Qualitative Analysis of the Major Constituents in Traditional Oriental Prescription Bang-poong-tong-sung-san by Liquid Chromatography/Ultraviolet Detector/Ion-Trap Time-of-Flight Mass Spectrometry

( Han Young Eom ),( Hyung Seung Kim ),( Sang Beom Han ) 한국질량분석학회 2014 Mass spectrometry letters Vol.5 No.1

An advanced and reliable high performance liquid chromatography (HPLC)/ultraviolet detector (UV)/ion-trap timeof- flight (IT-TOF) mass spectrometry was developed for the simultaneous quantification of 19 marker compounds in Bangpoong- tong-sung-san (BPTS), a traditional oriental prescription. Various parameters affecting HPLC separation and IT-TOF detection were investigated, and optimized conditions were identified. The separation was achieved on a Capcell PAK C18 column (1.5 mm × 250 mm, 5 μm particle size) using a gradient elution of acetonitrile and water containing 0.1% formic acid at a flow rate of 0.1 mL/min. The column temperature was maintained at 40oC and the injection volume was 2 μL. IT-TOF system was equipped with an electrospray ion source (ESI) operating in positive or negative ion mode. The optimized electrospray ionization parameters were as follows: ion spray voltage, +4.5 kV (positive ion mode), or -3.5 kV (negative ion mode); drying gas (N2), 1.5 L/min; heat block temperature, 200oC. Automatic MSn (n = 1~3) analyses were carried out to obtain structural information of analytes. Elemental compositions and their mass errors were calculated based on their accurate masses obtained from a formula predictor software. The marker compounds in BPTS were identified by comparisons between MSn spectra from standards and those from extracts. Moreover, the libraries of MS2 and MS3 spectra and accurate masses of parent and fragment ions for marker compounds were constructed. The developed method was successfully applied to the BPTS extracts and identified 17 out of 19 marker compounds in the BPTS extracts.

Charge-Directed Peptide Backbone Dissociations of o-TEMPO-Bz-C(O)-Peptides

( Aeran Jeon ),( Ji Hye Lee ),( Hyuk Su Kwon ),( Hyung Soon Park ),( Bong Jin Moon ),( Han Bin Oh ) 한국질량분석학회 2013 Mass spectrometry letters Vol.4 No.4

In the present study, we report that the charge-directed (assisted) peptide dissociation products, such as b- and y-type peptide backbone fragments, were the major products in MS/MS and MS3 applications of some o-TEMPO-Bz-C(O)-peptide ions, while radical-driven dissociation products, such as a/x and c/z-type fragments, were previously shown to be the major products in the free radical initiated peptide sequencing mass spectrometry (FRIPS MS). Those o-TEMPO-Bz-C(O)-peptides share a common feature in their sequences, that is, the peptides do not include an arginine residue that has the highest proton affinity among free amino acids. The appearance of b- and y-type fragments as major products in FRIPS MS can be understood in terms of the so-called “mobile-proton model”. When the proton is highly mobilized by the absence of arginine, the chare-directed peptide dissociation pathways appear to be more competitive than the radical-driven dissociation pathways, in our FRIPS experiments.

Analysis of Arginine, Glucose, Sucrose, and Polyethylene Glycols using a Wood Charcoal Matrix for MALDI-MS

( Sunyoung Lee ),( Jinhee Kim ),( Hyo-jik Yang ),( Seongjae Shin ),( Jangmi Hong ),( Jeongkwon Kim ) 한국질량분석학회 2010 Mass spectrometry letters Vol.1 No.1

Wood charcoal was investigated to determine its potential as an alternative matrix for matrix-assisted laser desorption/ ionization of various samples. Wood charcoal was an effective matrix for analyzing glucose, sucrose, arginine, and polyethylene glycols (PEGs), with detection levels of 100 pmol for glucose, 1 nmol for sucrose, 100 pmol for arginine, 100 pmol for PEG 400, 1 pmol for PEG 1540, and 10 pmol for PEG 3350. No analyte signal was observed for peptides or proteins.

