State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Engineering Research Center for Protein Drugs, National Center for Protein Sciences, Beijing Institute of Radiation Medicine, Beijing, P. R. China.
Proteomics. 2013 Aug;13(15):2229-37. doi: 10.1002/pmic.201200591. Epub 2013 Jul 9.
Reversed phase microcolumns have been widely used for peptide pretreatment to desalt and remove interferences before tandem LC-MS in proteomics studies. However, few studies have characterized the effects of experimental parameters as well as column characteristics on the composition of identified peptides. In this study, several parameters including the concentration of ACN in washing buffer, the microcolumn's purification effect, the peptide recovery rate, and the dynamic-binding capacity were characterized in detail, based upon stable isotope labeling by amino acids in a cell culture quantitative approach. The results showed that peptide losses can be reduced with low ACN concentration in washing buffers resulting in a recovery rate of approximately 82%. Furthermore, the effects of ACN concentration and loading amount on the properties of identified peptides were also evaluated. We found that the dynamic-binding capacity of the column was approximately 26 μg. With increased loading amounts, more hydrophilic peptides were replaced by hydrophobic peptides.
反相微柱已广泛用于肽预处理，在蛋白质组学研究中的串联 LC-MS 之前脱盐和去除干扰。然而，很少有研究描述实验参数和柱特性对鉴定肽组成的影响。在这项研究中，我们根据稳定同位素标记的氨基酸细胞培养定量方法，详细描述了包括洗涤缓冲液中 ACN 浓度、微柱的纯化效果、肽回收率和动态结合容量等参数。结果表明，低浓度 ACN 的洗涤缓冲液可以减少肽的损失，回收率约为 82%。此外，还评估了 ACN 浓度和上样量对鉴定肽性质的影响。我们发现柱的动态结合容量约为 26 μg。随着上样量的增加，更多的亲水肽被疏水性肽取代。
