Lim Jung Mi, Levine Rodney L
Laboratory of Biochemistry, National Heart, Lung, and Blood Institute, Building 50, Room 2347, 50 South Drive, MSC 8012, Bethesda, 20892-8012, Maryland, USA.
Laboratory of Biochemistry, National Heart, Lung, and Blood Institute, Building 50, Room 2351, 50 South Drive, MSC 8012, Bethesda, 20892-8012, Maryland, USA.
Anal Biochem. 2025 Sep;704:115892. doi: 10.1016/j.ab.2025.115892. Epub 2025 May 8.
Cysteine alkylation is widely used in mass-spectrometric based proteomic studies. The oxidation state of each cysteine can be determined by labeling free thiols with one alkylating agent and disulfides with a second alkylating agent that differs in mass from the first. We have developed an improved method utilizing biotin-conjugated maleimides to specifically label cysteine residues in the thiol state and in disulfide linkage. The biotin tag effectuates very efficient enrichment of cysteine containing peptides, greatly increasing sensitivity for those peptides. We also achieve very high recovery of the biotinylated peptides from an avidin column by elution with hexafluoro-2-propanol (HFIP). The method offers improved mapping of the cysteine proteome and its oxidation state.
半胱氨酸烷基化在基于质谱的蛋白质组学研究中被广泛应用。每个半胱氨酸的氧化状态可通过用一种烷基化试剂标记游离巯基，并用与第一种质量不同的第二种烷基化试剂标记二硫键来确定。我们开发了一种改进方法，利用生物素共轭马来酰亚胺特异性标记处于巯基状态和二硫键连接状态的半胱氨酸残基。生物素标签能非常有效地富集含半胱氨酸的肽段，极大地提高了这些肽段的灵敏度。我们还通过用六氟 - 2 - 丙醇（HFIP）洗脱，从抗生物素蛋白柱中实现了生物素化肽段的非常高的回收率。该方法改进了对半胱氨酸蛋白质组及其氧化状态的图谱绘制。
