Journal of Chromatography B (v.855, #1)

New challenge of mass spectrometry technique by Toshimitsu Niwa; Akira Shimizu (1).

Tandem time-of-flight (TOF/TOF) mass spectrometry and the curved-field reflectron by Robert J. Cotter; Wendell Griffith; Christine Jelinek (2-13).
The curved-field reflectron (CFR), when used as the second mass analyzer in a tandem time-of-flight mass spectrometer, provides a design that enables the use of very high energy collision-induced dissociation (CID). Specifically, this is because the wide energy bandwidth of the CFR obviates the need for floating the collision region to decelerate the precursor ions and subsequently reaccelerating product ions to enable reflectron focusing. Here we describe the evolution of tandem instruments based on the CFR, from its introduction in 1993 to the current commercial TOF2 mass spectrometer from Shimadzu Corporation, and briefly review the history of TOF/TOF instruments. A number of applications are also described. One is the characterization of a C-terminal cleavage of cystatin C that appears to be associated with patients with remitting relapse multiple sclerosis (RRMS). Both surface-enhanced laser desorption/ionization (SELDI) and MALDI were used on a high performance TOF instrument operating in the MS and MS/MS modes. Tandem TOF mass spectrometry has also been used to determine the acetylation sites on histones and on the enzyme, histone acetyl transferase (HAT), responsible for the modification. Acetylation has been determined quantitatively for multiple sites on histone H3 and H4 using a deuteroacetylation method. For a number of closely spaced sites on the histone tail regions, MS/MS enables us to then determine both the order and distribution of acetylation.
Keywords: Tandem mass spectrometry; Time-of-flight; Curved-field reflectron; Matrix-assisted laser desorption/ionization (MALDI); Surface-enhanced laser desorption/ionization (SELDI); Multiple sclerosis; Cystatin; Histone acetyl transferase (HAT); Histones; Acetylation;

An overview is provided of six strategies for relative or absolute quantitation of protein abundances that are widely used in proteomic studies. Strengths and limitations are discussed. Four of these involve stable isotope labeling and isotope ratio measurements by mass spectrometry. In another, mass spectra are used to deconvolute overlapping peptide HPLC peaks to provide relative quantitation based on peak areas. The sixth provides relative abundances of proteins based on 2-D gel arrays. It should be noted that these strategies measure peptide and protein abundances, and cannot directly assess changes in regulation or expression.
Keywords: Proteomics; Mass spectrometry; 2-D gel electrophoresis; Stable isotopes relative abundance; Absolute abundance; Metabolic labeling; SILAC; O-18 labeling; ICAT; GIST; iTRAQ; AQUA; NBS;

Chemical proteomics is an effective approach to focused proteomics, having the potential to find specific interactors in moderate-scale comprehensive analysis. Unlike chemical genetics, chemical proteomics directly and comprehensively identifies proteins that bind specifically to candidate compounds by means of affinity chromatographic purification using the immobilized candidate, combined with mass spectrometric identification of interacting proteins. This is an effective approach for discovering unknown protein functions, identifying the molecular mechanisms of drug action, and obtaining information for optimization of lead compounds. However, immobilized-small molecule affinity chromatography always suffers from the problem of non-specific binders. Although several approaches were reported to reduce non-specific binding proteins, these are mainly focused on the use of low-binding-affinity beads or insertion of a spacer between the bead and the compound. Stable isotope labeling strategies have proven particularly advantageous for the discrimination of true interactors from many non-specific binders, including carrier proteins, such as serum albumin, and are expected to be valuable for drug discovery.
Keywords: Affinity chromatography; Drug discovery; Proteomics;

Cardiovascular proteomic analysis by Toru Suzuki; Ryozo Nagai (28-34).
Here, we report on our proteomic studies in the field of cardiovascular medicine. Our research has been focused on understanding the role of proteins in cardiovascular disease with a particular focus on epigenetic regulation and biomarker discovery, with the objective of better understanding cardiovascular pathophysiology to lead to the development of new and better diagnostic and therapeutic methods. We have used mass spectrometry for over 5 years as a viable method to investigate protein–protein interactions and post-translational modifications in cellular proteins as well as a method to investigate the role of extra-cellular proteins. Use of mass spectrometry not only as a research tool but also as a potential diagnostic tool is a topic of interest. In addition to these functional proteomics studies, structural proteomic studies are also done with expectations to allow for pinpoint drug design and therapeutic intervention. Collectively, our proteomics studies are focused on understanding the functional role and potential therapeutically exploitable property of proteins in cardiovascular disease from both intra-cellular and extra-cellular aspects with both functional as well as structural proteomics approaches to allow for comprehensive analysis.
Keywords: Proteomic analysis; Cardiovascular;

