University of Cincinnati/Agilent Technologies Metallomics Center of the Americas, Department of Chemistry, University of Cincinnati, Cincinnati, OH 45221-0172, USA.
J Am Soc Mass Spectrom. 2012 Jun;23(6):1053-61. doi: 10.1007/s13361-012-0366-2. Epub 2012 Mar 27.
A method for the selective detection and quantification of peptide:oligonucleotide heteroconjugates, such as those generated by protein:nucleic acid cross-links, using capillary reversed-phase high performance liquid chromatography (cap-RPHPLC) coupled with inductively coupled plasma mass spectrometry detection (ICPMS) is described. The selective detection of phosphorus as (31)P(+), the only natural isotope, in peptide-oligonucleotide heteroconjugates is enabled by the elemental detection capabilities of the ICPMS. Mobile phase conditions that allow separation of heteroconjugates while maintaining ICPMS compatibility were investigated. We found that trifluoroacetic acid (TFA) mobile phases, used in conventional peptide separations, and hexafluoroisopropanol/triethylamine (HFIP/TEA) mobile phases, used in conventional oligonucleotide separations, both are compatible with ICPMS and enable heteroconjugate separation. The TFA-based separations yielded limits of detection (LOD) of ~40 ppb phosphorus, which is nearly seven times lower than the LOD for HFIP/TEA-based separations. Using the TFA mobile phase, 1-2 pmol of a model heteroconjugate were routinely separated and detected by this optimized capLC-ICPMS method.
一种使用毛细管反相高效液相色谱（cap-RPHPLC）与电感耦合等离子体质谱检测（ICPMS）相结合，选择性检测和定量肽-寡核苷酸杂合体的方法，如由蛋白质-核酸交联产生的杂合体。通过 ICPMS 的元素检测能力，可以选择性地检测磷作为（31）P（+），这是唯一的天然同位素。考察了允许在保持 ICPMS 兼容性的同时分离杂合体的流动相条件。我们发现，三氟乙酸（TFA）流动相，用于常规肽分离，和六氟异丙醇/三乙胺（HFIP/TEA）流动相，用于常规寡核苷酸分离，都与 ICPMS 兼容，并能实现杂合体分离。基于 TFA 的分离方法的检测限（LOD）约为 40 ppb 磷，比基于 HFIP/TEA 的分离方法的 LOD 低近七倍。使用 TFA 流动相，通过这种优化的 capLC-ICPMS 方法，可以常规地分离和检测 1-2 pmol 的模型杂合体。
