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>

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>

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>

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>

Isotope-Coded Carbamidomethylation for Quantification of N-Glycoproteins with Online Microbore Hollow Fiber Enzyme Reactor-Nanoflow Liquid Chromatography-Tandem Mass Spectrometry

Kim, Jin Yong,Oh, Donggeun,Kim, Sook-Kyung,Kang, Dukjin,Moon, Myeong Hee American Chemical Society 2014 ANALYTICAL CHEMISTRY - Vol.86 No.15

<P>This paper introduces a simple, inexpensive, and robust quantitative proteomic method for quantifying N-linked glycoproteins based on isotope-coded carbamidomethylation (iCCM) incorporated into an online microbore hollow fiber enzyme reactor and nanoflow liquid chromatography-tandem mass spectrometry (mHFER-nLC-MS/MS). The iCCM quantitation uses carbamidomethylation (CM; a routine protection of thiol groups before proteolysis) of the Cys residue of proteins with iodoacetamide (IAA) or its isotope (IAA-<SUP>13</SUP>C<SUB>2</SUB>,D<SUB>2</SUB>: 4 Da difference). CM-/iCCM-labeled proteome samples are mixed for proteolysis; then, online enrichment of N-glycopeptides using lectin affinity is carried out in an mHFER before nLC-MS/MS for quantification using multiple reaction monitoring (MRM). Initial evaluation of the iCCM method varying the mixing ratio of CM-/iCCM-labeled bovine serum albumin (BSA) standards yielded successful quantification of 18 peptides with less than 2% variation in the calculated ratio of light/heavy-labeled peptides. The iCCM quantitation with mHFER-nLC-MS/MS was evaluated with three standard glycoproteins (α-1-acid glycoproteins, fetuin and transferrin) and then applied to serum glycoproteins from liver cancer patients and controls, resulting in successful quantification of 73 N-glycopeptides (from 49 N-glycoproteins), among which 19 N-glycopeptides from 14 N-glycoproteins showed more than a 2.5-fold aberrant change in liver cancer patients’ sera compared with the pooled control. Although iCCM quantitation with mHFER-nLC-MS/MS applies only to glycopeptides with Cys residue, the method can offer several advantages over other labeling methods when applied to targeted glycoproteins: The iCCM method does not require an additional labeling reaction under special conditions nor complicated procedures to purify labeled products using additional columns. Isotope labeling at the protein level can minimize potential uncertainty originating from unequal efficiencies in protein digestion in separate vials and retrieval of each labeled peptide when labeling takes place at the peptide level. In addition, the labeling reagents for the iCCM method are readily obtained at a reasonable cost, which can make protein quantification easily accessible.</P><P><B>Graphic Abstract</B> <IMG SRC='http://pubs.acs.org/appl/literatum/publisher/achs/journals/content/ancham/2014/ancham.2014.86.issue-15/ac501544r/production/images/medium/ac-2014-01544r_0001.gif'></P><P><A href='http://pubs.acs.org/doi/suppl/10.1021/ac501544r'>ACS Electronic Supporting Info</A></P>

갑상선 수술 후 합병증인 애성, 저칼슘혈증 및 혈종에 대한 임상적 연구

범우성,문덕진,김준식,박범석,Wooseong Beom,M.D.,Dukjin Moon,M.D.,Junsik Kim,M.D. and Bumsuk Park,M.D. 대한갑상선-내분비외과학회 2007 The Koreran journal of Endocrine Surgery Vol.7 No.4

Purpose: The use of thyroidectomy has increased as a diagnostic technique for thyroid disease. However, performance of a, thyroidectomy is accompanied with complications. Post-thyroidectomy complications include recurrent laryngeal nerve palsy, hypocalcemia, hematoma, infection, and thyroid storm. The aim of this study was to determine the clinical incidence and to evaluate complications after a thyroidectomy, including recurrent laryngeal nerve palsy, hypocalcemia, hematoma, and scaring, following a retrospective review of cases. Methods: From July 2004 to May 2006, 661 consecutive patients that had undergone a thyroidectomy were identified. Through a retrospective review, we evaluated the incidence and type of complications, including recurrent laryngeal nerve palsy, hypocalcemia, hematoma, and postoperative scaring. Results: 1) Recurrent laryngeal nerve palsy was a very serious complication, but had a very low incidence. Eight cases out of 661 cases developed and most of the cases developed after a total thyroidectomy. 2) Hypocalcemia was the most common complication. Each incidence of hypocalcemia of methods of thyroid surgery was significant (P= 0.019) but, thyroid disease did not have significant difference (P=0.071). 3) The incidence of postoperative hematoma was 2.74% (18/655). Graves' disease was more predominant than other diseases. Conclusion: Post-thyroidectomy complications and cosmetic problems include recurrent laryngeal nerve palsy, hypocalcemia, hematoma, and postoperative scar. An understanding of the incidence and review of complications after a thyroidectomy may reduce their incidence. (Korean J Endocrine Surg 2007;7:252-256)

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>

A Simple Carbamidomethylation-Based Isotope Labeling Method for Quantitative Shotgun Proteomics

Oh, Donggeun,Lee, Sun Young,Kwon, Meehyang,Kim, Sook-Kyung,Moon, Myeong Hee,Kang, Dukjin Korean Society for Mass Spectrometry 2014 Mass spectrometry letters Vol.5 No.3

