three columns display the activity of the gel extraction section of the 57 kDa protein (lane 4, 57 kDa), the gel extraction section of proteins <35 kDa (lane 5, <35 kDa) and the negative control. M represents marker (n = 3).

Figure 3

Homology alignment of P. infestans (putative phosphoenolpyruvate carboxykinase; PISP34) and P. halstedii 57 kDa protein. Partial alignment of PISP34 protein of P. infestans to the 57 kDa elicitor protein of P. halstedii is shown. Identity between both proteins amounts to 359 of 636 amino acids (56.4%). Asterisks (*) denotes homology and colon (:) denotes similarity of amino acids. Boxed letters (grey) mark de novo sequenced peptide candidates as shown in table 2.

Figure 4

Scheme of PCR experiments. The alignment shows the three amplicons, which led to the ORF and untranslated regions (UTRs) of the 57 kDa protein of P. halstedii. The internal amplicon was revealed with the primer pairs F1+R1 and F2 + R2 (nested PCR). The 3'-RACE-PCR amplicon was revealed with the gene-specific primer PhE-F1 and PhE-F2 (semi-nested PCR). The 5'-RACE-PCR amplicon was revealed with the gene-specific primer PhE-R1, PhE-R2 (semi-nested PCR) and PhE-R3 (semi-nested PCR). In all RACE experiments the provided adaptor primer of the SMART™ RACE cDNA amplification kit was used.