Dasgupta Sayani, Castro Leandro M, Tashima Alexandre K, Fricker Lloyd
Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, NY, USA.
Bioscience Institute, São Paulo State University, São Vicente, SP, Brazil.
Methods Mol Biol. 2018;1719:161-174. doi: 10.1007/978-1-4939-7537-2_10.
A number of different approaches have been used for quantitative peptidomics. In this protocol we describe the method in which peptides are reacted with formaldehyde and sodium cyanoborohydride, which converts primary and secondary amines into tertiary amines. By using different combinations of regular reagents, deuterated reagents (H), and reagents containing deuterium and C, it is possible to produce five isotopically distinct forms of the methylated peptides which can be quantified by mass spectrometry. Peptides with free N-termini that are primary amines incorporate two methyl groups using this procedure, which differ by 2 Da for each of the five isotopic combinations. Peptides that contain unmodified lysine residues incorporate additional pairs of methyl groups, leading to larger mass differences between isotopic forms. The reagents are commercially available, relatively inexpensive, and chemically stable.
已经有多种不同的方法用于定量肽组学。在本方案中，我们描述了一种肽与甲醛和氰基硼氢化钠反应的方法，该反应将伯胺和仲胺转化为叔胺。通过使用常规试剂、氘代试剂（H）以及含氘和碳的试剂的不同组合，可以产生五种同位素不同形式的甲基化肽，这些肽可以通过质谱进行定量。具有游离N端（即伯胺）的肽使用该方法会引入两个甲基，对于五种同位素组合中的每一种，这两个甲基的质量相差2 Da。含有未修饰赖氨酸残基的肽会引入额外的甲基对，导致同位素形式之间的质量差异更大。这些试剂可从商业渠道获得，相对便宜且化学性质稳定。
