Mass spectrometrical analysis of the mitochondrial carrier Aralar1 from mouse hippocampus.
Aralar1 is a mitochondrial aspartate/glutamate carrier and a key component of the malate-aspartate NADH shuttle system. An analytical approach to obtain high sequence coverage is important to predict conformation, identify splice variants and binding partners or generate specific antibodies. Moreover, a method allowing determination of Aralar1 from brain samples is a prerequisite for evaluating a biological role. Sucrose gradient ultracentrifugation was applied to enrich native membrane protein fractions and these were run on blue-native PAGE, followed by multidimensional gel electrophoresis. Spots from the third-dimensional gel electrophoresis were in-gel digested with trypsin, chymotrypsin and subtilisin. Subsequently, peptides were analyzed by nano-ESI-LC-MS/MS using collision-induced dissociation and electron transfer dissociation modes. Modiro v1.1 along with Mascot v2.2 software was used for data handling. Aralar1 could be clearly separated, unambiguously identified and characterized from protein extracts of mouse hippocampus by the use of the multidimensional gel electrophoretic steps. The combined sequence coverage of Aralar1 from trypsin, chymotrypsin and subtilisin digestions was 99.85%. The results provide the basis for future studies of Aralar1 at the protein chemical rather than at the immunochemical level in the brain and thus challenge and enable determination of Aralar1 levels required for understanding biological functions in health and disease.
Heo
Seok
S
Department of Pediatrics and Adolescent Medicine, Medical University of Vienna, Vienna, Austria.
Kang
Sung Ung
SU
Oehler
Rudolf
R
Pollak
Arnold
A
Lubec
Gert
G
D000818:Animals; D002462:Cell Membrane / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D006624:Hippocampus / Q000737:chemistry*; D057927:Hydrophobic and Hydrophilic Interactions; D013058:Mass Spectrometry / Q000379:methods*; D026901:Membrane Transport Proteins / Q000032:analysis* / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D033681:Mitochondrial Membrane Transport Proteins; D024101:Mitochondrial Proteins / Q000032:analysis* / Q000378:metabolism; D010446:Peptide Fragments / Q000032:analysis / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D020539:Sequence Analysis, Protein; D014461:Ultracentrifugation
D026901:Membrane Transport Proteins; D033681:Mitochondrial Membrane Transport Proteins; D024101:Mitochondrial Proteins; D010446:Peptide Fragments; C491956:Slc25a12 protein, mouse