LC-MSMS based identification of Transferrin glycosylation varients for diagnosis of CDG Authors: Aruna Jyothi. M, Sanovar Bhargava, Hima Bindu. A, Subbarayudu. S, Radha Rama Devi and Madhurarekha. Ch Institution: Sandor Lifesciences Pvt. Ltd., 8-2-326/5, Road No- 3, Banjara Hills, Hyderabad - 500034 Abstract No: INTRODUCTION: Congenital disorders of glycosylation (CDG) form a group of metabolic disorders caused by deficient glycosylation of proteins and/or lipids. The quantification of human transferrin (TF) is based on several techniques. We have standardised the protocols for fast purification of TF from human serum and also developed methods for screening of CDG samples using IEF, 2D and MALDI. Liquid chromatography coupled with Electron Spray Ionization - Mass Spectrometry (LC-MSMS) assay is more reliable for TF studies even at low concentrations (Nano level). In this study, a LC-MSMS based proteomics assay was developed for the determination of TF isoform levels in human serum, N-glycan attachment sites, peptide sequencing for diagnosis of CDG. Control & Test-1 b-y ion fragmentation of the Glycosylated peptide Glycosylation pattern CASE HISTORY Transferrin 1 2 3 TF-Test-1 TF-Test-2 TF-Control Direct Serum Marker SDS-PAGE Gel pI 4 5 6 7 8 9 10 202KD 113 KD 81 KD 46 KD 33 KD 26 KD 6 KD Purified Transferrin 2D Images Control Test-1 (Tetrasialo) Test-2 (Asialo) Marker (TF Isoforms) IEF GEL -ve +ve Test-2 Test-1 Normal Sample: Control Serum Test Sample-1: Fawad, 3yrs/M Test Sample-2: Snija, 2yrs/F, was investigated and reported as Type-I CDG by Exome Sequencing Ref Dr: Dr. Radha Rama Devi, Rainbow Hospitals, Hyderabad The incomplete addition of sialic acid to a proportion of serum transferrin molecule is the most commonly used marker for initial diagnosis of CDG. Two diagnostic types of abnormal profiles can be distinguished as CDG Type-I, characterized by an increase of di- and/or asialotransferrin and CDG Type-II, characterized by an increase of tri-, di-, mono-and/or asialotransferrin. Here we present CDG screening in suspected samples by LC-MSMS using ESI-QTOF for identification of transferrin glycosylation varients and diagnoses of CDG. Key Words: CDG; Transferrin; LC-ESI-QTOF; Glycosylation; Bioinformatics Glycosylation pattern b-y ion fragmentation of the Glycosylated peptide Test-2 Chromatogram Carbohydrate Profiling MS Spectrum CONCLUSION METHODOLOGY Purification of Transferrin was carried out from Serum in a single step using Rivanol Purification was confirmed by SDS PAGE, 2D and MALDI The Sequence of the glycosylated peptide that was monitored in the runs was CGLVPVLAENYNK LC-MSMS of CDG suspected samples Test-1 showed a tetrasialo-form indicating a normal sample and Test-2 showed an asialo form indicating CDG-I Carbohydrate profiling was also carried out using LC-MSMS Serum Purification of Transferrin Bio-informatics MassLynx – For acquiring chromatogram and spectra Trypsin Digestion Iso-Electric Focussing 2D –Gel Electrophoresis PNGase Treatment Intact Mass Analysis PLGS (Protein Lynx Global Server) – For identification of protein and glycosylated sites REFERENCES (In-Gel & In- Solution) H Arefanian, et.al., A New Protocol for Isolation and Purification of Transferrin from Human Serum; Iranian J. Publ. Health, Vol. 31, Nos. 1-2, PP. 15-18, 2002 Sakari Joenvaara, et. Al., N-Glycoproteomics – An automated workflow approach; Glycobiology vol. 18 no. 4 pp. 339–349, 2008 Ajit Varki, et. al., Essentials of Glycobiology, NCBI Bookshelf Yu Y, et. al., Quantification of human serum transferrin using liquid chromatography-tandem mass spectrometry based targeted proteomics; J Chromatogr B Analyt Technol Biomed Life Sci, 2012. Dirk J.Lefeber, et.al. (2011) How to find and diagnose a CDG due to defective N-Glycosylation. J Inherit Metab Dis. 34(4): 849–852 Ion-Mobility Separation for detection of isoforms Carbohydrate Profiling NIST Library Search – For structural analysis of carbohydrates Identification using LC-MSMS Characterization of CDG