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

Dukjin Kang, Sunok Oh, Pierluigi Reschiglian, Myeong Hee Moon

Research output: Contribution to journalArticle

41 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)505-515
Number of pages11
JournalAnalyst
Volume133
Issue number4
DOIs
Publication statusPublished - 2008 Apr 1

Fingerprint

Field Flow Fractionation
Mitochondria
proteomics
mitochondrion
Fractionation
Proteomics
flow field
Flow fields
fractionation
Proteins
protein
Electrophoresis, Gel, Two-Dimensional
peptide
Peptides
Gels
gel
Mitochondrial Size
analysis
Electrospray ionization
Electrospray Ionization Mass Spectrometry

All Science Journal Classification (ASJC) codes

  • Analytical Chemistry
  • Biochemistry
  • Environmental Chemistry
  • Spectroscopy
  • Electrochemistry

Cite this

Kang, Dukjin ; Oh, Sunok ; Reschiglian, Pierluigi ; Moon, Myeong Hee. / Separation of mitochondria by flow field-flow fractionation for proteomic analysis. In: Analyst. 2008 ; Vol. 133, No. 4. pp. 505-515.
@article{9a3d4dba7a7f488b87d9e9076cd13432,
title = "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.",
author = "Dukjin Kang and Sunok Oh and Pierluigi Reschiglian and Moon, {Myeong Hee}",
year = "2008",
month = "4",
day = "1",
doi = "10.1039/b716851a",
language = "English",
volume = "133",
pages = "505--515",
journal = "The Analyst",
issn = "0003-2654",
publisher = "Royal Society of Chemistry",
number = "4",

}

Separation of mitochondria by flow field-flow fractionation for proteomic analysis. / Kang, Dukjin; Oh, Sunok; Reschiglian, Pierluigi; Moon, Myeong Hee.

In: Analyst, Vol. 133, No. 4, 01.04.2008, p. 505-515.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Separation of mitochondria by flow field-flow fractionation for proteomic analysis

AU - Kang, Dukjin

AU - Oh, Sunok

AU - Reschiglian, Pierluigi

AU - Moon, Myeong Hee

PY - 2008/4/1

Y1 - 2008/4/1

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=41149109568&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=41149109568&partnerID=8YFLogxK

U2 - 10.1039/b716851a

DO - 10.1039/b716851a

M3 - Article

C2 - 18365121

AN - SCOPUS:41149109568

VL - 133

SP - 505

EP - 515

JO - The Analyst

JF - The Analyst

SN - 0003-2654

IS - 4

ER -