Tskgel g2000swxl

The YCH was structurally characterized using circular dichroism (CD) spectroscopy using a MOS-500 CD spectrometer (Bio-Logic Science Instruments). The YCH solution (0.1 mg/mL) was prepared in deionized water. Parameters were as follows: wavelength range, 190-250 nm; scan speed, 100 nm/min; path length, 1 mm. Ellipticity (θ) was measured in millidegrees (mdeg).
The conformational change of the YCH was assessed by ANS fluorescence spectroscopy. The YCH was dissolved in phosphate buffer (pH 7.0, 10 mM) at a concentration of 1 μg/mL. Then, 20 μL of 8 mM ANS was mixed with 1.5 mL of the YCH solution, and the fluorescence spectrum was recorded using an F-2500 fluorescence spectrometer (Hitachi Ltd.) in a wavelength range from 400 to 700 nm.
The molecular weight distribution of the YCH was further analyzed, as described previously (Sun et al., 2021) (link). In brief, the mobile phase was composed of acetonitrile, water, and trifluoroacetic acid (45/55/0.1, vol/vol/vol). The YCH was dissolved in the mobile phase and filtered through a 0.45-μm filter. Then, the YCH solution was loaded on a TSK gel G2000 SWXL (300 × 7.8 mm internal diameter) column (Tosoh) for separation at a flow rate of 0.5 mL/min under a detection wavelength of 220 nm. The standards of cytochrome C, aprotinin, bacitracin, Gly-Gly-Tyr-Arg, and Gly-Gly-Gly were used to prepare a calibration curve of molecular weight.

Semi-confluent retinal pericytes were washed twice with pre-warmed Dulbecco's Modified Eagle Medium (DMEM)/Ham's F-12 medium without phenol red. Subsequently, the cells were cultured in the same medium for 24 h. After incubation, the medium was collected and filtered through a 0.22 µm filter. The solution was then acidified to pH 3.0 with trifluoroacetic acid (TFA) and subjected to a C18 solid phase extraction cartridge (Empore 10 mm/6 mL; 3M Company, St. Paul, MN) equilibrated with 2% acetonitrile and 0.1% TFA (buffer A). The cartridge was washed with buffer A, and the fraction of crude peptides was eluted with 80% acetonitrile and 0.1% TFA (buffer B). The eluent was evaporated in a vacuum concentrator and dissolved in 100 µL of 30% acetonitrile and 0.1% TFA (buffer C). The sample was separated by gel filtration column chromatography (TSKgel G2000SWXL; Tosoh, Tokyo, Japan) in buffer C at a flow rate of 1 mL/min. Corresponding peptide fractions (from the 8th to 11th fractions) were collected, combined, and then loaded onto a strong cation exchange spin column (MonoSpin SCX; GL Science, Tokyo, Japan). The peptide sample was washed twice with buffer B, eluted with 4% NH 4 OH in MeOH, and evaporated to dryness. Samples were reconstituted in 50 mM ammonium bicarbonate and analyzed for total peptide concentration using BCA protein assay kit (ThermoFisher Pierce, Rockford, IL).

The MW distributions of PCHs were determined by high-performance gel-ltration chromatography (HPLC) using a TSK gel G2000 SWXL (300 mm × 7.8, TOSOH, Tokyo, Japan) (Eric et al. 2013) (link). HPLC analysis was performed on a Waters e2695 Alliance HPLC system (Water Co., Milford, MA, USA), which was equipped with a 2487 UV detector and Empower workstation. The ow rate was set at 0.5 mL/min with the mobile phase consisting of acetonitrile/water/tri uoroacetic acid (45/55/0.1, v/v/v). Sample (10.0 μL) was injected into the system, and the column was maintained at a temperature of 30 ℃. The following standards: cytochrome C (12,500 Da), aprotinin (6,500 Da), bacitracin (1,450 Da), Gly-Gly-Tyr-Arg (451 Da), and Gly-Gly-Gly (189 Da) were used to build the calibration curve used to evaluate the MW of the sample. UV absorbance was recorded at 200 nm, and data were processed with gel-permeation chromatography (GPC) software (Waters Co., Milford, US).