Nowalk Andrew J, Nolder Christi, Clifton Dawn R, Carroll James A
Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA.
Proteomics. 2006 Apr;6(7):2121-34. doi: 10.1002/pmic.200500187.
Borrelia burgdorferi, the cause of Lyme disease, produces excessive amounts of membrane lipoproteins such as outer surface protein A (OspA) when grown in vitro, and consequently many low or moderately abundant proteins are underrepresented when cell lysates are examined by 2-DE. We analyzed the B. burgdorferi B31 proteome computationally and by IPG or modified NEPHGE after subcellular fractionation into membrane-associated and soluble proteins. The B. burgdorferi B31 theoretical proteome is comprised of 1623 proteins and has a mean pI of 8.36 and a median pI of 9.03 with 68% of the proteome possessing a pI >/=7.5. Separation of soluble proteins by IPG resulted in 205 individual spots and identification of 78 protein spots by MALDI-TOF MS. Separation by modified NEPHGE routinely resulted in approximately 185 soluble and 160 membrane protein spots with the identification of 88 individual protein spots combined by MALDI-TOF MS. Homologues to GroEL and aminopeptidase I were present in greater amounts in the membrane faction, with enolase at nearly equivalent amounts in the soluble and membrane fractions. Identification of proteins isolated and separated by such methods will enable future determination of proteome changes in membrane and soluble protein fractions as spirochetes adapt to their changing environments.
莱姆病的病原体伯氏疏螺旋体在体外生长时会产生过量的膜脂蛋白，如外表面蛋白A（OspA），因此，当通过双向电泳检测细胞裂解物时，许多低丰度或中等丰度的蛋白质代表性不足。我们通过计算以及在将细胞亚细胞分级分离为膜相关蛋白和可溶性蛋白后，采用固相pH梯度等电聚焦（IPG）或改良的非平衡pH梯度电泳（NEPHGE）对伯氏疏螺旋体B31的蛋白质组进行了分析。伯氏疏螺旋体B31的理论蛋白质组由1623种蛋白质组成，平均等电点为8.36，中位数等电点为9.03，其中68%的蛋白质组等电点≥7.5。通过IPG分离可溶性蛋白质得到了205个独立的斑点，并通过基质辅助激光解吸电离飞行时间质谱（MALDI-TOF MS）鉴定了78个蛋白质斑点。通过改良的NEPHGE分离通常会得到大约185个可溶性蛋白斑点和160个膜蛋白斑点，通过MALDI-TOF MS联合鉴定了88个独立的蛋白质斑点。伴侣蛋白GroEL和氨肽酶I的同源物在膜组分中的含量更高，烯醇化酶在可溶性组分和膜组分中的含量几乎相等。通过这些方法分离和鉴定的蛋白质，将有助于未来确定当螺旋体适应不断变化的环境时，膜蛋白组分和可溶性蛋白组分中蛋白质组的变化情况。
