Hynek R, Kasicka V, Kucerová Z, Kás J
Department of Biochemistry and Microbiology, Institute of Chemical Technology, Prague, Czech Republic.
J Chromatogr B Biomed Appl. 1996 May 31;681(1):37-45. doi: 10.1016/0378-4347(95)00462-9.
A combination of reversed-phase high-performance liquid chromatography (RP-HPCL) and capillary zone electrophoresis (CZE) was used for the characterization of peptide maps of swine pepsin after its digestion with alpha-chymotrypsin. Peptide maps obtained by both methods were compared and five selected chromatographic peaks were identified on an electrophoreogram. The different order of peaks found in RP-HPLC compared to CZE confirmed the complementarity of these two methods. More peptide fragments were resolved by RP-HPLC, which was also found to be less sensitive to salt content in peptide mixtures, than by CZE, but only CZE was able to separate and identify phosphorylated and dephosphorylated peptide fragments of swine pepsin digest. CZE provides faster separation than RP-HPLC, however, the salts have to be removed by ultrafiltration or by RP-HPLC pre-separation prior to CZE analysis. Combined use of RP-HPLC and CZE for peptide mapping makes it possible to distinguish between the phosphorylated and dephosphorylated forms of swine pepsin. This is important from a diagnostic point of view, because pepsin phosphorylation may be associated with gastric cancer.
采用反相高效液相色谱（RP-HPCL）和毛细管区带电泳（CZE）相结合的方法，对猪胃蛋白酶经α-胰凝乳蛋白酶消化后的肽图进行表征。比较了两种方法获得的肽图，并在电泳图上鉴定了五个选定的色谱峰。与CZE相比，RP-HPLC中峰的顺序不同，证实了这两种方法的互补性。与CZE相比，RP-HPLC能分离出更多的肽片段，且对肽混合物中的盐含量不太敏感，但只有CZE能够分离和鉴定猪胃蛋白酶消化产物中的磷酸化和去磷酸化肽片段。CZE比RP-HPLC分离速度更快，然而，在CZE分析之前，必须通过超滤或RP-HPLC预分离去除盐分。RP-HPLC和CZE联合用于肽图分析，可以区分猪胃蛋白酶的磷酸化和去磷酸化形式。从诊断的角度来看，这很重要，因为胃蛋白酶磷酸化可能与胃癌有关。
