A STRATEGY TO ENRICH FOR UBIQUITINATED PEPTIDES

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 由于泛素修饰的调节几乎在每一个细胞通路中起作用，因此确定泛素附着位点至关重要。 这些位点已通过质谱法基于携带来自泛素部分的两个额外甘氨酸残基的胰蛋白酶肽的分子量增加而鉴定。 然而，这种具有GG位移的肽一直难以发现。 我们设计了一种在质谱分析之前通过两个步骤富集GG-肽的方法。 首先，在亲和纯化泛素之前，我们通过用2-硝基-5-硫氰基苯甲酸（NTCB）处理来降低蛋白质组的复杂性; NTCB在半胱氨酸残基处切割，而泛素不含半胱氨酸。 其次，我们通过强阳离子交换（SCX）色谱分离高度带电的GG-肽。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. As regulation by ubiquitin modification plays a role in nearly every cellular pathway, it is critical to identify sites of ubiquitin attachment. These sites have been identified by mass spectrometry based on the increase in molecular mass of a tryptic peptide carrying two additional glycine residues from the ubiquitin moiety. However, such peptides with GG shifts have been difficult to discover. We devised a method to enrich for GG-peptides prior to mass spectrometry by two steps. First, before affinity purification of ubiquitin, we reduce the complexity of the proteome by treatment with 2-nitro-5-thiocyanobenzoic acid (NTCB); NTCB cleaves at cysteine residues and ubiquitin contains no cysteine. Second, we separate the highly charged GG-peptides by strong cation exchange (SCX) chromatography.