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

Kang, Dukjin,Oh, Sunok,Reschiglian, Pierluigi,Moon, Myeong Hee Royal Society of Chemistry 2008 The Analyst Vol.133 No.4

<P>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.</P> <P>Graphic Abstract</P><P>Flow field-flow fractionation (FlFFF) is utilized for the size separation of rat liver mitochondria, and proteins of differently sized mitochondria are examined by shotgun proteomics and 2D-PAGE. <IMG SRC='http://pubs.rsc.org/services/images/RSCpubs.ePlatform.Service.FreeContent.ImageService.svc/ImageService/image/GA?id=b716851a'> </P>

Deuterium Oxide Labeling for Global Omics Relative Quantification: Application to Lipidomics

Kim, Jonghyun,Kang, Dukjin,Lee, Sung Ki,Kim, Tae-Young American Chemical Society 2019 ANALYTICAL CHEMISTRY - Vol.91 No.14

<P>A novel quantitative mass spectrometric method based on partial metabolic deuterium oxide (D<SUB>2</SUB>O) labeling, named “Deuterium Oxide Labeling for Global Omics Relative Quantification (DOLGOReQ)”, was developed for relative quantification of lipids on a global scale. To assess the precision and robustness of DOLGOReQ, labeled and unlabeled lipids from HeLa cells were mixed in various ratios based on their cell numbers. Using in-house software developed for automated high-throughput data analysis of DOLGOReQ, the number of detectable mass isotopomers and the degree of deuterium labeling were exploited to filter out low quality quantification results. Quantification of an equimolar mixture of HeLa cell lipids exhibited high reproducibility and accuracy across multiple biological and technical replicates. Two orders of magnitude of effective dynamic range for reasonable relative quantification could be established with HeLa cells mixed from 10:1 to 1:10 ratios between labeled and unlabeled samples. The quantification precision of DOLGOReQ was also illustrated with lipids commonly detected in both positive and negative ion modes. Finally, quantification performance of DOLGOReQ was demonstrated in a biological sample by measuring the relative change in the lipidome of HeLa cells under normal and hypoxia conditions.</P> [FIG OMISSION]</BR>

Miniaturized asymmetrical flow field-flow fractionation: Application to biological vesicles

Oh, Sunok,Kang, Dukjin,Ahn, Sung-Min,Simpson, Richard J.,Lee, Bong-Hee,Moon, Myeong Hee Wiley - VCH Verlag GmbH & Co. KGaA 2007 Journal of Separation Science Vol.30 No.7

<P>Asymmetrical flow field-flow fractionation (AFlFFF) has been carried out in a miniaturized channel by reducing the channel dimensions. Performance of the miniaturized AFlFFF (mAFlFFF) channel was evaluated with standard proteins and polystyrene latex spheres from nanometer to micrometer size. By reducing the channel dimension, proteins or particulate materials can be separated within a few minutes without a significant loss in resolution. The mAFlFFF channel was applied for the separation of exosomes harvested from immortalized human mesenchymal stem cell line. It shows a potential to fractionate exosome vesicles according to sizes which can be useful for proteomic studies in relation to immunotherapeutic applications.</P>

Enrichment Strategies for Identification and Characterization of Phosphoproteome

( Sun Young Lee ),( Dukjin Kang ),( Jongki Hong ) 한국질량분석학회 2015 Mass spectrometry letters Vol.6 No.2

Phosphorylation upon protein is well known to a key regulator that implicates in modulating many cellular processes like growth, migration, and differentiation. Up to date, grafting of multidimensional separation techniques onto advanced mass spectrometry (MS) has emerged as a promising tool for figuring out the biological functions of phosphorylation in a cell. However, advanced MS-based phosphoproteomics is still challenging, due to its intrinsic issues, i.e., low stoichiometry, less susceptibility in positive ion mode, and low abundance in biological sample. To overcome these bottlenecks, diverse techniques (e.g., SCX, HILIC, ERLIC, IMAC, TiO2, etc.) are continuously developed for on-/off-line enrichment of phosphorylated protein (or peptide) from biological samples, thereby helping qualitative/quantitative determination of phosphorylated protein and its phosphorylated sites. In this review, we introduce to the overall views of enrichment tools that are universally used to selectively isolate targeted phosphorylated protein (or peptide) from ordinary ones before MS-based phospoproteomic analysis.

