Levernæs Maren Christin Stillesby, Broughton Marianne Nordlund, Reubsaet Léon, Halvorsen Trine Grønhaug
Department of Pharmaceutical Chemistry, School of Pharmacy, University of Oslo, PO Box 1068 Blindern, 0316 Oslo, Norway.
Department of Medical Biochemistry, Norwegian Radium Hospital, Oslo University Hospital, PO Box 4953 Nydalen, 0424 Oslo, Norway.
J Chromatogr B Analyt Technol Biomed Life Sci. 2017 Jun 15;1055-1056:51-60. doi: 10.1016/j.jchromb.2017.03.044. Epub 2017 Apr 14.
Immunocapture-based bottom-up LC-MS is a promising technique for the quantification of low abundant proteins. Magnetic immunocapture beads provide efficient enrichment from complex samples through the highly specific interaction between the target protein and its antibody. In this article, we have performed the first thorough comparison between digestion of proteins while bound to antibody coated beads versus after elution from the beads. Two previously validated immunocapture based MS methods for the quantification of pro-gastrin releasing peptide (ProGRP) and human chorionic gonadotropin (hCG) were used as model systems. The tryptic peptide generation was shown to be protein dependent and influenced by protein folding and accessibility towards trypsin both on-beads and in the eluate. The elution of proteins bound to the beads was also shown to be incomplete. In addition, the on-beads digestion suffered from non-specific binding of the trypsin generated peptides. A combination of on-beads digestion and elution may be applied to improve both the quantitative (peak area of the signature peptides) and qualitative yield (number of missed cleavages, total number of identified peptides, coverage, signal intensity and number of zero missed cleavage peptides) of the target proteins. The quantitative yield of signature peptides was shown to be reproducible in all procedures tested.
基于免疫捕获的自下而上液相色谱-质谱联用技术是一种很有前景的低丰度蛋白质定量技术。磁性免疫捕获珠通过靶蛋白与其抗体之间的高度特异性相互作用，从复杂样品中实现高效富集。在本文中，我们首次对结合在抗体包被磁珠上的蛋白质消化与从磁珠上洗脱后的蛋白质消化进行了全面比较。两种先前经过验证的基于免疫捕获的质谱方法，用于促胃泌素释放肽（ProGRP）和人绒毛膜促性腺激素（hCG）的定量分析，被用作模型系统。结果表明，胰蛋白酶肽段的生成取决于蛋白质，并受到蛋白质折叠以及在磁珠上和洗脱液中对胰蛋白酶的可及性的影响。结合在磁珠上的蛋白质洗脱也不完全。此外，磁珠上的消化存在胰蛋白酶生成肽段的非特异性结合问题。磁珠上消化和洗脱相结合的方法，可用于提高靶蛋白的定量（特征肽段的峰面积）和定性产率（漏切数、鉴定肽段总数、覆盖率、信号强度和零漏切肽段数）。在所有测试步骤中，特征肽段的定量产率都具有可重复性。
