Department of Molecular and Structural Biochemistry, NC State University, Raleigh, North Carolina 27695-7253, USA.
J Proteome Res. 2010 Oct 1;9(10):5370-81. doi: 10.1021/pr1006069.
On the basis of the complete genome sequence of the root-knot nematode Melodogyne hapla, we have deduced and annotated the entire proteome of this plant-parasite to create a database of 14,420 proteins. We have made this database, termed HapPep3, available from the Superfamily repository of model organism proteomes (http://supfam.mrc-lmb.cam.ac.uk/SUPERFAMILY). To experimentally confirm the HapPep3 assignments using proteomics, we applied a data-independent LC/MS(E) analysis to M. hapla protein extracts fractionated by SDS-PAGE. A total of 516 nonredundant proteins were identified with an average of 9 unique peptides detected per protein. Some proteins, including examples with complex gene organization, were defined by more than 20 unique peptide matches, thus, providing experimental confirmation of computational predictions of intron/exon structures. On the basis of comparisons of the broad physicochemical properties of the experimental and computational proteomes, we conclude that the identified proteins reflect a true and unbiased sampling of HapPep3. Conversely, HapPep3 appears to broadly cover the protein space able to be experimentally sampled. To estimate the false discovery rate, we queried human, plant, and bacterial databases for matches to the LC/MS(E)-derived peptides, revealing fewer than 1% of matches, most of which were to highly conserved proteins. To provide a functional comparison of the acquired and deduced proteomes, each was subjected to higher order annotation, including comparisons of Gene Ontology, protein domains, signaling, and localization predictions, further indicating concordance, although those proteins that did deviate seem to be highly significant. Approximately 20% of the experimentally sampled proteome was predicted to be secreted, and thus potentially play a role at the host-parasite interface. We examined reference pathways to determine the extent of proteome similarity of M. hapla to that of the free-living nematode, Caenorhabditis elegans, revealing significant similarities and differences. Collectively, the analyzed protein set provides an initial foundation to experimentally dissect the basis of plant parasitism by M. hapla.
基于根结线虫 Melodogyne hapla 的全基因组序列，我们推断并注释了该植物寄生线虫的整个蛋白质组，创建了一个包含 14420 个蛋白质的数据库。我们将这个数据库命名为 HapPep3，可从模式生物蛋白质组超家族数据库（http://supfam.mrc-lmb.cam.ac.uk/SUPERFAMILY）中获得。为了使用蛋白质组学实验验证 HapPep3 的分配，我们对 SDS-PAGE 分离的 M. hapla 蛋白提取物进行了无依赖数据的 LC/MS(E)分析。共鉴定到 516 个非冗余蛋白质，每个蛋白质平均检测到 9 个独特肽段。一些蛋白质，包括具有复杂基因组织的例子，由超过 20 个独特肽段匹配定义，从而为计算预测的内含子/外显子结构提供了实验验证。基于对实验和计算蛋白质组广泛的物理化学性质的比较，我们得出结论，鉴定到的蛋白质反映了 HapPep3 的真实和无偏抽样。相反，HapPep3 似乎广泛涵盖了可实验采样的蛋白质空间。为了估计假阳性率，我们查询了人类、植物和细菌数据库中与 LC/MS(E)衍生肽段的匹配，发现匹配不到 1%，其中大多数是高度保守的蛋白质。为了对获得的和推断的蛋白质组进行功能比较，我们对每个蛋白质组进行了更高阶的注释，包括对基因本体论、蛋白质结构域、信号和定位预测的比较，进一步表明了一致性，尽管那些偏离的蛋白质似乎具有重要意义。大约 20%的实验采样蛋白质组被预测为分泌蛋白，因此可能在宿主-寄生虫界面发挥作用。我们检查了参考途径，以确定 M. hapla 与自由生活线虫 Caenorhabditis elegans 的蛋白质组的相似程度，发现存在显著的相似性和差异。总的来说，分析的蛋白质组提供了一个初步的基础，用于实验剖析 M. hapla 对植物寄生的基础。