Enrichment Strategies for Identification and Characterization of Phosphoproteome

Lee, Sun Young,Kang, Dukjin,Hong, Jongki Korean Society for Mass Spectrometry 2015 Mass spectrometry letters Vol.6 No.2

Phosphorylation upon protein is well known to a key regulator that implicates in modulating many cellular processes like growth, migration, and differentiation. Up to date, grafting of multidimensional separation techniques onto advanced mass spectrometry (MS) has emerged as a promising tool for figuring out the biological functions of phosphorylation in a cell. However, advanced MS-based phosphoproteomics is still challenging, due to its intrinsic issues, i.e., low stoichiometry, less susceptibility in positive ion mode, and low abundance in biological sample. To overcome these bottlenecks, diverse techniques (e.g., SCX, HILIC, ERLIC, IMAC, TiO₂, etc.) are continuously developed for on-/off-line enrichment of phosphorylated protein (or peptide) from biological samples, thereby helping qualitative/quantitative determination of phosphorylated protein and its phosphorylated sites. In this review, we introduce to the overall views of enrichment tools that are universally used to selectively isolate targeted phosphorylated protein (or peptide) from ordinary ones before MS-based phospoproteomic analysis.

Performance of hollow-fiber flow field-flow fractionation in protein separation

Park, Ilyong,Paeng, Ki-Jung,Kang, Dukjin,Moon, Myeong Hee WILEY-VCH Verlag 2005 Journal of separation science Vol.28 No.16

<P>Since hollow-fiber flow field-flow fractionation (HF FlFFF) utilizes a cylindrical channel made of a hollow-fiber membrane, which is inexpensive and simple in channel assembly and thus disposable, interests are increasing as a potential separation device in cells, proteins, and macromolecules. In this study, performance of HF FlFFF of proteins is described by examining the influence of flow rate conditions and length of fiber (polyacrylonitrile or PAN in this work) on sample recovery as well as experimental plate heights. The interfiber reproducibility in terms of separation time and recovery was also studied. Experiments showed that sample recovery was consistent regardless of the length of fiber when the effective field strength (equivalent to the mean flow velocity at the fiber wall) and the channel void time were adjusted to be equivalent for channels of various fiber lengths. This supported that the majority of sample loss in HF FlFFF separation of apoferritin and their aggregates may occur before the migration process. It is finally demonstrated that HF FlFFF can be applied for characterizing the reduction in Stokes' size of low density lipoproteins from blood plasma samples obtained from patients having coronary artery disease and from healthy donors.</P>

Dual Lectin-Based Size Sorting Strategy to Enrich Targeted N-Glycopeptides by Asymmetrical Flow Field-Flow Fractionation: Profiling Lung Cancer Biomarkers

Kim, Jin Yong,Kim, Sook-Kyung,Kang, Dukjin,Moon, Myeong Hee American Chemical Society 2012 ANALYTICAL CHEMISTRY - Vol.84 No.12

<P>A dual lectin-based size sorting and simultaneous enrichment strategy for selectively isolating N-linked glycopeptides was developed using asymmetrical flow field-flow fractionation (AF4). AF4 is an elution-based method for separating biological macromolecules that has been utilized for the separation of lectin–glycopeptide complexes formed by mixing serum peptides with lectin cocktails according to the difference in diffusion coefficients. It has also been used for simultaneous depletion of nonglycosylated peptides. The dual lectin-based enrichment method was applied to proteolytic peptides from lung cancer serum samples with two lectins (WGA, GlcNAc-specific, and SNA, Sia-specific), and the whole mixture was separated by AF4. The lectin–glycopeptide complex fractions collected during AF4 separation were endoglycosidically digested with PNGase F. The resulting deamidated glycopeptides were analyzed by nanoflow liquid chromatography-electrospray ionization-tandem mass spectrometry (nLC-ESI-MS-MS) to semiquantitatively profile the N-linked glycopeptides from the sera of lung cancer patients and healthy controls. The AF4 enrichment strategy coupled with nLC-ESI-MS-MS identified 16/24 (up/down-regulated by at least 10-fold compared to normal sera) N-linked glycopeptides from a WGA complex fraction of lung cancer sera and 18/3 from a SNA fraction.</P><P><B>Graphic Abstract</B> <IMG SRC='http://pubs.acs.org/appl/literatum/publisher/achs/journals/content/ancham/2012/ancham.2012.84.issue-12/ac300772w/production/images/medium/ac-2012-00772w_0007.gif'></P><P><A href='http://pubs.acs.org/doi/suppl/10.1021/ac300772w'>ACS Electronic Supporting Info</A></P>

Effect of fibroblast co-culture on the proliferation, viability and drug response of colon cancer cells

