Red blood cell N-glycan profiling by MALDI-MS

Protein glycosylation is one of the most common posttranslational modifications in mammalian cells, estimated to be found on over 50% of all human proteins. Glycosylation is found on cell surfaces and in extracellular matrices creating the initial point of contact in cellular interactions. Protein glycans play important roles in biological function/activity, protein folding and molecular recognition. Changes in N-linked glycosylation have long been associated with disease development, and acquired glycan modifications have been described in multifactor diseases such as cancer and inflammatory disorder (Barone et al., 2009). Red blood cell (RBC) glycosylated membrane proteins are easily isolated from blood, purified and readily available. Therefore, glycosylation analysis of erythrocyte membrane glycoproteins might be the key for detection of glycobiomarker to be used for diagnostic and therapeutic purposes. The characterization of glycosylation is a challenge due to the heterogeneity of glycoforms. The main methods of glycosylation analysis involve the separation of released glycan by chromatographic techniques, and detection/identification by mass spectrometry (MS). MS-based methods are capable of structural characterization of unknown glycans and high- throughput analysis of known glycan structures. Due to the advancement in MS techniques, matrix-assisted lasers desorption ionization (MALDI) time-of-fly (TOF) mass spectrometry has become an eligible technique for the study of glycan populations derived from biological samples (Barone et al., 2009; North et al., 2012). There are few studies that investigated N-glycosylation of erythrocyte membrane glycoproteins using MS. In order to elucidated the glycosylation defects in congenital dyserythropoietic anemia type II (CDA II), also called hereditary erythroblastic multinuclearity with positive acidified-serum test (HEMPAS), Fukunda et al. (1987) developed a method based on fast-atom bombardment (FAB) mass spectrometry. HEMPAS patients show morphological changes in red cell membrane and modifications of band 3 and brand 4.5 membrane glycoproteins. Whereas in normal human erythrocyte, band 3 and 4.5 contain N-linked complex glycans with long side chain of lactosamine repeating units, in HEMPAS the same bands present species at lower molecular weight and suggest an incomplete glycosylation. Recently, Denecke et al. (2008) compared erythrocyte band 3 mass mapping from HEMPAS with that from control by MALDI-TOF MS following SDS-PAGE and lectin-binding strategies. Unprocessed oligosaccharides (high mannose, hybrid, and truncated complex species) were found in the HEMPAS patient. Structural data of erythrocyte N-glycans implicate that HEMPAS is not a distinct glycosylation disorder but caused by a defect disturbing Golgi processing in erythroblasts. In the present study, we reported the application of high-sensitivity mass spectrometric-based glycomic methodologies to the analysis of N-linked glycans derived from human erythrocyte membrane proteins. Erythrocytes were isolated from whole blood sample by centrifugation. Subsequently, cells were lysed by the addition of hypotonic buffer, and extraction of membrane glycoproteins was performed using a denaturant agent. N-linked glycans release was then achieved by treatment with peptide N-glycosidase F (PNGase F). Once purified, the pool of N-glycans was derivatized by means of chemical permethylation in order to enhanced the sensitivity of mass spectrometric detection. MALDI-TOF-MS profiling was utilized in order to produce a fingerprint of erythrocyte derivatized N-glycans. The spectrum contains a full complement of high-mannose type structures and a series of complex type glycans comprising a mixture of bi- tri- and tetra-antennary str