Identification of New Hematopoietic Cell Subsets with a Polyclonal Antibody Library Specific for Neglected Proteins

The identification of new markers, the expression of which defines new phenotipically and functionally distinct cell subsets, is a main objective in cell biology. We have addressed the issue of identifying new cell specific markers with a reverse proteomic approach whereby approximately 1700 human open reading frames encoding proteins predicted to be transmembrane or secreted have been selected in silico for being poorly known, cloned and expressed in bacteria. These proteins have been purified and used to immunize mice with the aim of obtaining polyclonal antisera mostly specific for linear epitopes. Such a library, made of about 1600 different polyclonal antisera, has been obtained and screened by flow cytometry on cord blood derived CD34+CD45dim cells and on peripheral blood derived mature lymphocytes (PBLs). We identified three new proteins expressed by fractions of CD34+CD45dim cells and eight new proteins expressed by fractions of PBLs. Remarkably, we identified proteins the presence of which had not been demonstrated previously by transcriptomic analysis. From the functional point of view, looking at new proteins expressed on CD34+CD45dim cells, we identified one cell surface protein (MOSC-1) the expression of which on a minority of CD34+ progenitors marks those CD34+CD45dim cells that will go toward monocyte/granulocyte differentiation. In conclusion, we show a new way of looking at the membranome by assessing expression of generally neglected proteins with a library of polyclonal antisera, and in so doing we have identified new potential subsets of hematopoietic progenitors and of mature PBLs.鉴定可通过表达特征区分表型与功能均独特的细胞亚群的新型标志物，是细胞生物学领域的核心研究目标之一。本研究采用反向蛋白质组学方法，旨在鉴定新型细胞特异性标志物：通过计算机辅助筛选，选取约1700个编码预测为跨膜或分泌型蛋白的人类开放阅读框，将其克隆并在细菌中表达。将上述蛋白纯化后免疫小鼠，以获取主要针对线性表位的多克隆抗血清库。本研究成功构建了包含约1600种不同多克隆抗血清的库，并通过流式细胞术对脐带血来源的CD34+CD45dim细胞以及外周血来源的成熟淋巴细胞（PBLs）进行筛选。研究共鉴定出3种可在部分CD34+CD45dim细胞上表达的新型蛋白，以及8种可在部分PBLs上表达的新型蛋白。值得注意的是，部分此前未被转录组分析检测到的蛋白也被本研究成功鉴定。从功能角度分析针对CD34+CD45dim细胞的新型蛋白时，本研究鉴定出一种细胞表面蛋白MOSC-1：该蛋白在少数CD34+造血祖细胞上的表达，可标记出将向单核细胞/粒细胞方向分化的CD34+CD45dim细胞亚群。综上，本研究通过多克隆抗血清库检测通常被忽视的蛋白的表达情况，为膜蛋白质组研究提供了全新的研究思路；借此方法，本研究还鉴定出了潜在的新型造血祖细胞亚群与成熟PBLs亚群。