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Abstract No.: MoP-222
Session: Proteomics: General
Presentation date: Mon, Aug 28, 2006
Presentation time: 14:30 – 16:00

The Effect of Minimum Labeling with Fluorescent Dyes on the Quality and Reliability of Mass Spectrometry Based Protein Identification in 2D-Gel Electrophoretic Proteome Analysis

Thomas Hrebicek1, Karin Duerrschmid2, Norbert Auer2, Karl Bayer2, Andreas Rizzi1

1 Institute of Analytical Chemistry, University of Vienna, Vienna, Austria
2 Dep. of Biotechnology, University of Natural Resources and Applied Life Science, Vienna, Austria

Correspondence address: Thomas Hrebicek, Institute of Analytical Chemistry, University of Vienna, Waehringer Strasse 38, Vienna, 1090 Austria.

Keywords: Dyes; Electrophoresis, 2D; MALDI; Proteomic.

Novel aspect: Monitoring of differential electrophoretic migration of labeled and unlabeled proteins in DIGE, the consequence of this effect on the reliability of MS-based protein identification as well as a mathematical approach for prediction of migration.

 

Introduction
Quantification of differential protein expression in the context of proteome research is frequently carried out by 2D gel electrophoresis and fluorescent staining. When using the ETTAN™ DIGE system, the sample is covalently labeled prior to separation in a sub-stoichiometric amount. Therefore, every protein exists in a labeled and an unlabeled form. This paper shows the extent of the different electrophoretic migration of these two species and the consequence of this effect on the reliability of mass spectrometry based protein identification.

Methods
All experiments were performed with E.coli, expressing a heterologous model protein, i.e., recombinant superoxide dismutase. After cell lysis the proteins were covalently labeled with CyDyes. After 2D-GE the minimum-labeled protein species were visualized by a laser scanner. For visualizing the non-derivatized major part of the protein amount, an additional post staining with Deep Purple was performed. An automated spot picker excised the spots from the gels according to the coordinates of the different staining procedures. Various spots covering the whole mass range were chosen. The tryptic peptides of the excised proteins were analyzed by MALDI-TOF and MALDI-QIT-rTOF and the proteins identified by peptide-mass-fingerprinting (PMF) and sequence-tag-determination, respectively.

Results
Derivatization by fluorescent dyes causes a significant increase in the molecular mass. Due to minimal labeling only 1-2% of the proteins were involved in the reaction and, thus, two different species per protein were present. The coordinates of these species could be distinguished easily. As expected, the different migration was more distinct in the lower molecular mass range but also visible at higher masses. Automated spot picking according to the coordinates of the covalently attached label often did not include the major part of the protein. A second, post-staining step was required in order to increase the amino acid coverage and the reliability of the PMF based protein identification. A mathematical approach was tested which corrects for the label-induced mass shift and allows to localize the center of protein accumulation without requiring a post-staining procedure.

Conclusions
The covalently bound label of the DIGE system causes a different electrophoretic migration in comparison to the unlabeled one in the low as well as high mass range. This effect compromises the quality of the mass spectra and the reliability of protein identification. A mathematical approach has been developed to correct for this effect.