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ISSN: 2329-6798
Modern Chemistry & Applications
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Trends in Characterization of PEGylated Proteins by Mass Spectrometry

Daniela Hutanu and Costel C Darie*
Biochemistry & Proteomics Group, Department of Chemistry & Biomolecular Science, Clarkson University, USA
Corresponding Author : Costel C Darie
Biochemistry & Proteomics Group
Department of Chemistry & Biomolecular Science
Clarkson University, 8 Clarkson Avenue Potsdam
NY, 13699-5810, USA
Tel: 315-268-7763
Fax: 315-268-6610
E-mail: [email protected]
Received May 29, 2013; Accepted June 28, 2014; Published July 03, 2014
Citation: Hutanu D, Darie CC (2014) Trends in Characterization of PEGylated Proteins by Mass Spectrometry. Mod Chem appl 2:128. doi: 10.4172/2329-6798.1000128
Copyright: © 2014 Hutanu D, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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Characterization of PEGylated proteins in crucial in the biotechnology industry for quality control and formulation purposes. This mini review lists several Mass Spectrometry (MS) methods employed in the past 20 years for PEGylated protein analysis. The trend seems to shift from predominantly qualitative MALDI, to liquid chromatography coupled with MS for quantitative or conformational studies.

Mass spectrometry; Proteomics; Proteins; PEGylated proteins
PEGylation is a particular crucial step in the formulation of biotherapeutic protein drugs, as the attachment of a Polyethylene Glycol (PEG) leads to increased circulating life of the drug and the dosage interval, improved pharmacokinetic profile, while at the same time it improves the drug solubility and stability to proteolitic enzymes. Attachment of PEGs to proteins occurs on the N-terminus site, carbohydrate, sulfhydryl, and on aminoacids Lys, His, Arg, Tyr, Ser, Thr, Cys, Asp, Glu. The heterogeneity of the PEGylation product, the degree of PEGylation, coupled with the complex protein structure, make the characterization of PEGylated proteins analytically challenging.
MS [1-5] is a robust and accurate analytical tool used widely in characterization of proteins [5-10], protein post-translational modifications [11-15] and protein protein interactions [16,17]. MS is also used in characterization of biotherapeutics (and PEGylated proteins; see below). For decades, MALDI-TOF MS has been employed as the technique of choice for characterization PEGylated proteins in terms of accurate average molecular weight and degree of PEGylation. Table 1 lists several PEGylated proteins reported in publications the past 20 years as analyzed by MALDI. Different types of PEG derivatives were attached to the proteins, ranging in size in ~ 5-50 kDa, mono and heterofunctional, linear of branched, monodisperse or polydisperse. PEGylation derivatization agents include a 12 kDa monomethoxyPEG [18], 20 kDa methoxy-PEG propionaldehyde [19], 20 kDa (mPEG) (2)-Lys-NHS [20], or a monodisperse Boc-PEG-NH2 [21] , 20 kDa and 40 kDa two-branched PEGs , or trimer-structured PEGs with MW of 23.5, 43.5 and 47 kDa [22]. Regardless of the type of PEG used for polymer modification of the protein, MALDI provided excellent information on molecular weight ID, and heterogeneity, specifically on the total amount and distribution of PEG on protein, or site specific information on PEGylation coupling site.
Electrospray ionization MS (ESI-MS) has started to gain popularity too in the past decade for analysis of PEGylated proteins. ESI-MS is preferred to MALDI due to automated workflow and reduced sample preparation time. The overlapping protein charge pattern and the polydispersity of the PEgylation derivatives complicate the ESI-MS spectrum, so various techniques have been employed by research groups to reduce these drawbacks. Table 2 lists different liquid chromatography MS methods employed for analysis of several PEGylated proteins, the majority of the work reported in the literature the past seven years.
In several instances, qualitative MS methods were easily implemented for quantitative analysis of PEGylated compounds using analytical standards. Surrogate peptides from tryptic dygests were employed by Wu et al. [23] in protein quantification, while isotopically labeled internal standards of de-PEGylated proteins allowed precise and accurate quantification of PEGylated proteins [24,25]. Quantitative analysis of biotherapeutic PEGylated proteins is especially useful in the determination of freely circulating drug in biological fluids. In addition to quantitative and qualitative information on the protein linear structure, MS approaches exist for probing the secondary conformation and the protein dynamics, hydrogen/deuterium exchange MS is a valuable orthogonal MS tool for more in depth exploration of the PEGylated protein structure. Hydrogen/deuterium exchange has been used successfully by the Wei et al. [19] to examine changes in the protein conformation and dynamics induced by the PEGylation process in the structure of the granulocyte colony stimulating factor.
In conclusion, different orthogonal MS approaches are needed to fully characterize PEGylated proteins since PEGylation introduces challenges to the routine MS analysis of a protein, such as increased charging, heterogeneity, stability, and conformational changes.
This work was supported in part by the David A. Walsh fellowship, the U.S. Army research office (DURIP grant #W911NF-11-1-0304) and the generosity of SciFund Challenge 3 Donors.

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