Separation of mitochondria by flow field-flow fractionation for proteomic analysis

Abstract

Flow field-flow fractionation (FlFFF) has been utilized for size-based separation of rat liver mitochondria. Collected fractions of mitochondria of various sizes were examined by confocal microscopy, and mitochondria of each fraction were lysed and analyzed by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) for the comparison of protein patterns in differently sized mitochondria by densitometric measurements, and for protein characterization of some gel spots with nanoflow liquid chromatography-electrospray ionization-tandem mass spectrometry (nLC-ESI-MS-MS). FlFFF fractions of the mitochondria were also tryptically digested for shotgun proteomic characterization of mitochondrial proteins/peptides by nLC-ESI-MS-MS. Peak area (integrated ion counts) of some peptides extracted from LC-MS chromatograms were examined at different fractions for the quantitative comparison. Among 130 proteins, 105 unique proteins were found to be mitochodrial from the off-line combination of FlFFF and nLC-ESI-MS-MS analysis. It also showed that 23 proteins were found in all fractions but some proteins were found exclusively in certain fractions. Among 25 proteins listed from other subcellular species, seven proteins were known to exist in mitochondria as well as in other subcellular locations, which may support the possible translocation or multiple localizations of proteins among organelles. This study demonstrated effective use of FlFFF for the isolation and/or enrichment of intact mitochondria isolated from cells, as well as its potential use for the fractionation of other subcellular components in the framework of subcellular functional proteomics.

Asymmetrical flow field-flow fractionation technique for separation and characterization of biopolymers and bioparticles

“…Uranium carbonate and hydroxide passed through the FFF membrane. AsFlFFF was also used to fractionate micron sized bioparticles and utilised as pre-analytical technique for the characterization of size-dependent proteome patterns [31,33,52]. Additional studies on the applicability of AsFlFFF in proteomics can be found elsewhere [11,53].…”

“…The system was applied to the analysis and characterization of intact proteins. At the moment the interest in the off-line coupling of conventional or miniaturized AsFlFFF with nanoflow LC-ESI-MS-MS is emerging progressively [11,31,[51][52][53][54]. The protein fractions collected off-line can be digested followed by peptide analysis carried out by nLC-ESI-MS-MS for shotgun analysis.…”

From bioseparation to artificial micro-organs: microfluidic chip based particle manipulation techniques

“…Besides affinity based methods to remove high abundance proteins from a particular sample, subcellular fractionation has been used to reduce complexity of a sample while at the same time retaining information about the cellular location of proteins identified from such an organelle fraction. Current fractionation technologies include density gradient centrifugation and free flow electrophoresis (FFE) 85,86 , which are tedious to perform, require high investments or dilute the sample in an in-acceptable way. Chip-based isoelectric separation was reported, yet amount of organelle was too low for further analysis with 2D-PAGE 10 .…”

Review on recent advances in the analysis of isolated organelles

“…However, it may also be accomplished by magnetic capture [30–37], free flow electrophoresis [38, 39], flow field-flow fractionation [40], fluorescent organelle sorting [41, 42], laser capture microdissection [43], and dielectrophoresis [44]. Because fractionation protocols have been reviewed recently [1, 7, 45], here we include only reports that were not included in these reviews.…”