离线pH梯度-强阳离子交换色谱法分离肽段混合物

Separation of Peptides Using Off-Line pH Gradient-Strong Cation Exchange Chromatography

复杂肽段混合物的有效分离是高覆盖率地鉴定蛋白质混合物的前提。Shotgun蛋白质组学研究通常采用二维液相色谱(强阳离子交换色谱-反相色谱)分离后接串联质谱检测的方法。但由于离子交换色谱体系中含有高浓度的盐,使得在线分析的难度较大;而在离线分析时,也常因需要对高盐组分进行脱盐处理而易引起样品损失。因此,该文尝试用pH梯度替代盐梯度,实现pH梯度-强阳离子交换色谱方法应用于复杂肽段混合物的分离。通过对缓冲体系pH值的计算,优化了乙酸-乙酸铵体系线性pH梯度配合盐梯度的离子交换色谱方法,以及柠檬酸-氨水体系线性pH梯度的离子交换色谱方法。将这两种方法应用于牛血清白蛋白酶切产物的分离取得了与常规强阳离子交换色谱相似的分离效果。乙酸-乙酸铵体系采用的是低浓度的可挥发性铵盐,采用真空冻干的方法可以有效除盐,基质辅助激光解吸质谱靶上自然挥发也可以达到较好的脱盐效果,简化了常规方法繁琐费时的脱盐步骤及避免了由此造成的样品损失。柠檬酸-氨水体系采用pH梯度洗脱替代盐梯度洗脱,大大降低了体系中的盐浓度。这两种方法在复杂体系蛋白质组研究的样本预分离中具有较好的应用前景。

On the basis of theoretical pH calculation for buffer systems, an off-line linear pH gradient-strong cation exchange chromatographic method was developed for peptides separation. For the acetate buffer system, the peptides from tryptic digest of BSA were eluted by the linear pH gradient （ pH 3.7 - 6.0） with a low concentration of ammonium acetate salt gradient, whose salt is volatile and can be easily removed by lyophilization. As for the citrate buffer system, the peptides were eluted by a wider range of linear pH gradient （ pH 3.0 - 8.5 ） with a even lower concentration of ammonium citrate salt gradient. Both methods are effective for the peptides separation and before mass spectrometric detection can simplify the desalting step, which is labor-intensive and may incur significant sample loss for the routine strong cation exchange chromatography.