Zhiyuan Yu

Reversed-Phase-Reversed-Phase Liquid Chromatography Approach with High Orthogonality for Multidimensional Separation of Phosphopeptides

Protein phosphorylation regulates a series of important biological processes in eukaryotes. However, the phosphorylation sites found up to now are far below than that actually exists in proteins due to the extreme complexity of the proteome sample. Here a new reversed-phase-reversed-phase liquid chromatography (RP-RPLC) approach was developed for multidimensional separation of phosphopeptides. In this approach, a large number of fractions were collected from the first dimensional RPLC separation at high pH. And then these fractions were pooled every two fractions with equal time interval, one from the early eluted section and another one from the later eluted section. The pooled fractions were finally submitted to RPLC-tandem mass spectrometry (MS/MS) analysis at low pH. It was found the resulting 2D separation was highly orthogonal and yielded more than 30% phosphopeptide identifications over the conventional RP-RPLC approach. This study provides a powerful approach for efficient separation of phosphopeptides and global phosphorylation analysis, where the orthogonality of 2D separation is greatly improved and the first dimensional separation is of high resolution.

Preparation of monodisperse immobilized Ti4+ affinity chromatography microspheres for specific enrichment of phosphopeptides

This study presented an approach to prepare monodisperse immobilized Ti(4+) affinity chromatography (Ti(4+)-IMAC) microspheres for specific enrichment of phosphopeptides in phosphoproteome analysis. Monodisperse polystyrene seed microspheres with a diameter of ca. 4.8mum were first prepared by a dispersion polymerization method. Monodisperse microspheres with a diameter of ca. 13mum were prepared using the seed microspheres by a single-step swelling and polymerization method. Ti(4+) ion was immobilized after chemical modification of the microspheres with phosphonate groups. The specificity of the Ti(4+)-IMAC microspheres to phosphopeptides was demonstrated by selective enrichment of phosphopeptides from mixture of tryptic digests of alpha-casein and bovine serum albumin (BSA) at molar ratio of 1 to 500 by MALDI-TOF MS analysis. The sensitivity of detection for phosphopeptides determined by MALDI-TOF MS was as low as 5fmol for standard tryptic digest of beta-casein. The Ti(4+)-IMAC microspheres were compared with commercial Fe(3+)-IMAC adsorbent and homemade Zr(4+)-IMAC microspheres for enrichment of phosphopeptides. The phosphopeptides and non-phosphopeptides identified by Fe(3+)-IMAC, Zr(4+)-IMAC and Ti(4+)-IMAC methods were 26, 114, 127 and 181, 11, 11 respectively for the same tryptic digest samples. The results indicated that the Ti(4+)-IMAC had the best performance for enrichment of phosphopeptides.

Preparation of polyamine-functionalized copper specific adsorbents for selective adsorption of copper

The level of serum free copper is greatly elevated in Wilsons disease. For patients with acute Wilsons disease, liver transplantation is the only lifesaving treatment. Plasma exchange or albumin dialysis is often used as a bridge to liver transplantation to maintain a stable clinical status for patients. Hemoperfusion is another effective therapy in removing toxins from the plasma. However, hemoperfusion has not been reported to remove copper due to lack of copper specific adsorbent. In this work, copper chelating agents, triethylenetetramine and tetraethylenepentamine, were covalently immobilized onto macroporous poly(glycidyl methacrylate-co-trimethylolpropane trimethacrylate) microspheres to prepare copper specific adsorbents. The resulting adsorbents demonstrated good adsorption capacities of 63.44 and 58.48 mg/g, respectively, for Cu2+ ion. Additionally, with the interference of other metal ions such as Fe2+, Mg2+, Zn2+ and Ca2+, the prepared copper adsorbents still demonstrated good specificity toward Cu2+ ion. These results indicate that the adsorbents are promising adsorbents in hemoperfusion therapy for selective removal of copper for patients with severe Wilsons disease.