Quantitative measurement of collagen methylation by capillary electrophoresis

Abstract

Collagen methylation has been exploited in various applications involving living cells. We have observed correlation between the collagen methylation with the rate of cell proliferation in three-dimensional (3-D) microenvironment. To quantify the degree of collagen methylation, we have developed a capillary zone electrophoresis method. Using a polyvinyl alcohol-coated fused-silica capillary and UV detection at 200 nm, we have optimized pH and separated the native collagen into three major bands in phosphate buffer (50 mM, pH 2.5) with 0.05% hydroxypropylmethylcellulose. Under these conditions, the methylated collagens were separated into four major bands, which changed with different methylation reaction conditions. We propose an index to quantify the degree of collagen methylation that also correlates with their effects on cell proliferation.

Optimization of 3-D hepatocyte culture by controlling the physical and chemical properties of the extra-cellular matrices

“…The characterization of the degree of methylation involved separations on a CE-L1 System (CE Resources Pte Ltd.) with 0.05% hydroxypropyl methyl cellulose at pH=2.5 and 21 1C, which resolved the methylated collagen into four major peaks. An increase in the degree of methylation was correlated to an increase in the ratio of the areas under the last two peaks over the first two, defined as g [18]. Collagen methylated at 4 1C, 6 days had a lower g of 1.4 and was denoted as slightly methylated collagen, while collagen methylated at 23 1C, 1 day (g ¼ 1:9) was denoted as highly methylated collagen.…”

“…We investigated the effects of varying the degree of collagen methylation on the formation of nano-fibres in the microcapsule. The degree of collagen methylation under different reaction conditions was characterized by a capillary electrophoresis method based on an empirical ratio g [18] (see methods). Collagen methylated under milder conditions of 4 1C, 6 days had a lower g of 1.4 and was denoted as slightly methylated collagen, while collagen methylated at 23 1C, 1 day with g ¼ 1:9 was denoted as highly methylated collagen.…”

“…The degree of methylation was controlled by adjusting the time and temperature of the reaction and characterized by a capillary electrophoresis method [18]. Briefly, the methylation reaction involved stirring the precipitated collagen in acidified methanol for 4 1C, 6 days and 23 1C, 1 day respectively, followed by dialysis to pH 7.4, freeze-drying.…”

Electrostatic self-assembly of multilayer copolymeric membranes on the surface of porous tantalum implants for sustained release of doxorubicin

“…17 The degree of methylation was controlled by adjusting the time and temperature of the reaction. 18 The precipitated collagen type I was dissolved in 0.1 M HCl containing methanol for 6 days at 4°C. At the end of the reaction, the solution was dialyzed against deionized water at 4°C using dialysis tubing with molecular weight cut-off (MWCO) of 8000-15,000 until the pH of the external reservoir reached 6.4, followed by freeze-drying.…”

Capillary electrophoresis of proteins 2003–2005

“…coccus [248], conformational changes in HSA [249], metal-binding proteins [250], ricine and its subunits [251], capsid proteins from human rhinovirus [252,253], antithrombin III from human plasma [254], prostate-specific antigen isoforms [255], human fibrinopeptides [256], ribonuclease H [257], endogenous extracellular signalregulated protein kinase [258], collagen methylation [259], half antibody in IgG(4) [260], and detect bone morphogenic protein-2 in rat vascular smooth muscle cells [261,262].…”