Isotope-Dilution Mass Spectrometry for Quantification of Urinary Active Androgens Separated by Gas Chromatography

( Su Hyeon Lee ),( Man Ho Choi ),( Won-yong Lee ),( Bong Chul Chung ) 한국질량분석학회 2010 Mass spectrometry letters Vol.1 No.1

Cross reacting antibodies can cause an overestimation of the results of immunoassays. Therefore, alternative methods are needed for the accurate quantification of steroids. Gas chromatography combined with isotope-dilution mass spectrometry (GC-IDMS) is developed to quantify urinary active androgens, testosterone, epitestosterone and dihydrotestosterone, which are clinically relevant androgens to both hair-loss and prostate diseases. The method devised involves enzymatic hydrolysis with β- glucuronidase, solid-phase extraction, liquid-liquid extraction using methyl tert-butyl ether and subsequent conversion to pentafluorophenyldimethylsilyl- trimethylsilyl (flophemesyl-TMS) derivatives for sensitive and selective analysis in selected-ion monitoring mode. Flophemesyl-TMS derivatization not only eliminates matrix interference but also has a good peak resolution within a 6 min-run. A selective and sensitive GC technique with flophemesyl-TMS derivatives also allows accurate quantitative analysis of three active androgens when combined with IDMS. The limit of quantification of the three analytes was <50 pg/mL, and extraction recoveries ranged from 91.9 to 102.1%. The precision and accuracy were 1.2~6.5% and 89.0~106.7%, respectively. This GC-IDMS method can be useful for evaluating the drug efficacy and monitoring the biological processes responsible for male-pattern baldness and prostate diseases.

Differential Protein Quantitation in Mouse Neuronal Cell Lines using Amine- Reactive Isobaric Tagging Reagents with Tandem Mass Spectrometry

( Kun Cho ),( Gun Wook Park ),( Jin Young Kim ),( Sang Kwang Lee ),( Han Bin Oh ),( Jong Shin Yoo ) 한국질량분석학회 2010 Mass spectrometry letters Vol.1 No.1

The high-throughput identification and accurate quantification of proteins are essential strategies for exploring cellular functions and processes in quantitative proteomics. Stable isotope tagging is a key technique in quantitative proteomic research, accompanied by automated tandem mass spectrometry. For the differential proteome analysis of mouse neuronal cell lines, we used a multiplexed isobaric tagging method, in which a four-plex set of amine-reactive isobaric tags are available for peptide derivatization. Using the four-plex set of isobaric tag for relative and absolute quantitation (iTRAQ) reagents, we analyzed the differential proteome in several stroke time pathways (0, 4, and 8 h) after the mouse neuronal cells have been stressed using a glutamate oxidant. In order to obtain a list of the differentially expressed proteins, we selected those proteins which had apparently changed significantly during the stress test. With 95% of the peptides showing only a small variation in quantity before and after the test, we obtained a list of eight up-regulated and four down-regulated proteins for the stroke time pathways. To validate the iTRAQ approach, we studied the use of oxidant stresses for mouse neuronal cell samples that have shown differential proteome in several stroke time pathways (0, 4, and 8 h). Results suggest that histone H1 might be the key protein in the oxidative injury caused by glutamate-induced cytotoxicity in HT22 cells.

Dynamic MRM Measurements of Multi-Biomarker Proteins by Triple- Quadrupole Mass Spectrometry with Nanoflow HPLC-Microfluidics Chip

( Eun Sun Ji ),( Mi Hee Cheon ),( Ju Yeon Lee ),( Jong Shin Yoo ),( Hyun-jin Jung ),( Jin Young Kim ) 한국질량분석학회 2010 Mass spectrometry letters Vol.1 No.1

The development of clinical biomarkers involves discovery, verification, and validation. Recently, multiple reaction monitoring (MRM) coupled with stable isotope dilution mass spectrometry (IDMS) has shown considerable promise for the direct quantification of proteins in clinical samples. In particular, multiple biomarkers have been tracked in a single experiment using MRM-based MS approaches combined with liquid chromatography. We report here a highly reproducible, quantitative, and dynamic MRM system for validating multi-biomarker proteins using Nanoflow HPLC-Microfluidics Chip/Triple-Quadrupole MS. In this system, transitions were acquired only during the retention window of each eluting peptide. Transitions with the highest MRM-MS intensities for the five target peptides from colon cancer biomarker candidates were automatically selected using Optimizer software. Relative to the corresponding non-dynamic system, the dynamic MRM provided significantly improved coefficients of variation in experiments with large numbers of transitions. Linear responses were obtained with concentrations ranging from fmol to pmol for five target peptides.

