反相C18固相萃取小柱用于蛋白质组分析中少量样品预处理

Application of reverse phase C18 tip in pretreatment of small amount complex proteomic samples

摘要:

针对少量且复杂蛋白质组样品，开发一种耗时短、操作简便的分离方法。以人的脑海马组织蛋白样品为研究对象，采用反相C18固相萃取小柱，采用不同乙腈浓度对酶切后样品进行梯度洗脱，与反相高效液相色谱法对比分离效果。通过比较不同乙腈梯度洗脱方案所鉴定到的谱图数、非冗余多肽数、蛋白数和各洗脱组分间重复率分析，确定一种样品量少、简单易行、分离效果好的实验方案。虽然反相C18固相萃取小柱法鉴定蛋白总数占高效液相色谱法的85.5%，但其操作简单，耗时少。通过4种不同乙腈浓度方案比较，确定乙腈洗脱浓度优化为5%、15%、20%和90%时，分离30 μg人的海马多肽混合物可以得到较优的分离效果。其蛋白鉴定数为反相液相色谱法的67.0%，且重复性良好。该结果证实反相C18固相萃取小柱分离效果比反相高效液相色谱法稍差，但此方法可以分离少量复杂蛋白质组样品。该方法分离样品充分，操作易行，耗时短，是进行蛋白质组学分析中少量样品的一个简便预处理方法。

Abstract:

To develop a timesaving and easy operating Reverse Phase (RP) chromatography method, we adopted Thermo Pierce? RP C18 Tip to separate small amount hippocampus peptide mixtures and to compare with high performance liquid chromatography (HPLC). According to the separation performance of 4 ACN gradient optimization methods, we determined the best ACN concentration gradient. The results showed that, the experiment took only 10 min by separating with eight ACN concentration gradient, which accounted 1/4 for HPLC. But as for the identified proteins, RP C18 Tip accounted 85.5% for HPLC. ACN gradient of 5%, 15%, 20% and 90% had best repeatability (P=0.429) and result for separating 30 μg peptides. This method is easy to operate, timesaving and has low cost. It could be used into pretreatment of small amount complex proteomic samples.