1. 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., /5, Road No- 3, Banjara Hills, Hyderabad
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 , 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