title: Transglutaminase-mediated PEGylation of proteins: direct identification of the sites of protein modification by mass spectrometry using a novel monodisperse PEG.
abstract: Poly(ethylene glycol) (PEG) has been widely used to prolong the residence time of proteins in blood and to decrease their immunogenicity and antigenicity. A drawback of this polymer lies in its polydispersity that makes difficult the identification of the sites of protein modification. This is a mandatory requirement if a PEGylated protein should be approved as a drug. Here, a fast and reliable method is proposed to characterize proteins conjugated at the level of glutamine (Gln) residues using microbial transglutaminase (TGase). The novelty resides in the use of a monodisperse Boc-PEG-NH(2) for the derivatization that allows the direct identification of the sites of PEGylation by electrospray ionization mass spectrometry (ESI-MS). The procedure has been tested on three model proteins, namely, human granulocyte colony-stimulating factor, human growth hormone, and horse heart apomyoglobin. The Gln residues linked to the polymer chain were easily identified by ESI-MS and tandem MS analyses, demonstrating the advantage of using a monodisperse polymer in combination with mass spectrometry for an easy characterization of conjugated proteins. Interestingly, the PEGylation reaction led to the production only of mono- and bis-derivative products, indicating that the TGase-mediated PEGylation can be extremely selective and thus very useful for the derivatization of protein drugs.
authors.affiliation: CRIBI Biotechnology Centre, University of Padua, Viale G. Colombo 3, 35121 Padua, Italy.
authors.forename: Anna
authors.initials: A
authors.lastname: Mero
authors.forename: Barbara
authors.initials: B
authors.lastname: Spolaore
authors.forename: Francesco M
authors.initials: FM
authors.lastname: Veronese
authors.forename: Angelo
authors.initials: A
authors.lastname: Fontana
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D006801:Humans; D008969:Molecular Sequence Data; D011092:Polyethylene Glycols / Q000737:chemistry* / Q000378:metabolism; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism*; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization; D013302:Streptomyces / Q000201:enzymology*; D053719:Tandem Mass Spectrometry; D011503:Transglutaminases / Q000378:metabolism*
chemical_list: D011506:Proteins; D011092:Polyethylene Glycols; D011503:Transglutaminases