Černigoj Urh, Gašperšič Jernej, Fichtenbaum Andreas, Lendero Krajnc Nika, Vidič Jana, Mitulović Goran, Štrancar Aleš
BIA Separations d.o.o., Mirce 21, SI-5270 Ajdovščina, Slovenia.
Medical University of Vienna, Clinical Department of Laboratory Medicine, Waehringer Guertel 18-20, A-1090 Wien, Austria.
Anal Chim Acta. 2016 Oct 26;942:146-154. doi: 10.1016/j.aca.2016.08.044. Epub 2016 Sep 19.
Metal oxide affinity chromatography has been one of the approaches for specific enrichment of phosphopeptides from complex samples, based on specific phosphopeptide adsorption forming bidentate chelates between phosphate anions and the surface of a metal oxide, such as TiO, ZrO, FeO, and AlO. Due to convective mass transfer, flow-independent resolution and high dynamic binding capacity, monolith chromatographic supports have become important in studies where high resolution and selectivity are required. Here, we report the first synthesis and characterization of immobilisation of rutile TiO nanoparticles onto organic monolithic chromatographic support (CIM-OH-TiO). We demonstrate the specificity of CIM-OH-TiO column for enrichment of phosphopeptides by studying chromatographic separation of model phosphorylated and nonphosphorylated peptides as well as proving the phosphopeptide enrichment of digested bovine α-casein. The work described here opens the possibility for a faster, more selective enrichment of phosphopeptides from biological samples that will enable future advances in studying protein phosphorylation.
金属氧化物亲和色谱法一直是从复杂样品中特异性富集磷酸化肽段的方法之一，其基于特定磷酸化肽段的吸附，在磷酸根阴离子与金属氧化物（如TiO₂、ZrO₂、Fe₂O₃和Al₂O₃）表面形成双齿螯合物。由于具有对流传质、与流速无关的分离度和高动态结合容量，整体式色谱载体在需要高分辨率和高选择性的研究中变得至关重要。在此，我们报道了将金红石型TiO₂纳米颗粒固定在有机整体式色谱载体（CIM-OH-TiO₂）上的首次合成与表征。通过研究模型磷酸化和非磷酸化肽段的色谱分离以及证明经酶解的牛α-酪蛋白中磷酸化肽段的富集情况，我们展示了CIM-OH-TiO₂柱对磷酸化肽段富集的特异性。本文所述工作为从生物样品中更快、更具选择性地富集磷酸化肽段开辟了可能性，这将推动未来蛋白质磷酸化研究的进展。
