Analysis of N-terminal sequencing results of N-terminal site-specific mono-PEGylated recombinant human granulocyte colony-stimulating factor
10.13200/j.cnki.cjb.004538
- VernacularTitle:N端定点单修饰聚乙二醇化人粒细胞刺激因子N端测序结果分析
- Author:
SHI Xinchang
- Publication Type:Journal Article
- Keywords:
N-terminal site-specific mono-modification;
PEGylation;
Recombinant human granulocyte colony-stimulating factor(rh G-CSF);
N-terminal sequencing;
Modified site;
N-terminal blocking
- From:
Chinese Journal of Biologicals
2025;38(08):969-975
- CountryChina
- Language:Chinese
-
Abstract:
Objective To investigate the effect of occupation of modified site of N-terminal site-specific mono-PEGylated recombinant human granulocyte colony-stimulating factor(rh G-CSF)(PEG-G for short) on N-terminal amino acid sequencing based on Edman degradation method.MethodsAccording to the standard sequencing procedure of the N-terminal amino acid sequencer, the N-terminal amino acid sequence of the reference substance for N-terminal site-specific mono-PEGylation of human granulocyte stimulating factor was detected for four cycles, with a sample loading amount of 500 fmol. The waste liquid and mobile phase from the N-terminal amino acid sequencer were collected and evaporated to dryness under low pressure. After dissolving in 2 m L of water, PEG was detected by reversed-phase high performance liquid chromatography(RPHPLC) combined with an evaporative light scattering detector(ELSD).[conditions: mobile phase A, 0. 1% trifluoroacetic acid(TFA)-aqueous solution; mobile phase B, 0. 1% TFA-acetonitrile solution; chromatographic column: C4 column; flow rate: 1. 0 m L/min; injection volume: 100 μL; column temperature: 25 ℃; elution gradient: 0 min→20 min with mobile phase B of 5%→95%, 20 min→30 min with mobile phase B of 95%, 30 min→40 min with mobile phase B of 5%. ELSD conditions: default parameters for carrier gas; temperature 90 ℃; gain 100]. After confirming the PEG retention time, the mobile phase was collected according to the retention time and mass spectrometry analysis was performed.(mass spectrometry collection mode: cation mode; m/z: 3 800-6 000; deconvolution mass tolerance: 20 ppm). N-terminal amino acid sequencing analysis was performed on PEG-G from other companies.ResultsIn the cleaning waste liquid of the N-terminal sequencer, an evaporative light detection chromatographic peak with a retention time close to HPLC was detected. After mass spectrometry analysis, the peak exhibited typical PEG mass spectrometry characteristics, proving that the N-terminal cleaning program(i.e. the first cycle) could cleave the first peptide bond at the N-terminus of some PEG-G, causing the modified PEG to detach. In the sequencing of similar products from other enterprises, it was found that the cracking efficiency was related to the sulfur(S) element near the modified site.ConclusionThe cleaning and sequencing program of N-terminal sequencer can break the N-terminal site-specific PEGylated molecules, which provides technical supports for the establishment of the detection method for N-terminal modified sites in N-terminal site-specific single-modified PEG-G.
