Otto A, Thiede B, Müller E C, Scheler C, Wittmann-Liebold B, Jungblut P
Max-Delbrück-Centrum für, Molekulare Medizin, Berlin-Buch, Germany.
Electrophoresis. 1996 Oct;17(10):1643-50. doi: 10.1002/elps.1150171027.
Peptide mass fingerprinting is a powerful tool for the identification of proteins separated by two-dimensional gel electrophoresis (2-DE). The identification of in-gel digested proteins by peptide mass fingerprinting was significantly improve in comparison to blot-digests by using a peptide-collecting device. This device allows the effective purification and concentration of enzymatic digests of low-intensity spots without expensive equipment and is described in detail. Sensitivity in the fmol range was demonstrated by unequivocal identification of bovine serum albumin after sodium dodecyl sulfate--polyacrylamide gel electrophoresis. Furthermore the high performance liquid chromatography pattern of in-gel digests indicated a 2- to 3-fold higher yield of the separated peptides. Therefore, a higher amount of the peptides was available to perform N-terminal sequencing. The identification of 16 proteins from a high-resolution 2-DE gel map of human myocardium tissue has been achieved by means of this technique. Three of these proteins were associated with changes in spot intensity with dilated cardiomyopathy.
肽质量指纹图谱是鉴定通过二维凝胶电泳（2-DE）分离的蛋白质的有力工具。与印迹消化法相比，使用肽收集装置可显著提高通过肽质量指纹图谱鉴定胶内消化蛋白质的效率。该装置无需昂贵设备就能有效纯化和浓缩低强度斑点的酶解消化物，并对其进行了详细描述。在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳后，通过明确鉴定牛血清白蛋白证明了其在飞摩尔范围内的灵敏度。此外，胶内消化物的高效液相色谱图谱表明，分离出的肽产量提高了2至3倍。因此，有更多的肽可用于进行N端测序。通过该技术已从人心肌组织的高分辨率2-DE凝胶图谱中鉴定出16种蛋白质。其中三种蛋白质与扩张型心肌病斑点强度的变化有关。
