Department of Biochemistry, Escola Paulista de Medicina, Federal University of Sao Paulo, Sao Paulo, SP, 04023-901, Brazil.
Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, NY, 10461, USA.
J Am Soc Mass Spectrom. 2018 May;29(5):866-878. doi: 10.1007/s13361-017-1852-3. Epub 2017 Dec 12.
Quantitative peptidomics and proteomics often use chemical tags to covalently modify peptides with reagents that differ in the number of stable isotopes, allowing for quantitation of the relative peptide levels in the original sample based on the peak height of each isotopic form. Different chemical reagents have been used as tags for quantitative peptidomics and proteomics, and all have strengths and weaknesses. One of the simplest approaches uses formaldehyde and sodium cyanoborohydride to methylate amines, converting primary and secondary amines into tertiary amines. Up to five different isotopic forms can be generated, depending on the isotopic forms of formaldehyde and cyanoborohydride reagents, allowing for five-plex quantitation. However, the mass difference between each of these forms is only 1 Da per methyl group incorporated into the peptide, and for many peptides there is substantial overlap from the natural abundance of C and other isotopes. In this study, we calculated the contribution from the natural isotopes for 26 native peptides and derived equations to correct the peak intensities. These equations were applied to data from a study using human embryonic kidney HEK293T cells in which five replicates were treated with 100 nM vinblastine for 3 h and compared with five replicates of cells treated with control medium. The correction equations brought the replicates to the expected 1:1 ratios and revealed significant decreases in levels of 21 peptides upon vinblastine treatment. These equations enable accurate quantitation of small changes in peptide levels using the reductive methylation labeling approach. Graphical abstract ᅟ.
定量肽组学和蛋白质组学通常使用化学标记物，通过与试剂共价修饰肽，这些试剂在稳定同位素的数量上有所不同，从而可以根据每种同位素形式的峰高，对原始样品中相对肽水平进行定量。已经使用了不同的化学试剂作为定量肽组学和蛋白质组学的标记物，每种试剂都有其优缺点。最简单的方法之一是使用甲醛和氰基硼氢化钠将胺甲基化，将伯胺和仲胺转化为叔胺。根据甲醛和氰基硼氢化试剂的同位素形式，最多可以生成五种不同的同位素形式，允许进行五重定量。然而，这些形式之间的质量差异仅为每个肽中结合的甲基基团的 1 Da，对于许多肽来说，由于 C 和其他同位素的自然丰度，存在大量重叠。在这项研究中，我们计算了 26 种天然肽的天然同位素的贡献，并推导出了校正峰强度的方程。这些方程应用于使用人胚肾 HEK293T 细胞的研究数据，其中五个重复样本用 100 nM 长春新碱处理 3 小时，并与用对照培养基处理的五个重复样本进行比较。校正方程将重复样本调整到预期的 1:1 比例，并显示长春新碱处理后 21 种肽的水平显著降低。这些方程使得使用还原甲基化标记方法可以准确定量肽水平的微小变化。
