Université de Pau et des Pays de l'Adour /CNRS UMR 5254, Laboratoire de Chimie Analytique Bio-Inorganique et Environnement/IPREM, 64053 Pau, France.
Anal Chem. 2013 Mar 19;85(6):3064-70. doi: 10.1021/ac303618v. Epub 2013 Feb 27.
Low background signals are an indispensable prerequisite for accurate quantification in bioanalytics. This poses a special challenge when using derivatized samples, where excess reagent concentrations are increasing the background signal. Precleaning steps often are time-consuming and usually lead to analyte losses. In this study, a set of labeled model peptides and a protein digest was analyzed using inductively coupled plasma mass spectrometry (ICPMS), coupled to nano ion pairing reversed-phase high-performance liquid chromatography (nano-IP-RP-HPLC). In addition, matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) was used for peptide identification. Peptides were labeled with lanthanide metals using bifunctional DOTA-based (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) reagents. The resulting metal excess was removed online during nano-HPLC, by trapping the labeled peptides on a C18-precolumn and washing them prior to their elution to the analytical column. Different ion pairing reagents like TFA (trifluoroacetic acid) and HFBA (heptafluorobutyric acid) were used in the study to enhance interactions of the different peptide species with the C18 material of the precolumn. HFBA even allowed the detection of a highly hydrophilic peptide that was not retained using TFA. It was shown that for the mixture of labeled model peptides, even a short 3 min washing step already enhanced the removal of the excess reagents significantly, whereas peptide losses were observable starting with a 10 min washing time. A 6 min washing time was determined to be the best parameter for lowering the lanthanide metal background while maintaining maximum peptide recovery. Alternative precleaning setups using EDTA to enhance the removal of free metal or an offline approach using solid phase extraction did not show promising results. The application of the optimized method to labeled peptides in a lysozyme digest showed results comparable to those obtained with model peptides.
低背景信号是生物分析中准确定量的不可或缺的前提条件。当使用衍生化样品时，这带来了特殊的挑战，因为过量的试剂浓度会增加背景信号。预清洁步骤通常很耗时，并且通常会导致分析物损失。在这项研究中，使用电感耦合等离子体质谱（ICP-MS），与纳米离子对反相高效液相色谱（nano-IP-RP-HPLC）联用，分析了一组标记的模型肽和蛋白质消化物。此外，基质辅助激光解吸电离质谱（MALDI-MS）用于肽鉴定。使用基于双功能 DOTA 的（1,4,7,10-四氮杂环十二烷-1,4,7,10-四乙酸）试剂将肽标记为镧系金属。在 nano-HPLC 过程中，通过将标记的肽在线捕获到 C18 预柱上，并在洗脱至分析柱之前对其进行洗涤，从而在线去除多余的金属。在研究中使用了不同的离子对试剂，如 TFA（三氟乙酸）和 HFBA（七氟丁酸），以增强不同肽物种与预柱 C18 材料的相互作用。HFBA 甚至允许检测到使用 TFA 未保留的高度亲水肽。结果表明，对于标记的模型肽混合物，即使是 3 分钟的短洗涤步骤也能显著增强多余试剂的去除效果，而从 10 分钟的洗涤时间开始则会观察到肽损失。确定 6 分钟的洗涤时间是降低镧系金属背景的最佳参数，同时保持最大的肽回收率。使用 EDTA 增强游离金属去除的替代预清洁设置或使用固相萃取的离线方法并未显示出有希望的结果。优化方法在溶菌酶消化物中标记肽的应用结果与模型肽的结果相当。
