Identification of post-translational modifications on Odorant-Binding Protein isoforms from pig olfactory secretome by high-resolution mass spectrometry: O-beta-N-acetylglucosaminylation and phosphorylation

Odorant-Binding Proteins (OBP) are players of perireceptor events in olfaction. A molecular mechanism explaining their specificity for odors and pheromones has still to be proposed. A new track comes from the analysis of pig olfactory secretome, which is mainly composed of OBP isoforms, generated from 3 gene products by PTM, phosphorylation and O‐β‐N‐acetylglucosaminylation (O-GlcNAcylation), which are unusual for secreted proteins. These diverse isoforms could display different binding properties towards ligands. Before testing such a function, both post-translational modifications, although assessed by specific antibodies, have to be identified by mass spectrometry. We report here for the first time, the identification of phosphorylation and O-GlcNAcylation on peptides coming from trypsin digestion of OBP by ESI-HCD-MS/MS. PEAKS software analysis of raw MS data allowed selecting spectra that were analyzed manually to identify PTM. Four peptides corresponding to two different portions of OBP sequence were modified either by a phosphate group or by a hexNAc moiety. Due to the high energy used in HCD, the data did not allow precise localization of the modified sites. We suggest that these two PTM, by generating multiple isoforms, should extend the binding repertoire of secreted OBPs

气味结合蛋白（Odorant-Binding Proteins, OBP）是嗅觉通路中参与受体旁事件的关键分子。目前学界尚未提出可解释其对气味与信息素结合特异性的分子机制。一项全新的研究方向源自猪嗅觉分泌组（porcine olfactory secretome）的分析：该分泌组主要由OBP亚型构成，这些亚型由3种基因产物经翻译后修饰（Post-translational modification, PTM）、磷酸化及O-β-N-乙酰葡糖胺基化（O-GlcNAcylation）产生，而这类修饰模式在分泌蛋白中较为罕见。这些多样化的OBP亚型可能对不同配体展现出差异化的结合特性。在验证这一功能假说前，尽管已有特异性抗体对这两类翻译后修饰完成初步检测，但仍需通过质谱技术实现精准鉴定。本研究首次报道了利用电喷雾碰撞诱导解离串联质谱（Electrospray Ionization Higher-energy Collisional Dissociation Tandem Mass Spectrometry, ESI-HCD-MS/MS），对OBP经胰蛋白酶酶解所得肽段进行磷酸化与O-β-N-乙酰葡糖胺基化修饰鉴定的研究成果。通过PEAKS软件对原始质谱数据进行分析，筛选出有效质谱图并开展人工解析以鉴定修饰位点。最终在对应OBP序列两个不同区域的四条肽段上，分别发现了磷酸基团或己糖胺（hexNAc）修饰基团的存在。由于HCD碎裂过程采用了较高的碰撞能量，现有数据无法实现修饰位点的精确定位。本研究推测，这两类翻译后修饰通过产生多种OBP亚型，可拓展分泌型OBP的配体结合谱。