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Abstract No.: MoP-038
Session: Food and Nutrition
Presentation date: Mon, Aug 28, 2006
Presentation time: 14:30 – 16:00

Gel Filtration - Electrospray Ionization Mass Spectrometry Analysis of Wheat Gluten

Timo Mauriala1, Jarkko Venalainen1, Pekka Mannisto1, Seppo Auriola1

1 University of Kuopio, Kuopio, Finland

Correspondence address: Timo Mauriala, University of Kuopio, Department of Pharmacautical Chemistry, P.O Box 1627, Kuopio, 70211 Finland.

Keywords: Digests,; Electrospray Ionization (ESI); Peptide; Sample Preparation.

Novel aspect: Identification of wheat gluten peptides by SEC-UV-MS method.

 

Introduction
Celiac Sprue, also known as Celiac Disease, is an autoimmune disease of the small intestine caused by the intake of gluten proteins from common food sources like wheat. The toxic components of wheat gluten belong to proline and glutamin-rich prolamin family called gliadins produced during endoluminal proteolytic digestion. It has been proposed1,2 that 33mer peptide from gliadin is the principal contributor to this protein's immunotoxicity. In this study a size-exclusion chromatography-ultraviolet detection-mass spectrometry (SEC-UV-MS) method was developed to detect gliadin and its digested peptides in wheat extracts. SEC is widely used separation and characterization method for biological macromolecules, especially proteins. However, on-line coupling of SEC and mass spectrometer is not utilized in routine protein and peptide analysis.

Methods
The chromatographic separation was performed by linking TSKgel 4000 and TSKgel 2000 columns (TOSOH Corporation, Montgomeryville, PA, USA) together. Isocratic flow of 40 % acetonitrile with 0.1% TFA was used for separation. The flow (1 ml/min) was splitted (3:1) between UV detector and Finnigan LTQ mass spectrometer (Thermo, San Jose, CA, USA). LTQ was operated in positive full scan mode (m/z 400-2000) and tandem mass spectra (MS/MS) were collected for peptide identification.

Results
The mass spectrometry detection narrows down suitable mobile phases used in SEC. Only volatile buffers can be used and total salt concentration must be kept as low as possible. We found that 40% acetonitrile with TFA worked well in our study. Gliadin and its digested peptides including toxic 33mer were separated in 20 minutes run. Analytes were also separated from late eluting small molecules and salts. The detection with full scan mode was successful and peptides were identified with MS/MS mode.

References
1. T. Marti, O. Molberg, Q. Li, G. Gray, C. Khosla, J. Pharmacol. Exp. Ther. 312, 19 (2005).
2. L. Shan, S. Qiao, H. Arentz-Hansen, O. Molberg, G. Gray, L. Sollid, C. Khosla, J. Proteome Res. 4, 1732 (2005).