PDF(Pubmed)
Abstract：
Immuno-mass spectrometry (MS) is a powerful method for the quantitative analysis of low-abundance proteins in biological specimens. In these procedures, collecting specifically and efficiently the target protein antigens from the antigen-antibody complex generated on the surface of nanocarrier beads is crucial and can be performed by hydrolyzing the proteins directly on the beads or after elution. Herein, we optimized the conditions of the immunoaffinity purification via elution using serum α-fetoprotein (AFP) as a model and its specific antibody immobilized covalently on magnetic beads. Antibody-coated beads were incubated with human serum spiked with standard AFP for antigen-antibody reaction. AFP was then eluted from the beads using various eluents, including organic solvents, to optimize the elution conditions. After proteolytically hydrolyzing the eluted protein, stable isotope-labeled standard peptides were added to the hydrolysate to quantify the eluted AFP via liquid chromatography-tandem MS. Using an optimized workflow for quantitative analysis afforded a correlation between the amount of spiked AFP and heavy to light ratios calculated based on peptide ion peak areas, from which an endogenous AFP concentration of 2.3±0.6 ng/mL was determined in normal serum; this is consistent with previous reports using radioimmunoassay methods. The present immuno-MS workflow could apply to the detection and quantitation of other low-abundance biofluid biomarkers.
摘要：
免疫质谱（MS）是定量分析生物标本中低丰度蛋白质的有力方法。在这些程序中,从纳米载体珠表面上产生的抗原-抗体复合物中特异性和有效地收集靶蛋白抗原是至关重要的,并且可以通过直接在珠上或在洗脱后水解蛋白来进行。在这里，我们以血清甲胎蛋白（AFP）为模型，并将其特异性抗体共价固定在磁珠上，通过洗脱优化了免疫亲和纯化的条件。将抗体包被的珠子与掺入标准AFP的人血清一起孵育，以进行抗原-抗体反应。然后使用各种洗脱剂从珠子上洗脱AFP，包括有机溶剂,优化洗脱条件。蛋白水解后洗脱的蛋白质，将稳定同位素标记的标准肽添加到水解物中，以通过液相色谱-串联MS定量洗脱的AFP。使用优化的工作流程进行定量分析，提供了基于肽离子峰面积计算的AFP掺入量与重轻比之间的相关性。在正常血清中确定内源性AFP浓度为2.3±0.6ng/mL；这与以前使用放射免疫分析方法的报道一致。本免疫MS工作流程可应用于其他低丰度生物流体生物标志物的检测和定量。
