A mass spectrometry approach for the study of deglycosylated proteins Lancia N.F. Darville a , Mark E. Merchant b , Kermit K. Murray a, ⁎ a Department of Chemistry, Louisiana State University, Baton Rouge, LA 70803, United States b Department of Chemistry, McNeese State University, Lake Charles, LA 70609, United States abstract article info Article history: Received 22 May 2011 Accepted 26 May 2011 Available online 1 June 2011 Keywords: Proteomics Mass spectrometry Deglycosylation Mass spectrometry (MS) analysis, after enzymatic or chemical deglycosylation, requires preparatory steps to remove salts and buffers. In this work, the glycosylated protein fetuin and a lectin protein isolated from the serum of Alligator mississippiensis were used to evaluate methods for desalting samples after an enzymatic or chemical deglycosylation. Precipitation and dialysis were used to prepare the deglycosylated samples for MS analysis. Both the precipitation and dialysis methods were suitable for sample preparation prior to analysis by matrix assisted laser desorption ionization (MALDI) MS. © 2011 Elsevier B.V. All rights reserved. 1. Introduction Proteomics is the study of the structure and functions of proteins in an organism or tissue, and mass spectrometry is now a routinely used method for protein identification in complex mixtures [1]. Prior to a typical mass spectrometry analysis, separation techniques such as two-dimensional gel electrophoresis and high performance liquid chromatography (HPLC) are used to reduce the complexity of protein mixtures [1,2]. Some proteins are post-translationally modified to help regulate gene expression, protein turnover and cellular structure [3]. Glycosyl- ation is a common post-translational modification that is involved in biological functions such as immune recognition and inflammation [4]. There are two major types of protein carbohydrate linkages: N- and O- linked glycosylation. During N-glycosylation, the glycan is attached to an asparginine residue followed by any amino acid other than proline, which is linked to a serine or threonine residue. During O-glycosylation, the glycan is attached to a serine or threonine residue. Glycoproteins have been characterized by mass spectrometry, but this is challenging due to the heterogeneity of the carbohydrate moieties and the complexity of the resulting mass spectra [5]. Glycosylation of the protein may also limit the extent of proteolytic digestion. To avoid this, the N- and/or O-linked carbohydrates can be removed prior to further analysis [6,7]. A direct approach using enzymatic or chemical cleavage of the glycan can be used to enable the analysis of the protein without the oligosaccharides. An enzymatic approach using peptide: N-glycosidase (PNGase F) [8] and a chemical approach using trifluoromethanesulfonic acid (TFMS) have been used to remove the oligosaccharides [9,10]. PNGase F is an enzyme that removes N-linked oligosaccharides, leaving both the protein and oligosaccharides intact for further analysis. To date, there have been no enzymatic techniques reported that cleave O-linked oligosaccharides. TFMS is a non-specific deglycosylating agent that cleaves both N-linked and O-linked oligosaccharides to leave the intact protein [11]. The proteins used in this study were enzymatically or chemically deglycosylated, followed by matrix assisted laser desorption ionization (MALDI) MS. The steps for both chemical and enzymatic deglycosylation approaches are fully established and has been reported previously [9,12,13], but the deglycosylated product requires cleanup prior to MALDI mass spectrometry. We have highlighted important sample desalting procedures that follow deglycosylation prior to mass spectrometry analysis, which are not clearly outlined in many pro- cedures. This study focuses on the treatment of deglycosylated proteins for mass spectrometry analysis. 2. Experimental 2.1. Materials HPLC grade acetonitrile, formic acid, and trifluoroacetic acid (TFA) were obtained from Sigma-Aldrich (St. Louis, MO, USA) and used without further purification. Fetuin, a plasma glycoprotein produced by the liver, was obtained from Sigma-Aldrich and the lectin protein was isolated from the blood plasma of Alligator mississippiensis. Blood from seven American alligators was collected; anticoagulated with heparin, and the blood was allowed to clot overnight at ambient temperature. The serum was collected and the lectin was isolated using mannan-agarose affinity chromatography. Microchemical Journal 99 (2011) 309–311 ⁎ Corresponding author at: Louisiana State University, 337 Choppin Hall, Baton Rouge, LA 70803, United States. Tel.: +1 225 578 3417; fax: +1 225 578 3458. E-mail address: kkmurray@lsu.edu (L.N.F. Darville). 0026-265X/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.microc.2011.05.020 Contents lists available at ScienceDirect Microchemical Journal journal homepage: www.elsevier.com/locate/microc