Koh, Byumseok,Jeon, Hyojin,Kim, Dahee,Kang, Dukjin,Kim, Kwang Rok D.A. Spandidos 2019 Oncology letters Vol.17 No.2

<P>Interactions between cancer cells and the surrounding fibroblasts serve an important role in cancer proliferation. Colon cancer co-culture model with colon fibroblasts and two metastatic models with lung and skin fibroblasts were established, and the co-culture effects on colon cancer cell proliferation, apoptosis and drug response were evaluated. Co-culture with CCD-18Co and BJ reduces SW480 cell proliferation by 4.2 and 5.3%, respectively, while WI-38 acts as a positive regulator and increases SW480 cell proliferation by 36%. CCD-18Co and BJ co-culture can also enhance XAV939 potency against SW480 cells by 16.8 and 27.3%; however, WI-38 co-culture reduces the effect of XAV939 by 38.2%. The present results suggest that, depending on fibroblast type, co-culture can have a positive/negative influence on colon cancer growth; therefore, care should be taken when considering fibroblasts as a target for future cancer therapies.</P>

TEMPO-Assisted Free Radical-Initiated Peptide Sequencing Mass Spectrometry (FRIPS MS) in Q-TOF and Orbitrap Mass Spectrometers: Single-Step Peptide Backbone Dissociations in Positive Ion Mode

Jang, Inae,Lee, Sun Young,Hwangbo, Song,Kang, Dukjin,Lee, Hookeun,Kim, Hugh I.,Moon, Bongjin,Oh, Han Bin Springer US 2017 Journal of the American Society for Mass Spectrome Vol.28 No.1

<P>The present study demonstrates that one-step peptide backbone fragmentations can be achieved using the TEMPO [2-(2,2,6,6-tetramethyl piperidine-1-oxyl)]-assisted free radical-initiated peptide sequencing (FRIPS) mass spectrometry in a hybrid quadrupole time-of-flight (Q-TOF) mass spectrometer and a Q-Exactive Orbitrap instrument in positive ion mode, in contrast to two-step peptide fragmentation in an ion-trap mass spectrometer (reference Anal. Chem. <B>85</B>, 7044-7051 ((30))). In the hybrid Q-TOF and Q-Exactive instruments, higher collisional energies can be applied to the target peptides, compared with the low collisional energies applied by the ion-trap instrument. The higher energy deposition and the additional multiple collisions in the collision cell in both instruments appear to result in one-step peptide backbone dissociations in positive ion mode. This new finding clearly demonstrates that the TEMPO-assisted FRIPS approach is a very useful tool in peptide mass spectrometry research.</P> [FIG OMISSION]</BR>

Development of an infant formula certified reference material for the analysis of organic nutrients

Lee, Joonhee,Kim, Byungjoo,Lee, Sun Young,Choi, Jongoh,Kang, Dukjin,Lee, Hwasim,Choi, KiHwan,Lee, Hyeyoung,Sim, Hee-Jung,Baek, Song-Yee,Lee, Honghee,Hyung, Seok-Won,Ahn, Seonghee,Seo, Dongwon,Hwang, J Applied Science Publishers 2019 Food chemistry Vol.298 No.-

<P><B>Abstract</B></P> <P>Infant formula certified reference material (CRM, KRISS CRM 108-02-003) were developed for the analysis of organic nutrients. The CRM is a milk-based infant formula powder, packaged at 14 g per unit. Ten thousand units were prepared and stored at −70 °C. For the certification of each nutrient, ten units were analyzed for simultaneous value-assignment and homogeneity test. Analytical methods used were isotope dilution mass spectrometry (IDMS) based on liquid chromatography mass spectrometer (LC/MS) or gas chromatography mass spectrometer (GC/MS) as higher-order reference methods.13 vitamins, 3 fatty acids, and total cholesterol were certified. The between-unit relative standard deviation of measurement results for each nutrient ranged 0.2% to 2.5%, showing very good homogeneity. The expanded relative uncertainties of the certified values ranged from 1% to 8%, indicating that they have higher-order metrological quality. The values of proximates (proteins, lipids, carbohydrates, water, and ash) were assigned through inter-laboratory comparisons.</P> <P><B>Highlights</B></P> <P> <UL> <LI> An infant formula CRMs for the analysis of organic nutrients was developed. </LI> <LI> Organic nutrients were certified by IDMS approaches as higher-order reference methods. </LI> <LI> Homogeneities and stability of the CRM were evaluated by IDMS approaches. </LI> <LI> Metrological qualities of the certified values were proved by their small uncertainties. </LI> <LI> Five proximates were value-assigned by interlaboratory comparison. </LI> </UL> </P>