Study of posttranslational modifications in lenticular alphaA-Crystallin of mice using proteomic analysis techniques.
In the present work the complexity in the 2D-gel protein pattern of murin lenticular alphaA-Crystallin was analyzed. An in depth study of the different protein isoforms was done combining different proteomic tools. Lens proteins of four different ages, from embryo to 100-week-old mice, were separated by large 2D-PAGE, revealing an increase in the number and intensity of the spots of alphaA-Crystallin during the process of aging. For further analyses the oldest mice were chosen. Comparison and evaluation of two different staining methods proved Imidazole-Zinc to be a good alternative to the generally used Coomassie stain. The characterization of the different alphaA-Crystallin protein species was done using nanoLC-ESI-MS/MS (liquid chromatography electrospray ionisation tandem mass spectrometry). Data interpretation was done by database searching, manual validation and a new MS/MS-interpretation tool for posttranslational modifications--the PTM-Explorer. Using this way, eight different phosphorylation sites were identified and localized; the identification of four of them was not published so far. Furthermore, quantitative N-terminal acetylation of alphaA-Crystallin and variable C-terminal truncation was observed, also not published in this extent yet. The results of the mass spectrometric analysis were validated by immunoblotting experiments using two different alphaA-Crystallin specific antibodies. In addition, a fluorescent phospho-specific stain was used to detect the protein spots including phosphorylation groups. Re-separation 2D-PAGE was done to round off the present study and explain the appearance of some of the protein spots in the gel as artifacts of the 2D-PAGE separation.
Schaefer
Heike
H
Medizinisches Proteom-Center, Ruhr-Universitaet Bochum, Universitaetsstr.150, 44801 Bochum, Germany.
Chamrad
Daniel C
DC
Herrmann
Marion
M
Stuwe
Janine
J
Becker
Gabriele
G
Klose
Joachim
J
Blueggel
Martin
M
Meyer
Helmut E
HE
Marcus
Katrin
K
D000375:Aging; D000595:Amino Acid Sequence; D000818:Animals; D002853:Chromatography, Liquid; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D007093:Imidazoles; D015151:Immunoblotting; D007908:Lens, Crystalline / Q000196:embryology / Q000254:growth & development / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000032:analysis; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D040901:Proteomics / Q000379:methods*; D012394:Rosaniline Dyes; D021241:Spectrometry, Mass, Electrospray Ionization; D013194:Staining and Labeling; D053719:Tandem Mass Spectrometry; D015032:Zinc; D038202:alpha-Crystallin A Chain / Q000378:metabolism*
D007093:Imidazoles; D010750:Phosphoproteins; D012394:Rosaniline Dyes; D038202:alpha-Crystallin A Chain; C048139:Coomassie blue; C029899:imidazole; D015032:Zinc