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Two dimensional blue native/SDS-PAGE to identify mitochondrial complex I subunits modified by 4-hydroxynonenal (HNE).

Wu J, Luo X, Yan LJ - Front Physiol (2015)

Bottom Line: Mitochondrial complex I (NADH-ubiquinone oxidoreductase), containing at least 45 subunits in mammalian cells, sits in a lipid-rich environment and is thus very susceptible to HNE modifications.HNE-positive bands were then excised and the proteins contained in them were identified by mass spectrometric peptide sequencing.The method was successfully applied for the identification of two complex I subunits that showed enhanced HNE-modifications in diabetic kidney mitochondria.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutical Sciences, UNT System College of Pharmacy, University of North Texas Health Science Center Fort Worth, TX, USA.

ABSTRACT
The lipid peroxidation product 4-hydroxynonenal (HNE) can form protein-linked HNE adducts, thereby impacting protein structure and function. Mitochondrial complex I (NADH-ubiquinone oxidoreductase), containing at least 45 subunits in mammalian cells, sits in a lipid-rich environment and is thus very susceptible to HNE modifications. In this paper, a procedure for the identification of HNE-modified complex I subunits is described. Complex I was isolated by first dimensional non-gradient blue native polyacrylamide gel electrophoresis (BN-PAGE). The isolated complex I band, visualized by either Coomassie blue staining or silver staining, was further analyzed by second dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). HNE-modified proteins were visualized by Western blotting probed with anti-HNE antibodies. HNE-positive bands were then excised and the proteins contained in them were identified by mass spectrometric peptide sequencing. The method was successfully applied for the identification of two complex I subunits that showed enhanced HNE-modifications in diabetic kidney mitochondria.

No MeSH data available.


Mass spectrometric peptide sequencing of band 1 and band 2. Shown are (A) 21 peptides that matched to NDUFS1, (B) 17 peptides that matched NDUFS2, and (C) the identity of each protein indicated in Figure 5A.
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Figure 6: Mass spectrometric peptide sequencing of band 1 and band 2. Shown are (A) 21 peptides that matched to NDUFS1, (B) 17 peptides that matched NDUFS2, and (C) the identity of each protein indicated in Figure 5A.

Mentions: To investigate which complex I subunits could undergo HNE modifications, we then switched to kidney mitochondria isolated from STZ diabetic rats. This switch was due to our observation that there were no detectable changes in cardiac complex I activity in STZ diabetic rats 4 weeks post STZ injection. The idea was to explore which subunits underwent enhanced HNE modifications under diabetic conditions as it is possible that kidney mitochondrial proteins may exhibit enhanced HNE modifications in diabetes (Sivitz and Yorek, 2010). Results are shown in Figures 4, 5. Figure 4A shows a first dimensional in-gel complex I activity staining between control and diabetes. Figure 4B shows densitometric quantification of complex I activity in each group whereby complex I activity in STZ diabetes was higher than that in the control group. Figure 5A shows anti-HNE immunostaining of complex I subunits, in which two prominent bands could be visualized to show a basal level of HNE modification that was enhanced by STZ-induced diabetes. HNE content in each band was also higher in STZ diabetes group than in control group (Figures 5B,C). These two bands were excised and subjected to mass spectrometric peptide sequencing. As shown in Figure 6, there were 21 peptides in band 1 (Figure 6A) that matched to the NADH-ubiquinone oxidoreductase 75 kDa subunit (NDUFS1) and 17 peptides in band 2 (Figure 6B) that matched to the NADH dehydrogenase iron–sulfur protein 2 (NDUFS2). Hence, the two proteins identified were NDUFS1 (75 kDa) and NDUFS2 (53 kDa), respectively (Figure 6C). Both have been reported to undergo HNE modifications under different experimental conditions (Choksi et al., 2008; Zhao et al., 2014). It should be noted that the reason that these two proteins could be positively identified is likely due to the fact that they are the most abundant subunits in complex I, which is the drawback of all gel-based proteomic approaches for identification of posttranslationally modified proteins.


