Optimized proteomic analysis of a mouse model of cerebellar dysfunction using amine-specific isobaric tags.
Recent proteomic applications have demonstrated their potential for revealing the molecular mechanisms underlying neurodegeneration. The present study quantifies cerebellar protein changes in mice that are deficient in plasma membrane calcium ATPase 2 (PMCA2), an essential neuronal pump that extrudes calcium from cells and is abundantly expressed in Purkinje neurons. PMCA2-null mice display motor dyscoordination and unsteady gait deficits observed in neurological diseases such as multiple sclerosis and ataxia. We optimized an amine-specific isobaric tags (iTRAQ)-based shotgun proteomics workflow for this study. This workflow took consideration of analytical variance as a function of ion signal intensity and employed biological repeats to aid noise reduction. Even with stringent protein identification criteria, we could reliably quantify nearly 1000 proteins, including many neuronal proteins that are important for synaptic function. We identified 21 proteins that were differentially expressed in PMCA2-null mice. These proteins are involved in calcium homeostasis, cell structure and chromosome organization. Our findings shed light on the molecular changes that underlie the neurological deficits observed in PMCA2-null mice. The optimized workflow presented here will be valuable for others who plan to implement the iTRAQ method.
Hu
Jun
J
Center for Advanced Proteomics Research and Department of Biochemistry and Molecular Biology, New Jersey Medical School, University of Medicine and Dentistry of New Jersey, Newark, NJ, USA.
Qian
Jin
J
Borisov
Oleg
O
Pan
Sanqiang
S
Li
Yan
Y
Liu
Tong
T
Deng
Longwen
L
Wannemacher
Kenneth
K
Kurnellas
Michael
M
Patterson
Christa
C
Elkabes
Stella
S
Li
Hong
H
D000588:Amines / Q000737:chemistry*; D000818:Animals; D015153:Blotting, Western; D064026:Calbindins; D000252:Calcium-Transporting ATPases / Q000172:deficiency / Q000235:genetics / Q000378:metabolism; D027682:Cation Transport Proteins / Q000172:deficiency / Q000235:genetics / Q000378:metabolism; D002526:Cerebellar Diseases / Q000235:genetics / Q000378:metabolism*; D002531:Cerebellum / Q000378:metabolism / Q000473:pathology; D002852:Chromatography, Ion Exchange / Q000379:methods; D019295:Computational Biology; D004195:Disease Models, Animal; D015180:Electrophoresis, Gel, Two-Dimensional; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D053499:Plasma Membrane Calcium-Transporting ATPases; D011506:Proteins / Q000032:analysis / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D015203:Reproducibility of Results; D064030:S100 Calcium Binding Protein G / Q000032:analysis / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D000588:Amines; D064026:Calbindins; D027682:Cation Transport Proteins; D011506:Proteins; D064030:S100 Calcium Binding Protein G; D053499:Plasma Membrane Calcium-Transporting ATPases; C506959:Atp2b2 protein, mouse; D000252:Calcium-Transporting ATPases