1. Preparation of mixed-mode stationary phase for separation of peptides and proteins in high performance liquid chromatography
Sarah Alharthi, Ashraf Ali, Muzaffar Iqbal, Aliya Ibrar, Bashir Ahmad, Sobia Nisa, Fazal Mabood Sci Rep. 2022 Mar 8;12(1):4061. doi: 10.1038/s41598-022-08074-7.
Porous silica particles were prepared by sol-gel method with some modification to get wide-pore particles. These particles were derivatized with N-phenylmaleimide-methylvinylisocyanate (PMI) and styrene by reversible addition fragmentation chain transfer (RAFT) polymerization to prepare N-phenylmaleimide embedded polystyrene (PMP) stationary phases. Narrow bore stainless steel column (100 × 1.8 mm i.d) was packed by slurry packing method. The chromatographic performance of PMP column was evaluated for the separation of synthetic peptides mixture composed of five peptides (Gly-Tyr, Gly-Leu-Tyr, Gly-Gly-Tyr-Arg, Tyr-Ile-Gly-Ser-Arg, Leucine enkephalin) and tryptic digest of human serum albumin (HAS) respectively. Number of theoretical plates as high as 280,000 plates/m were obtained for peptides mixture at optimum elution condition. Separation performance of the developed column was compared with commercial Ascentis Express RP-Amide column and it was observed that separation performance of PMP column was better than commercial column in terms of separation efficiency and resolution.
2. Preparation of well-dispersed gold/magnetite nanoparticles embedded on cellulose nanocrystals for efficient immobilization of papain enzyme
Khaled A Mahmoud, Edmond Lam, Sabahudin Hrapovic, John H T Luong ACS Appl Mater Interfaces. 2013 Jun 12;5(11):4978-85. doi: 10.1021/am4007534. Epub 2013 May 24.
A nanocomposite consisting of magnetite nanoparticles (Fe3O4NPs) and Au nanoparticles (AuNPs) embedded on cellulose nanocrystals (CNCs) was used as a magnetic support for the covalent conjugation of papain and facilitated recovery of this immobilized enzyme. Fe3O4NPs (10-20 nm in diameter) and AuNPs (3-7 nm in diameter) were stable and well-dispersed on the CNC surface. Energy-dispersive spectroscopy, X-ray diffraction, and Fourier transform infrared spectroscopy were used to evaluate the surface composition and structure of CNC/Fe3O4NPs/AuNPs. The nanocomposite was successfully used for the immobilization and separation of papain from the reaction mixture. The optimal enzyme loading was 186 mg protein/g CNC/Fe3O4NPs/AuNPs, significantly higher than the value reported in the literature. The activity of immobilized papain was studied by electrochemical detection of its specific binding to the Thc-Fca-Gly-Gly-Tyr-Arg inhibitory sequence bound to an Au electrode. The immobilized enzyme retained 95% of its initial activity after 35 days of storage at 4 °C, compared to 41% for its free form counterpart.
3. A Graphene Oxide-Based Fluorescent Platform for Probing of Phosphatase Activity
Ting Sun, Ning Xia, Lin Liu Nanomaterials (Basel). 2016 Jan 18;6(1):20. doi: 10.3390/nano6010020.
We presented a strategy for fabricating graphene oxide (GO)-based fluorescent biosensors to monitor the change of phosphorylation state and detect phosphatase activity. By regulating the interaction between the negatively charged phosphate group and the positively charged amino residue, we found that GO showed different quenching efficiency toward the phosphorylated and dephosphorylated dye-labeled peptides. To demonstrate the application of our method, alkaline phosphatase (ALP) was tested as a model enzyme with phosphorylated fluorescein isothiocyanate (FITC)-labeled short peptide FITC-Gly-Gly-Gly-Tyr(PO₃2-)-Arg as the probe. When the negatively charged phosphate group in the Tyr residue was removed from the peptide substrate by enzymatic hydrolysis, the resulting FITC-Gly-Gly-Gly-Tyr-Arg was readily adsorbed onto the GO surface through electrostatic interaction. As a result, fluorescence quenching was observed. Furthermore, the method was applied for the screening of phosphatase inhibitors.
