Cobb K A, Novotny M
Anal Chem. 1989 Oct 15;61(20):2226-31. doi: 10.1021/ac00195a003.
Procedures for the reduced-scale analysis of proteins by peptide mapping have been developed, allowing peptide maps to be obtained from picomole to femtomole quantities of protein. The use of trypsin immobilized on agarose gel and placed in a small reactor column has made it possible to reproducibility digest as little as 50 ng of protein. This represents a decrease in sample size of approximately 3 orders of magnitude from conventional tryptic digestion schemes. Separations of tryptic digests were accomplished by using either microcolumn high-performance liquid chromatography (HPLC) or capillary zone electrophoresis (CZE). Separations of 100 ng (4 pmol) of tryptic digest samples of beta-casein were achieved with microcolumn HPLC, while separations of approximately 2 ng (80 fmol) of beta-casein tryptic digest (from a total sample size of 50 ng) were possible with CZE. Peptide maps from phosphorylated and dephosphorylated forms of beta-casein were readily distinguishable using both separation methods, demonstrating an ability to detect a single amino acid modification in a protein. Relative standard deviations of peak retention or migration times were less than 3% for microcolumn HPLC and less than 1% for CZE.
已经开发出通过肽图对蛋白质进行微量分析的方法，使得能够从皮摩尔至飞摩尔量的蛋白质中获得肽图。使用固定在琼脂糖凝胶上并置于小型反应器柱中的胰蛋白酶，可重复性地消化低至50 ng的蛋白质。这表示与传统的胰蛋白酶消化方案相比，样品量减少了约3个数量级。胰蛋白酶消化产物的分离通过使用微柱高效液相色谱（HPLC）或毛细管区带电泳（CZE）来完成。使用微柱HPLC可实现对100 ng（4 pmol）β-酪蛋白胰蛋白酶消化样品的分离，而使用CZE可实现对约2 ng（80 fmol）β-酪蛋白胰蛋白酶消化产物（来自50 ng的总样品量）的分离。使用这两种分离方法都能很容易地区分β-酪蛋白磷酸化和去磷酸化形式的肽图，证明了检测蛋白质中单个氨基酸修饰的能力。微柱HPLC的峰保留时间或迁移时间的相对标准偏差小于3%，CZE的小于1%。
