Toward top-down determination of PEGylation site using MALDI in-source decay MS analysis

Abstract

A novel matrix assisted laser desorption/ionization (MALDI)-based mass spectrometric approach has been evaluated to rapidly analyze a custom designed PEGylated peptide that is 31 residues long and conjugated with 20 kDa linear polyethylene glycol (PEG) at the side chain of Lys. MALDI-TOF MS provided sufficiently high resolution to allow observation of each of the oligomers of the heterogeneous PEGylated peptide (m/⌬m of ca. 500), while a typical ESI-MS spectrum of this molecule was extremely complex and unresolved. Reflector in-source decay (reISD) analysis using MALDI-TOF MS was attempted to identify the PEGylation site at intact molecular level without any sample treatment. An reISD spectrum of the free peptide was observed with abundant c-, y-, and [z ϩ 2]-fragment ion series, whereas, in the fragmented PEGylated peptide, the fragment ion series were truncated at the residue where PEG was attached. Therefore, a direct comparison of these top-down reISD spectra suggested the location of the PEGylation site. Results from this study demonstrate a clear analytical utility of the ISD technique to characterize structural aspects of heterogeneous biomolecules. (J Am Soc Mass Spectrom 2009, 20, 326 -333) © 2009 American Society for Mass Spectrometry A significant challenge is associated with development of biomolecules into effective therapeutic use due to their limited chemical and physical stability. In particular, short plasma half-life of biotherapeutics may lead to poor efficacy in humans, and, therefore, developing methods to achieve longer circulation times at low dosage volumes is required. To obtain such improved stability, biomolecules can be chemically modified. One of the most successful approaches currently employed is to covalently attach inert, nontoxic, and nonantigenic polymers, such as polyethylene glycol (PEG), to active molecules, a strategy termed PEGylation [1][2][3][4][5][6]. Due to its favorable properties, PEG is approved by FDA [5] for oral, injection, and dermal administration to humans. PEGylation technology has been successfully developed and applied to achieve prolonged in vivo circulation of biomolecules in the body for effective potency, as demonstrated by a series of marketed PEGylated therapeutics, including Neulasta®, or pegfilgrastim [5,7,8].Typically, the conjugation of PEG of ca. 20 kDa or larger to biotherapeutics has proven efficient by providing desired stability [4,9,10]. However, tremendous analytical challenges are associated with studying such heterogeneous molecules [11], where confident determination of the PEGylation site is one of the important requirements to fully characterize and understand PEGylated molecules. Previously, several groups have reported on the identification of the site at which PEG was conjugated [12], where PEG of relatively small size were commonly used [13][14][15]. Due to a lower degree of polymerization associated with smaller PEG, an analysis was readily performed in these cases. Also, these studies mostly involved enzymatic digestions to sel...

MALDI In-Source Decay, from Sequencing to Imaging

“…Finally, Yoo and collaborators showed recently that MALDI-ISD was also an appropriate tool to localize PEGylation of protein [69], which is a chemical modification widely employed in the pharmaceutical field [70].…”

Advances and challenges in analytical characterization of biotechnology products: Mass spectrometry-based approaches to study properties and behavior of protein therapeutics

“…Identification of conjugation sites can be accomplished by comparing the peptide map of a particular form of the conjugated protein to that of the unmodified protein (Monkarsh et al 1997; Foser et al 2003; Arduini et al 2004). Recently, feasibility of the top-down approach to localization of PEGylation sites has also been demonstrated (Yoo et al 2009; Lu et al 2010). …”

Characterization of a monoPEG20000-Endostar

“…MALDI has been widely accepted as a useful means of studying various types of biomolecules due to its soft ioniza- tion properties [19]. MALDI is most conveniently combined with a time-of-flight (TOF) mass analyzer to resolve large heterogeneous molecules.…”

“…Such high molecular weight results in unresolved or poorly resolved ionic signal [21]. The heterogeneity associated with a large PEG further complicates the spectra [19]. The interpretation of ESI-MS spectra of PEGylated molecule is essentially unachievable.…”