Combination of Three Functionalized Temperature-Sensitive Chromatographic Materials for Serum Protein Analysis

Abstract

We have developed a methodology to capture acidic proteins, alkaline proteins, and glycoproteins separately in mouse serum using a combination of three functionalized temperature-responsive chromatographic stationary phases. The temperature-responsive polymer poly(N-isopropylacrylamide) was attached to the stationary phase, silica. The three temperature-responsive chromatographic stationary phase materials were prepared by reversible addition–fragmentation chain transfer polymerization. Alkaline, acidic, and boric acid functional groups were introduced to capture acidic proteins, alkaline proteins, and glycoproteins, respectively. The protein enrichment and release properties of the materials were examined using the acidic protein, bovine serum albumin; the alkaline protein, protamine; and the glycoprotein, horseradish peroxidase. Finally, the three materials were used to analyze mouse serum. Without switching the mobile phase, the capture and separation of mouse serum was achieved by the combination of three temperature-responsive chromatographic stationary phase materials. On the whole, 313 proteins were identified successfully. The number of different proteins identified using the new method was 1.46 times greater than the number of proteins that has been identified without applying this method. To our knowledge, this method is the first combinatorial use of three functionalized temperature-responsive chromatographic stationary phase silica materials to separate proteins in mouse serum.

Synthetic Thermo-Responsive Terpolymers as Tunable Scaffolds for Cell Culture Applications

“…Likewise, to promote cell attachment and subsequent detachment, Konishi et al formed annealed nanofibers based on a random crosslinked copolymer of NiPAAm with butyl acrylate (BA) and N-(2-hydroxyethyl) acrylamide (HEAAm) [ 48 ]. On the other hand, the selected monomers have been previously used as binary combinations for different applications, such as the fractional purification of polysaccharides [ 52 ] and the capture of glycoproteins and acidic/alkaline proteins [ 53 ].…”