Yen T Y, Joshi R K, Yan H, Seto N O, Palcic M M, Macher B A
Department of Chemistry and Biochemistry, San Francisco State University, San Francisco, California 94132, USA.
J Mass Spectrom. 2000 Aug;35(8):990-1002. doi: 10.1002/1096-9888(200008)35:8<990::AID-JMS27>3.0.CO;2-K.
Cysteine residues and disulfide bonds are important for protein structure and function. We have developed a simple and sensitive method for determining the presence of free cysteine (Cys) residues and disulfide bonded Cys residues in proteins (<100 pmol) by liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) in combination with protein database searching using the program Sequest. Free Cys residues in a protein were labeled with PEO-maleimide biotin immediately followed by denaturation with 8 M urea. Subsequently, the protein was digested with trypsin or chymotrypsin and the resulting products were analyzed by capillary LC/ESI-MS/MS for peptides containing modified Cys and/or disulfide bonded Cys residues. Although the MS method for identifying disulfide bonds has been routinely employed, methods to prevent thiol-disulfide exchange have not been well documented. Our protocol was found to minimize the occurrence of the thiol-disulfide exchange reaction. The method was validated using well-characterized proteins such as aldolase, ovalbumin, and beta-lactoglobulin A. We also applied this method to characterize Cys residues and disulfide bonds of beta 1,4-galactosyltransferase (five Cys), and human blood group A and B glycosyltransferases (four Cys). Our results demonstrate that beta 1,4-galactosyltransferase contains one free Cys residue and two disulfide bonds, which is in contrast to work previously reported using chemical methods for the characterization of free Cys residues, but is consistent with recently published results from x-ray crystallography. In contrast to the results obtained for beta 1,4-galactosyltransferase, none of the Cys residues in A and B glycosyltransferases were found to be involved in disulfide bonds.
半胱氨酸残基和二硫键对蛋白质的结构和功能至关重要。我们开发了一种简单且灵敏的方法，通过液相色谱/电喷雾电离串联质谱法（LC/ESI-MS/MS）并结合使用Sequest程序进行蛋白质数据库搜索，来测定蛋白质（<100 pmol）中游离半胱氨酸（Cys）残基和二硫键连接的Cys残基的存在情况。蛋白质中的游离Cys残基立即用聚乙二醇马来酰亚胺生物素进行标记，随后用8 M尿素变性。接着，用胰蛋白酶或糜蛋白酶消化该蛋白质，所得产物通过毛细管LC/ESI-MS/MS分析含有修饰Cys和/或二硫键连接的Cys残基的肽段。尽管用于鉴定二硫键的质谱方法已被常规使用，但防止硫醇-二硫键交换的方法尚未得到充分记载。我们的方案被发现可将硫醇-二硫键交换反应的发生降至最低。该方法使用醛缩酶、卵清蛋白和β-乳球蛋白A等特征明确的蛋白质进行了验证。我们还将此方法应用于表征β1,4-半乳糖基转移酶（五个Cys）以及人血型A和B糖基转移酶（四个Cys）的Cys残基和二硫键。我们的结果表明，β1,4-半乳糖基转移酶含有一个游离Cys残基和两个二硫键，这与先前使用化学方法表征游离Cys残基的工作结果相反，但与最近发表的X射线晶体学结果一致。与β1,4-半乳糖基转移酶的结果相反，在A和B糖基转移酶中未发现任何Cys残基参与二硫键的形成。
