Yu Ying-Qing, Fournier Jennifer, Gilar Martin, Gebler John C
Waters Corporation, Milford, MA 01757, USA.
J Sep Sci. 2009 Apr;32(8):1189-99. doi: 10.1002/jssc.200800652.
Identification of phosphopeptides by MS is challenging due to their relatively low abundance in proteomic samples and their limited ionization efficiency. Various affinity enrichment methods have been used in the literature. Titanium dioxide SPE devices have been recently proposed as an alternative to immobilized metal affinity chromatography for phosphopeptide enrichment. This study evaluates the TiO(2 )method using sorbent packed in a 96 well microscale extraction plate operated using a vacuum manifold. The phosphopeptide recovery and enrichment selectivity were investigated at various loading conditions. The effectiveness of organic additives such as dihydroxybenzoic acid derivatives and other nonaliphatic carboxylic acids on enrichment selectivity was examined. The performance of TiO(2) was compared to IMAC sorbent. The results suggest that various additives improve the enrichment selectivity by effectively interfering with the acidic peptides binding to TiO(2) sorbent. Interaction of phosphopeptides with sorbent is also affected, which leads to overall reduction in phosphopeptide recovery. The new SPE device was successfully utilized for the extraction of phosphopeptides from yeast lysate digest using 2,5-dihydroxybenzoic acid to minimize the interference from nonphosphorylated peptides.
由于磷酸化肽段在蛋白质组样品中的丰度相对较低且电离效率有限，通过质谱鉴定磷酸化肽段具有挑战性。文献中已使用了各种亲和富集方法。最近有人提出用二氧化钛固相萃取装置替代固定化金属亲和色谱法来富集磷酸化肽段。本研究评估了使用填充在96孔微量萃取板中的吸附剂并通过真空歧管操作的TiO₂方法。在各种上样条件下研究了磷酸化肽段的回收率和富集选择性。考察了二羟基苯甲酸衍生物等有机添加剂和其他非脂肪族羧酸对富集选择性的影响。将TiO₂的性能与固定化金属亲和色谱吸附剂进行了比较。结果表明，各种添加剂通过有效干扰酸性肽与TiO₂吸附剂的结合来提高富集选择性。磷酸化肽段与吸附剂的相互作用也受到影响，这导致磷酸化肽段回收率总体降低。使用2,5 - 二羟基苯甲酸，新的固相萃取装置成功用于从酵母裂解物消化物中提取磷酸化肽段，以尽量减少非磷酸化肽段的干扰。
