Proteom analysis of Blue Mussel tissue by two
dimensional gel electrophoresis and MALDI – MS
Heike Helmholz, Eva Rieker, Nicole Asendorf, Daniel Pröfrock, Stephan Lassen, Andreas Prange
Helmholtz-Zentrum Geesthacht, Institute of Coastal Research; Marine Bioanalytical Chemistry, Geesthacht, Germany
Introduction
„Environmental Proteomics“
Contaminants are continually entering the aquatic environment and hence the tissue of resident biota
such as mussels, which are used worldwide as sentinel bioindicator for chemical stress. In order to
detect early changes on the molecular level and to identify prognostic protein biomarker related to
the impact of hazardous substances the methodologies and tools of proteome investigation provide
interesting possibilities for environmental research. Proteomic techniques enable the detection of
changes of protein expression profiles, which allow a much better insiged into contaminat related
effects, simultaneously. In consequence a set of proteins can be identified as early – warning
indicators for environmental stress. To reduce the number of proteins to be analyzed, tissue specific
responses can be detected by dissecting organs involved in different metabolic pathways or different
exposure routes. In this study tissues of wild and caged Blue Mussels Mytilus edulis (L.) have been
analysed by two dimensional gel electrophoresis (2D GE) and by matrix assisted laser
desorption/ionisation – time of flight – mass spectrometry (MALDI-ToF-MS).
MALDI-MS/MS for
protein analysis
Transplanted Mussels
(Poster ET11P 1187)
Experimental
Tissue preparation, protein extraction and purification
Gel electrophoresis and mass spectrometry
Gills and digestive glands have been prepared, weighted and homogenized in 100 mM Tris
buffer pH 8.0 using a TubeDrive® homogenizer. Further cell disruption was achieved by
mixing the primary homogenate with a lysis buffer containing chaotrop salts and membrane
active substances and processing with ultrasound. Extracted proteins were obtained by
chemical precipitation utilising 50% trichloracetic acid and acetone.
Two dimensional gel electrophoresis was performed with self-casted and pre-casted gels of
various dimensions. Isoelectric focussing was performed in broad and narrow pH range
according to the recommended protocols (BioRad, D). Comassie or silver stained gels were
compared with Delta2d software followed by manual spot picking and in-gel tryptic digestion.
Peptide mass fingerprints and MS/MS sequence data have been obtained by MALDI-ToF.
Results and Discussion
Protein extraction and purification
protein (mg/g FW gland)
120
 The combination of several physical and chemical
100
tissue extraction steps is
necessary for a sufficient protein recovery
80
60
 The utilisation of chaotropic salts and surfactants enhances the quantitative yield
40
 Protein content is lower in gill tissue compared to digestive glands
 Due to interfering compounds further purification is necessary for digestive gland
20
0
1st Extraction * 2nd Extraction
(Ultrasound)
homogenates
Ultrasound +
Lysis **
Marker
Protein yield after several tissue extraction
steps (digestive gland n=3)
Gill*
Gill** Gland* Gland**
Process control – protein
extract complexity and yield
2D Gel electrophoresis
pH 10
pH 3
pH 5
pH 8
200 kDa
 TCA protein precipitation of mussel digestive gland tissue
200 kDa
116 kDa
116 kDa
67 kDa
67 kDa
homogenate prior to 2D GE
45 kDa
 A narrow pH range (5-8) is recommended for the isoelectric
focussing in the first dimension
29 kDa
45 kDa
29 kDa
 An AnykDa gradient gel or a 12% (20-100 kDa) separation gel
21kDa
is best suited for the second dimension
14.3 kDa
21kDa
14.3 kDa
6.5 kDa
6.5 kDa
IEF: pH 5-8 11 cm; Comassie staining
IEF: pH 3-10 11cm; Silver staining
Proteom analysis
pH 4
pH 7
~pI / ~MW
Selection of gill and digestive gland tissue prior to proteom
analysis is recommended
Example Spot 1: Tropomyosin
200 kDa
116 kDa
Spot 1
4,7 / 38 kDa
Spot 2
5,5 / 45 kDa
Spot 3
5,6 / 32 kDa
67 kDa
45 kDa
Comparing 2D gels from mussels obtained from differently
charged coastal regions hints for expression variations of
enzymes, cytoskeleton and stress proteins have been
obtained:
Peptide Mass
Fingerprint
 Cysteinyl-tRNA-Synthetase, Nitricoxide-Synthase,
Triosephosphate- Isomerase
2
 Actin, Tropomyosin, Paramyosin
1
 HSP 70
3
29 kDa
2D – gel overlay of gill tissue samples obtained from blue mussels
from differently charged coastal regions
 Further comparative and mass spectrometric analysis for
protein clarification for the identification of potential biomarker
for indicating environmental chemical stress will be performed
Helmholtz-Zentrum Geesthacht • Max-Planck-Straße 1 • 21502 Geesthacht • Phone +49 (0)4152 87-0 • Fax +49 (0)4152 87-1403 • www.hzg.de
Contact: Dr. Heike Helmholz
Verification of database hits
obtained from PMF by
Peptide Fragment Spectra