Bie Zijun, Chen Yang, Li Hengye, Wu Ronghu, Liu Zhen
State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093, China.
Department of Chemistry and Biochemistry, Georgia Institute of Technology, GA 30332, USA.
Anal Chim Acta. 2014 Jun 27;834:1-8. doi: 10.1016/j.aca.2014.04.035. Epub 2014 Apr 26.
Boronate affinity materials have attracted increasing attentions as sample enrichment platforms for glycoproteomic analysis in recent years. However, most of the boronate affinity materials that have already employed for proteomic analysis are suffering from apparent disadvantages, such as alkaline pH for binding, weak affinity, and relatively poor selectivity. Benzoboroxoles are a unique class of boronic acids which have showed excellent binding properties for the recognition of cis-diol-containing compounds. Recently, a 3-carboxy-benzoboroxole-functionalized monolithic column had been reported and it had exhibited the best selectivity and affinity as well as the lowest binding pH among all reported boronate affinity monolithic columns. In this study, an off-line hyphenation of this boronate affinity monolithic column-based extraction with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was developed and the powerfulness of this hyphenated approach in the analysis of glycoproteins and glycopeptides in complex samples was investigated. The approach was first applied to the analysis of glycopeptides in the tryptic digest of horseradish peroxidase (HRP). Totally 22 glycopeptides were identified. To the best of our knowledge, this is the best performance among all the boronic acid-functionalized materials. We further employed this approach to the analysis of intact proteins in human saliva. Totally 6 intact glycoproteins were successfully identified. As comparison, when the samples were analyzed without extraction, only a few glycopeptides were identified from the tryptic digest of HRP while no glycoproteins were found from the saliva samples.
近年来，硼酸酯亲和材料作为糖蛋白质组分析的样品富集平台受到了越来越多的关注。然而，大多数已用于蛋白质组分析的硼酸酯亲和材料存在明显的缺点，如结合时需要碱性pH值、亲和力弱以及选择性相对较差。苯并硼恶唑是一类独特的硼酸，已显示出对含顺式二醇化合物具有优异的识别结合特性。最近，有报道称一种3-羧基-苯并硼恶唑功能化的整体柱，在所有报道的硼酸酯亲和整体柱中，它表现出了最佳的选择性和亲和力，以及最低的结合pH值。在本研究中，开发了一种基于该硼酸酯亲和整体柱萃取与基质辅助激光解吸/电离飞行时间质谱（MALDI-TOF MS）的离线联用方法，并研究了这种联用方法在分析复杂样品中的糖蛋白和糖肽方面的强大功能。该方法首先应用于辣根过氧化物酶（HRP）胰蛋白酶消化物中糖肽的分析。共鉴定出22种糖肽。据我们所知，这是所有硼酸功能化材料中表现最佳的。我们进一步将该方法应用于人类唾液中完整蛋白质的分析。成功鉴定出6种完整的糖蛋白。作为比较，当不进行萃取分析样品时，从HRP的胰蛋白酶消化物中仅鉴定出少数糖肽，而从唾液样品中未发现糖蛋白。