A Technique to Minimize Impurity Signal from Blank Rhenium Filaments for Highly Accurate TIMS Measurements of Uranium in Ultra-Trace Levels

( Jong-ho Park ),( Inhee Choi ),( Kyuseok Song ) 한국질량분석학회 2010 Mass spectrometry letters Vol.1 No.1

As background significantly affects measurement accuracy and a detection limit in determination of the trace amounts of uranium, it is necessary to minimize the impurities in the filaments used for thermal ionization mass spectrometry (TIMS). We have varied the degassing condition such as the heating currents and duration times to reduce the backgrounds from the filaments prepared with zone-refined rhenium tape. The most efficient degassing condition of the heating current and the duration time was determined as 3.5 A and 60 min, respectively. The TIMS measurement combined with the isotope dilution mass spectrometry IDMS) technique showed that the uranium backgrounds were determined to be in a few fg level from blank rhenium filaments. The background minimized filaments were utilized to measure the uranium isotope ratios of a U030 (NIST) standard sample. The excellent agreement of the measurement with the certified isotope ratios showed that the degassing procedure optimized in this study efficiently reduced the impurity signals of uranium from blank rhenium filaments to a negligible level.

High-Throughput Active Compound Discovery using Correlations between Activity and Mass Profiles

( Kyu Hwan Park ),( Kyo Joong Yoon ),( Kyung-hoon Kwon ),( Hyun Sik Kim ) 한국질량분석학회 2010 Mass spectrometry letters Vol.1 No.1

The active components in a plant extract can be represented as mass profiles. We introduce here a new, multi-compound discovery method known as Scaling of Correlations between Activity and Mass Profiles (SCAMP). In this method, a correlation coefficient is used to quantify similarities between the extract activity and mass profiles. The method was evaluated by first measuring the anti-oxidation activity of eleven fractions of an Astragali Radix extract using DPPH assays. Next, 15 T Fouriertransform ion cyclotron resonance (FT-ICR) MS was employed to generate mass profiles of the eleven fractions. A comparison of correlation coefficients indicated two compounds at m/z 285.076 and 286.076 that were strong antioxidants. Principal component analyses of these profiles yielded the same result. FT-ICR MS, which offers a mass resolving power of 500,000, was used to discern isotopic fine structures and indicated that the molecular formula corresponding to the peak at m/z 285.076 was C16H13O5. SCAMP in combination with high-resolution MS can be applied to any type of mixture to study pharmacological activity and is a powerful tool for active compound discovery in plant extract studies.

Ditopic Binding of Alkali Halide Ions to Trimethylboroxine

( Kyung Hwan Jeong ),( Seung Koo Shin ) 한국질량분석학회 2010 Mass spectrometry letters Vol.1 No.1

Trimethylboroxine (TMB) is a six-membered ring compound containing Lewis acidic boron and Lewis basic oxygen atoms that can bind halide anion and alkali metal cation, respectively. We employed Fourier transform ion cyclotron resonance spectroscopy to study the gas-phase binding of LiBrLi+ and F-(KF)2 to TMB. TMB forms association complexes with both LiBrLi+ and F-(KF)2 at room temperature, providing direct evidence for the ditopic binding. Interestingly, the TMB·F-(KF)2 anion complex is formed 33 times faster than the TMB·Li+BrLi cation complex. To gain insight into the ditopic binding of an ion pair, we examined the structures and energetics of TMB·Li+, TMB·F-, TMB·LiF (the contact ion pair), and Li+·TMB·F- (the separated ion pair) using Hartree-Fock and density functional theory. Theory suggests that F- binds more strongly to TMB than Li+ and the contact ion-pair binding (TMB·LiF) is more stable than the separated ion-pair binding (Li+·TMB·F-).

Collisionally-Activated Dissociation of Peptides with a Disulfide Bond: Confirmation of the Mobile-Proton Model Based Explanation

( Younjin Lee ),( Han Bin Oh ) 한국질량분석학회 2010 Mass spectrometry letters Vol.1 No.1

In the present study, collisionally-activated dissociation (CAD) experiments were performed under low energy collision conditions in six peptides containing a disulfide bond. Fragments produced as a result of the cleavage of a disulfide bond were obtained after CAD in four peptides (bactenecin, TGF-α, cortistantin, and linearly linked peptide, Scheme 1) with basic amino acid residues. In contrast, the CAD analysis of two peptides with no basic residue (oxytocin and tocinoic acid) rarely produced fragments indicative of cleavage of a disulfide bond. These results are consistent with the mobile proton model suggested by the McLuckey and O’Hair groups (ref. 22 and 23); nonmobile protons sequestered at basic amino acid residues appear to promote the cleavage of disulfide bonds.