一种无抗体的骨桥蛋白样本预处理方法，结合 MALDI-TOF MS/MS 分析。

An antibody-free sample pretreatment method for osteopontin combined with MALDI-TOF MS/MS analysis.

机构信息

KTH Royal Institute of Technology, School of Engineering Sciences in Chemistry, Biotechnology and Health, Department of Chemistry, Division of Applied Physical Chemistry, Analytical Chemistry, Stockholm, Sweden.

出版信息

PLoS One. 2019 Mar 7;14(3):e0213405. doi: 10.1371/journal.pone.0213405. eCollection 2019.

Osteopontin is an osteoblast-secreted protein with an aspartic acid-rich, highly phosphorylated, and glycosylated structure. Osteopontin can easily bind to integrins, tumor cells, extracellular matrix and calcium, and is related to bone diseases, various cancers, inflammation etc. Here, DEAE-Cibacron blue 3GA was used to extract recombinant osteopontin from human plasma, and to deplete abundant plasma proteins with an antibody-free method. Using selected buffer systems, osteopontin and human serum albumin could be bound to DEAE-Cibacron blue 3GA, while immunoglobulin G was excluded. The bound osteopontin could then be separated from albumin by using different sequential elution buffers. By this method, 1 μg/mL recombinant osteopontin could be separated from the major part of the most abundant proteins in human plasma. After trypsin digestion, the extracted osteopontin could be successfully detected and identified by MALDI-TOF MS/MS using the m/z 1854.898 peptide and its fragments.

骨桥蛋白是一种由成骨细胞分泌的蛋白，具有富含天冬氨酸、高度磷酸化和糖基化的结构。骨桥蛋白可以轻易地与整合素、肿瘤细胞、细胞外基质和钙结合，并与骨疾病、各种癌症、炎症等有关。在这里，使用 DEAE-Cibacron blue 3GA 从人血浆中提取重组骨桥蛋白，并使用无抗体的方法去除丰富的血浆蛋白。使用选定的缓冲系统，骨桥蛋白和人血清白蛋白可以与 DEAE-Cibacron blue 3GA 结合，而免疫球蛋白 G 则被排除在外。然后，通过使用不同的顺序洗脱缓冲液，可以将结合的骨桥蛋白从白蛋白中分离出来。通过这种方法，可以从人血浆中最丰富的大多数蛋白质中分离出 1 μg/mL 的重组骨桥蛋白。经过胰蛋白酶消化后，使用 m/z 1854.898 肽及其片段，通过 MALDI-TOF MS/MS 可以成功地检测和鉴定提取的骨桥蛋白。