Mapping Tissue-Specific Expression of Extracellular Proteins Using Systematic Glycoproteomic Analysis of Different Mouse Tissues

Due to their easy accessibility, proteins outside of the plasma membrane represent an ideal but untapped resource for potential drug targets or disease biomarkers. They constitute the major biochemical class of current therapeutic targets and clinical biomarkers. Recent advances in proteomic technologies have fueled interest in analysis of extracellular proteins such as membrane proteins, cell surface proteins, and secreted proteins. However, unlike the gene expression analyses from a variety of tissues and cells using genomic technologies, quantitative proteomic analysis of proteins from various biological sources is challenging due to the high complexity of different proteomes and the lack of robust and consistent methods for analyses of different tissue sources, especially for specific enrichment of extracellular proteins. Since most extracellular proteins are modified by oligosaccharides, the population of glycoproteins therefore represents the majority of extracellular proteomes. Here, we quantitatively analyzed glycoproteins and determined the expression patterns of extracellular proteins from 12 mouse tissues using solid-phase extraction of N-linked glycopeptides and liquid chromatography−tandem mass spectrometry. We identified peptides enclosing 1231 possible N-linked glycosites from 826 unique proteins. We further determined the expression pattern of formerly N-linked glycopeptides and identified extracellular glycoproteins specifically expressed in each tissue. Furthermore, the tissue specificities of the overexpressed glycoproteins in a mouse skin tumor model were determined by comparing them to the quantitative protein expression from the different tissues. These skin tumor-specific extracellular proteins might serve as potential candidates for cell surface drug targets or disease-specific protein markers.

由于细胞膜外蛋白质易于获取，其作为潜在药物靶点或疾病生物标志物的资源价值尚未被充分挖掘，是一类理想的研究对象。这类蛋白质亦是当前治疗靶点与临床生物标志物的主要生化类别。近年来，蛋白质组学技术（proteomic technologies）的发展推动了学界对膜蛋白（membrane proteins）、细胞表面蛋白（cell surface proteins）及分泌蛋白（secreted proteins）等细胞外蛋白质的研究热情。然而，相较于依托基因组学技术（genomic technologies）开展的多组织、多细胞基因表达分析，对不同生物来源的蛋白质开展定量蛋白质组学分析（quantitative proteomic analysis）仍面临诸多挑战：一方面不同蛋白质组（proteomes）本身复杂度极高，另一方面针对不同组织来源（尤其是细胞外蛋白质的特异性富集）尚未建立稳健且统一的分析方法。由于绝大多数细胞外蛋白质均经寡糖（oligosaccharides）修饰，糖蛋白（glycoproteins）群体因此构成了细胞外蛋白质组的主体。本研究通过N-连接糖肽（N-linked glycopeptides）固相萃取结合液相色谱-串联质谱法（liquid chromatography−tandem mass spectrometry），对12种小鼠组织的糖蛋白进行定量分析，并解析了其细胞外蛋白质的表达谱。研究团队从826种独特蛋白质中鉴定出1231个潜在N-连接糖基化位点（glycosites）；进一步明确了原N-连接糖肽的表达模式，并筛选出在各组织中特异性表达的细胞外糖蛋白。此外，通过与不同组织的定量蛋白质表达数据比对，本研究明确了小鼠皮肤肿瘤模型（mouse skin tumor model）中过表达糖蛋白的组织特异性。这些皮肤肿瘤特异性细胞外蛋白质有望成为细胞表面药物靶点或疾病特异性蛋白质标志物的潜在候选者。