Two dimensional blue native/SDS-PAGE to identify mitochondrial complex I subunits modified by 4-hydroxynonenal (HNE).

Wu J, Luo X, Yan LJ - Front Physiol (2015)

Mass spectrometric peptide sequencing of band 1 and band 2. Shown are (A) 21 peptides that matched to NDUFS1, (B) 17 peptides that matched NDUFS2, and (C) the identity of each protein indicated in Figure 5A.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4374453&req=5

Figure 6: Mass spectrometric peptide sequencing of band 1 and band 2. Shown are (A) 21 peptides that matched to NDUFS1, (B) 17 peptides that matched NDUFS2, and (C) the identity of each protein indicated in Figure 5A.
Mentions: To investigate which complex I subunits could undergo HNE modifications, we then switched to kidney mitochondria isolated from STZ diabetic rats. This switch was due to our observation that there were no detectable changes in cardiac complex I activity in STZ diabetic rats 4 weeks post STZ injection. The idea was to explore which subunits underwent enhanced HNE modifications under diabetic conditions as it is possible that kidney mitochondrial proteins may exhibit enhanced HNE modifications in diabetes (Sivitz and Yorek, 2010). Results are shown in Figures 4, 5. Figure 4A shows a first dimensional in-gel complex I activity staining between control and diabetes. Figure 4B shows densitometric quantification of complex I activity in each group whereby complex I activity in STZ diabetes was higher than that in the control group. Figure 5A shows anti-HNE immunostaining of complex I subunits, in which two prominent bands could be visualized to show a basal level of HNE modification that was enhanced by STZ-induced diabetes. HNE content in each band was also higher in STZ diabetes group than in control group (Figures 5B,C). These two bands were excised and subjected to mass spectrometric peptide sequencing. As shown in Figure 6, there were 21 peptides in band 1 (Figure 6A) that matched to the NADH-ubiquinone oxidoreductase 75 kDa subunit (NDUFS1) and 17 peptides in band 2 (Figure 6B) that matched to the NADH dehydrogenase iron–sulfur protein 2 (NDUFS2). Hence, the two proteins identified were NDUFS1 (75 kDa) and NDUFS2 (53 kDa), respectively (Figure 6C). Both have been reported to undergo HNE modifications under different experimental conditions (Choksi et al., 2008; Zhao et al., 2014). It should be noted that the reason that these two proteins could be positively identified is likely due to the fact that they are the most abundant subunits in complex I, which is the drawback of all gel-based proteomic approaches for identification of posttranslationally modified proteins.

Bottom Line: Mitochondrial complex I (NADH-ubiquinone oxidoreductase), containing at least 45 subunits in mammalian cells, sits in a lipid-rich environment and is thus very susceptible to HNE modifications.HNE-positive bands were then excised and the proteins contained in them were identified by mass spectrometric peptide sequencing.The method was successfully applied for the identification of two complex I subunits that showed enhanced HNE-modifications in diabetic kidney mitochondria.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutical Sciences, UNT System College of Pharmacy, University of North Texas Health Science Center Fort Worth, TX, USA.

ABSTRACT
The lipid peroxidation product 4-hydroxynonenal (HNE) can form protein-linked HNE adducts, thereby impacting protein structure and function. Mitochondrial complex I (NADH-ubiquinone oxidoreductase), containing at least 45 subunits in mammalian cells, sits in a lipid-rich environment and is thus very susceptible to HNE modifications. In this paper, a procedure for the identification of HNE-modified complex I subunits is described. Complex I was isolated by first dimensional non-gradient blue native polyacrylamide gel electrophoresis (BN-PAGE). The isolated complex I band, visualized by either Coomassie blue staining or silver staining, was further analyzed by second dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). HNE-modified proteins were visualized by Western blotting probed with anti-HNE antibodies. HNE-positive bands were then excised and the proteins contained in them were identified by mass spectrometric peptide sequencing. The method was successfully applied for the identification of two complex I subunits that showed enhanced HNE-modifications in diabetic kidney mitochondria.

No MeSH data available.