Quantification of peptides using N-terminal isotope coding and C-terminal derivatization for sensitive analysis by micro liquid chromatography-tandem mass spectrometry.
Quantification of peptides using N-terminal isotope coding and C-terminal derivatization for sensitive analysis by micro liquid chromatography-tandem mass spectrometry.
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DOI：
10.1002/jms.3845
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发表时间：
2016-12
期刊：
影响因子：
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通讯作者：
Yohei Sakaguchi;T. Kinumi;A. Takatsu
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文献类型：
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作者：
Yohei Sakaguchi;T. Kinumi;A. Takatsu
稳定同位素编码与质谱联用是一种流行的定量蛋白质组学和多肽定量方法。然而，特定官能团的衍生化反应的效率，特别是在复杂结构中，可能会影响准确度。在这里，我们提出了一种生物活性多肽的双官能团衍生，随后进行了微液相色谱-串联质谱仪(LC-MS/MS)。通过分离增敏反应和同位素编码衍生化反应，可以选择合适的化学试剂。用乙醛或乙醛-d4还原烷基化多肽氨基，得到不同质量的N-烷基化产物。该工艺简单、快速、产率高，可实现对质量分化肽的准确比较分析。然后，用1-(2-嘧啶)哌嗪对羧基进行衍生化，以提高MS/MS的灵敏度。用微型LC-MS/MS在线固相萃取分析血管紧张素I-IV、缓激肽和神经降压素。在所有情况下，与未衍生多肽的分析相比，灵敏度提高了17倍以上。本方法与同位素标记血管紧张素I[Asp-Arg-(Val-d8)-Tyr-Ile-His-Pro-(Phe-d8)-His-Leu]的同位素稀释定量结果一致。版权所有©2016 John Wiley&Sons，Ltd.
Stable isotope-coding coupled with mass spectrometry is a popular method for quantitative proteomics and peptide quantification. However, the efficiency of the derivatization reaction at a particular functional group, especially in complex structures, can affect accuracy. Here, we present a dual functional-group derivatization of bioactive peptides followed by micro liquid chromatography-tandem mass spectrometry (LC-MS/MS). By separating the sensitivity-enhancement and isotope-coding derivatization reactions, suitable chemistries can be chosen. The peptide amino groups were reductively alkylated with acetaldehyde or acetaldehyde-d4 to afford N-alkylated products with different masses. This process is simple, quick and high-yield, and accurate comparative analysis can be achieved for the mass-differentiated peptides. Then, the carboxyl groups were derivatized with 1-(2-pyrimidinyl)piperazine to increase MS/MS sensitivity. Angiotensins I-IV, bradykinin and neurotensin were analyzed after online solid phase extraction by micro LC-MS/MS. In all instances, a greater than 17-fold increase in sensitivity was achieved, compared with the analyses of the underivatized peptides. Furthermore, the values obtained from the present method were in agreement with the result from isotope dilution quantification using isotopically labeled angiotensin I [Asp-Arg-(Val-d8 )-Tyr-Ile-His-Pro-(Phe-d8 )-His-Leu]. Copyright © 2016 John Wiley & Sons, Ltd.