Efficient separation and analysis of peroxisomal membrane proteins using free‐flow isoelectric focusing

Abstract

We have elaborated a protocol for the fractionation of both hydrophilic and hydrophobic proteins using as a model the matrix and membrane compartments of highly purified rat liver peroxisomes because of their distinct proteomes and characteristic composition with a high quota of basic proteins. To keep highly hydrophobic proteins in solution, an urea/thiourea/detergent mixture, as used in traditional gel-based isoelectric focusing (IEF), was added to the electrophoresis buffer. Electrophoresis was conducted in the ProTeam free-flow electrophoresis (FFE) apparatus of TECAN separating proteins into 96 fractions on a pH 3-12 gradient. Consecutive sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis demonstrated that both matrix and the integral membrane proteins of peroxisomes could be successfully fractionated and then identified by mass spectrometry. This is documented by the detection of PMP22, which is the most hydrophobic and basic protein of the peroxisomal membrane with a pI > 10. The identification of 96 prominent spots corresponding to polypeptides with different physical and chemical properties, e.g., the most abundant integral membrane polypeptides of peroxisomes and specific ones of the mitochondrial and microsomal membrane, reflects the fractionation potential of free-flow (FF)-IEF, accentuating its value in proteomic research as an alternative perhaps superior to gel-based IEF.

Biochemistry and genetics of inherited disorders of peroxisomal fatty acid metabolism

“…The fact that mouse peroxisomes contain another thioesterase, ACOT4, which is highly specifi c for succinyl-CoA, suggests that degradation can go even further. In humans, ACOT4 does not, however, display such specifi city and hydrolyzes a variety of other CoA-esters 17 . The shortened dicarboxylic acids are exported and excreted in the urine, with both even and odd numbers of atoms.…”

“…The other hydrolase, mouse NUDT7, cleaves CoA and oxidized CoA into 3 ′ ,5 ′ -ADP and the corresponding 4'-phosphopantetheine. More detailed 17 Human ACOT4 combines the activities of three separate peroxisomal murine esterase encoded by Acot3 , Acot4 , and Acot5 (174). investigations showed that NUDT7 acts also acyl-CoAs, both medium-and long-chain acyl-CoAs, even choloyl-CoA and pristanoyl-CoA ( 191,192 ).…”

“…Hence, compared with the inner mitochondrial membrane, mammalian peroxisomes contain only a limited number of solute transporters. Moreover, proteomics on purifi ed peroxisomal membranes ( 4,17 ) failed to reveal additional transporters. With regard to degradation of bulky cofactors, nudix hydrolases ( 18 ) acting on NAD(P) H and CoA have been shown to be present in peroxisomes.…”

Rat Liver Peroxisomes after Fibrate Treatment

“…Isolation and Subfractionation of Peroxisomes-Homogenization of the tissue and subcellular fractionation was performed according to Völkl and Fahimi (18) with a few modifications described by Weber and co-workers (19). Finally, purified peroxisomes were washed once with gradient buffer (5 mM MOPS, 1 mM EDTA, 0.1% ethanol, 2 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, 1 mM ⑀-aminocaproic acid, pH 7.2) and stored at Ϫ80°C.…”

Modeling the Isoelectric Focusing of Peptides in an OFFGEL Multicompartment Cell

“…3 But because further protein analysis and characterization by mass spectrometry 4 require tedious sample preparation, new IEF schemes and devices have been designed for prefractionation of proteins by IEF: 5,6 several teams have explored the use of free-flow electrophoresis for the fractionation of proteomic samples. [7][8][9][10][11][12] Righetti et al have introduced multicompartment electrolyzers, in which proteins are separated into different compartments separated by Immobiline membranes. [13][14][15] Wall et al have also validated the use of Rotofor for fractionation of proteins prior to RP-HPLC and MALDI-TOF analysis of intact proteins.…”