The hydroxyproline-containing proteins (HCPs) among secretory and vacuolar proteins play important roles in growth and development of higher plants. Many hydroxyproline-rich glycoproteins (HRGPs), including Arabinogalactan proteins (AGPs), extensins (EXTs), and proline-rich proteins (PRPs), are identified as HCPs by bioinformatics approaches. The experimental evidence for validation of novel proline hydroxylation sites is vital for understanding their functional roles. In this study, the 62 HCPs containing 114 hydroxyproline (O, Hyp) residues were identified, and it was found that hydroxylation of proline residues in the HCPs could either constitute attachment sites for glycans or have other biological function in rice. The glycomodules of AO, OA, OG, VO, LO, and OE were abundant in the 62 HCPs. Further analysis showed that the 22 of 62 HCPs contained both signal peptides and transmembrane domains, and the 19 HCPs only contained transmembrane domains, while 21 HCPs contained neither. This study indicated the feasibility of mass spectrometry-based proteomics combined with bioinformatics approaches for the large-scale characterization of Hyp sites from complex protein digest mixtures. Furthermore, the expression of AGPs in rice was detected by using β-GlcY reagent and JIM13 antibody. The results displayed that the AGPs were widely distributed in different tissues and organs of rice, especially expressed highly in lateral root, pollen and embryo. In conclusion, our study revealed that the HCPs and Hyp residues in rice were ubiquitous and that these Hyps could be candidates for linking to glycans, which laid the foundation for further studying the functions of HCPs and hydroxylation of proline residues in rice.

分泌蛋白和液泡蛋白中的含羟脯氨酸的蛋白（HCP）在高等植物的生长发育中起重要作用。通过生物信息学方法，许多富含羟基脯氨酸的糖蛋白（HRGP），包括阿拉伯半乳聚糖蛋白（AGP），延伸蛋白（EXTs）和富含脯氨酸的蛋白（PRP）被确定为HCP。验证新型脯氨酸羟基化位点的实验证据对于理解其功能至关重要。在这项研究中，鉴定了62个含有114个羟脯氨酸（O，Hyp）残基的HCP，发现HCP中脯氨酸残基的羟基化可以构成聚糖的连接位点或在水稻中具有其他生物学功能。在62个HCP中，AO，OA，OG，VO，LO和OE的糖模块丰富。进一步的分析表明，62个HCP中的22个同时包含信号肽和跨膜结构域，而19个HCP仅包含跨膜结构域，而21个HCP不包含跨膜结构域。这项研究表明基于质谱的蛋白质组学结合生物信息学方法从复杂的蛋白质消化混合物中大规模鉴定Hyp位点的可行性。此外，使用β-GlcY试剂和JIM13抗体检测了水稻中AGP的表达。结果表明，AGPs广泛分布在水稻的不同组织和器官中，尤其在侧根，花粉和胚中高表达。总之，我们的研究表明，水稻中的HCP和Hyp残留物无处不在，这些Hyps可能是连接聚糖的候选物，