Fig. 1: Comparative study of the random PEGylation and characterisation of BSA using defined and disperse PEG 5 kDa. | Nature Communications

Fig. 1: Comparative study of the random PEGylation and characterisation of BSA using defined and disperse PEG 5 kDa.

From: Exactly defined molecular weight poly(ethylene glycol) allows for facile identification of PEGylation sites on proteins

Fig. 1

a Conventional PEGylation: uses disperse PEGylation agent. Random PEGylation of the primary amine groups of BSA, using disperse 5 kDa linear methoxy PEG-succinimidyl propionate (mPEG-SP) (i). Size-exclusion chromatography (SEC) to isolate the mono-PEG BSA isomers (ii). Digestion: enzymatic digestion of the mono-PEG BSA results in a mixture of PEGylated and un-PEGylated peptide fragments (iii). The different peptide/ PEG-peptide species are then separated/ analysed via UPLC-MS (iv). In case of the disperse PEG the resulting data cannot be interpreted. b In this work a defined, 5 kDa PEGylation agent is used to randomly PEGylate BSA. After steps (i–iii), in-line ESI-MS allows for extraction of the masses for individual PEG-peptide fragments. The masses of the PEGylated peptide fragments can be calculated and (equivalent to a standard peptide mapping procedure) these can then be compared to an online digestion database to identify the PEGylated fragments.

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