Department of Radiobiology, Faculty of Military Health Sciences, University of Defense in Brno, 500 01 Hradec Kralove, Czech Republic.
Department of Molecular Biology and Pathology, Faculty of Military Health Sciences, University of Defense in Brno, 500 01 Hradec Kralove, Czech Republic.
Int J Mol Sci. 2020 Jun 1;21(11):3971. doi: 10.3390/ijms21113971.
Mass spectrometry (MS) is a powerful and sensitive method often used for the identification of phosphoproteins. However, in phosphoproteomics, there is an identified need to compensate for the low abundance, insufficient ionization, and suppression effects of non-phosphorylated peptides. These may hamper the subsequent liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) analysis, resulting in incomplete phosphoproteome characterization, even when using high-resolution instruments. To overcome these drawbacks, we present here an effective microgradient chromatographic technique that yields specific fractions of enriched phosphopeptides compatible with LC-MS/MS analysis. The purpose of our study was to increase the number of identified phosphopeptides, and thus, the coverage of the sample phosphoproteome using the reproducible and straightforward fractionation method. This protocol includes a phosphopeptide enrichment step followed by the optimized microgradient fractionation of enriched phosphopeptides and final LC-MS/MS analysis of the obtained fractions. The simple fractionation system consists of a gas-tight microsyringe delivering the optimized gradient mobile phase to reversed-phase microcolumn. Our data indicate that combining the phosphopeptide enrichment with the microgradient separation is a promising technique for in-depth phosphoproteomic analysis due to moderate input material requirements and more than 3-fold enhanced protein identification.
质谱（MS）是一种强大而敏感的方法，常用于鉴定磷酸化蛋白。然而，在磷酸化蛋白质组学中，人们已经认识到需要补偿低丰度、不足的离子化和非磷酸化肽的抑制作用。这些可能会干扰随后的液相色谱-质谱/质谱（LC-MS/MS）分析，导致磷酸蛋白质组特征不完全，即使使用高分辨率仪器也是如此。为了克服这些缺点，我们在这里提出了一种有效的微梯度色谱技术，该技术可产生与 LC-MS/MS 分析兼容的富含磷酸肽的特定级分。我们的研究目的是使用可重复和简单的分级方法增加鉴定的磷酸肽数量，从而增加样品磷酸蛋白质组的覆盖率。该方案包括磷酸肽富集步骤，然后对富集的磷酸肽进行优化的微梯度分级，最后对获得的级分进行 LC-MS/MS 分析。简单的分级系统由一个气密微注射器组成，该注射器将优化的梯度流动相输送到反相微柱中。我们的数据表明，由于对输入材料的要求适中，并且蛋白质鉴定增加了 3 倍以上，因此将磷酸肽富集与微梯度分离相结合是一种很有前途的深入磷酸蛋白质组分析技术。