In this study, we present a new isotope-coded carbamidomethylation (iCCM)-based quantitative proteomics, as a complementary strategy for conventional isotope labeling strategies, with providing the simplicity, ease of use, and robustness. In iCCM-based quantification, two proteome samples can be separately isotope-labeled by means of covalently reaction of all cysteinyl residues in proteins with iodoacetamide (IAA) and its isotope (IAA-$^{13}C_2$, $D_2$), denoted as CM and iCCM, respectively, leading to a mass shift of all cysteinyl residues to be + 4 Da. To evaluate iCCM-based isotope labeling in proteomic quantification, 6 protein standards (i.e., bovine serum albumin, serotransferrin, lysozyme, beta-lactoglobulin, beta-galactosidase, and alpha-lactalbumin) isotopically labeled with IAA and its isotope, mixed equally, and followed by proteolytic digestion. The resulting CM-/iCCM-labeled peptide mixtures were analyzed using a nLC-ESI-FT orbitrap-MS/MS. From our experimental results, we found that the efficiency of iCCM-based quantification is more superior to that of mTRAQ, as a conventional nonisobaric labeling method, in which both of a number of identified peptides from 6 protein standards and the less quantitative variations in the relative abundance ratios of heavy-/light-labeled corresponding peptide pairs. Finally, we applied the developed iCCM-based quantitative method to lung cancer serum proteome in order to evaluate the potential in biomarker discovery study.

A Simple Carbamidomethylation-Based Isotope Labeling Method for Quantitative Shotgun Proteomics

( Donggeun Oh ),( Sun Young Lee ),( Meehyang Kwon ),( Sook Kyung Kim ),( Myeong Hee Moon ),( Dukjin Kang ) 한국질량분석학회 2014 Mass spectrometry letters Vol.5 No.3

In this study, we present a new isotope-coded carbamidomethylation (iCCM)-based quantitative proteomics, as a complementary strategy for conventional isotope labeling strategies, with providing the simplicity, ease of use, and robustness. In iCCM-based quantification, two proteome samples can be separately isotope-labeled by means of covalently reaction of all cysteinyl residues in proteins with iodoacetamide (IAA) and its isotope (IAA-13C2, D2), denoted as CM and iCCM, respectively, leading to a mass shift of all cysteinyl residues to be + 4 Da. To evaluate iCCM-based isotope labeling in proteomic quantification, 6 protein standards (i.e., bovine serum albumin, serotransferrin, lysozyme, beta-lactoglobulin, beta-galactosidase, and alpha-lactalbumin) isotopically labeled with IAA and its isotope, mixed equally, and followed by proteolytic digestion. The resulting CM-/iCCM-labeled peptide mixtures were analyzed using a nLC-ESI-FT orbitrap-MS/MS. From our experimental results, we found that the efficiency of iCCM-based quantification is more superior to that of mTRAQ, as a conventional nonisobaric labeling method, in which both of a number of identified peptides from 6 protein standards and the less quantitative variations in the relative abundance ratios of heavy-/light-labeled corresponding peptide pairs. Finally, we applied the developed iCCM-based quantitative method to lung cancer serum proteome in order to evaluate the potential in biomarker discovery study

Development of an Online Microbore Hollow Fiber Enzyme Reactor Coupled with Nanoflow Liquid Chromatography-Tandem Mass Spectrometry for Global Proteomics

Kim, Jin Yong,Lee, Sun Young,Kim, Sook-Kyung,Park, Sang Ryoul,Kang, Dukjin,Moon, Myeong Hee American Chemical Society 2013 ANALYTICAL CHEMISTRY - Vol.85 No.11

<P>In this study, we report the development of a microbore hollow fiber enzyme reactor (mHFER) coupled to nanoflow liquid chromatography-tandem mass spectrometry (nLC-ESI-MS/MS) for the online digestion or selective enrichment of glycopeptides and analysis of proteins. With mHFER, enzymatic digestion of protein could be achieved by continuous flow within a very small volume (∼10 μL) of mHF inserted in a PEEK tube. Digested peptides exited through the pores of the hollow fiber membrane wall to external single or multiplexed trap columns for nLC-ESI-MS/MS analysis. Evaluation of online mHFER-nLC-ESI-MS/MS system was made with bovine serum albumin (BSA) by varying the temperature of digestion and the amount of protein injected. We evaluated the ability of the mHFER system to enrich glycopeptides by injecting a mixture of lectin (concanavalin A) and digested peptides from α-1-acid glycoprotein (AGP) into the mHFER, followed by delivery of PNGase F for endoglycosidic digestion. Nonglycosylated peptides unbound to lectins eluted at the first breakthrough run while N-linked glycopeptides eluted after the endoglycosidic digestion. The developed method was applied to urine samples from patients with prostate cancer and controls; 67 N-linked glycopeptides were identified and relative differences in glycopeptide content between patient and control samples were determined.</P><P><B>Graphic Abstract</B> <IMG SRC='http://pubs.acs.org/appl/literatum/publisher/achs/journals/content/ancham/2013/ancham.2013.85.issue-11/ac400625k/production/images/medium/ac-2013-00625k_0006.gif'></P><P><A href='http://pubs.acs.org/doi/suppl/10.1021/ac400625k'>ACS Electronic Supporting Info</A></P>