Application of proteomic technologies to discover and identify biomarkers for excessive alcohol consumption: A review by Fumio Nomura; Takeshi Tomonaga; Kazuyuki Sogawa; Di Wu; Tatsuya Ohashi (35-41).
Since currently available markers of alcohol abuse are not satisfactory, searches for novel markers are warranted. Proteomic analyses are promising tools to discover and identify novel biomarkers. Using two different proteomic technologies, surface enhanced laser desorption/ionization time-of-flight mass spectrometry and agarose fluorescent two-dimensional difference gel electrophoresis, we could detect and identify a total of 11 potential biomarkers of excessive alcohol consumption. It was noteworthy that the down regulation of the 5.9 kDa protein fragment was consistently seen in habitual drinkers and the diagnostic efficiency was greater than those of conventional markers such as gamma glutamyl transferase and carbohydrate deficient transferrin.
Keywords: Alcohol; SELDI; 2D-DIGE; CDT; GGT;

Early diagnosis and treatment are critical for patients with inborn errors of metabolism (IEMs). For most IEMs, the clinical presentations are variable and nonspecific, and routine laboratory tests do not indicate the etiology of the disease. A diagnostic procedure using highly sensitive gas chromatography–mass spectrometric urine metabolome analysis is useful for screening and chemical diagnosis of IEM. Metabolite analysis can comprehensively detect enzyme dysfunction caused by a variety of abnormalities. The mutations may be uncommon or unknown. The lack of coenzymes or activators and the presence of post-translational modification defects and subcellular localization abnormalities are also reflected in the metabolome. This noninvasive and feasible urine metabolome analysis, which uses urease-pretreatment, partial adoption of stable isotope dilution, and GC/MS, can be used to detect more than 130 metabolic disorders. It can also detect an acquired abnormal metabolic profile. The metabolic profiles for two cases of non-inherited phenylketonuria are shown. In this review, chemical diagnoses of hyperphenylalaninemia, phenylketonuria, hyperprolinemia, and lactic acidemia, and the differential diagnosis of β-ureidopropionase deficiency and primary hyperammonemias including ornithine transcarbamylase deficiency and carbamoylphosphate synthetase deficiency are described.
Keywords: Metabolome; Chemical diagnosis; GC–MS; Lactic acidemia; Hyperphenylalaninemia; Secondary phenylketonuria; Hyperprolinemia; Hyperammonemias; Ornithine transcarbamylase deficiency;

Analysis of DNA-bound advanced glycation end-products by LC and mass spectrometry by Clemens Bidmon; Matthias Frischmann; Monika Pischetsrieder (51-58).
Sugars and sugar degradation products readily react in vitro with guanine derivatives, resulting in the formation of DNA-bound advanced glycation end-products (DNA-AGEs). The two diastereomers of N 2-(1-carboxyethyl)-2′-deoxyguanosine (CEdGA,B) and the cyclic adduct of methylglyoxal and 2′-deoxyguanosine (mdG) (N 2-7-bis(1-hydroxy-2-oxopropyl)-2′-deoxyguanosine have also been detected in cultured cells and/or in vivo. LC–MS/MS methods have been developed to analyze sensitively DNA adducts in vitro and in vivo. In this paper, the chemical structures of possible DNA-AGEs and the application of LC–MS/MS to measure DNA-AGEs are reviewed.
Keywords: Advanced glycation end-products; N 2-(1-carboxyethyl)-2′-deoxyguanosine; CEdGA,B; DNA; LC–MS/MS;

Liquid chromatography/electrospray ionization-mass spectrometry (LC/ESI-MS) demonstrated that glutathionyl hemoglobin (Hb) levels are increased in patients with diabetes, hyperlipidemia, uremia and Friedreich's ataxia. Glutathionylation of Hb is enhanced by oxidative stress. High performance liquid chromatography (HPLC) and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) have also been developed for the quantification of glutathionyl Hb. Glutathionyl-lens proteins were detected in uremic patients and cataractous aged subjects. Glutathionylation of numerous enzymes is induced by oxidative stress, reduces their catalytic activities and may be involved in protection from the damaging effects of oxidative agents. Thioredoxin, glutaredoxin (thioltransferase) and protein disulfide isomerase are the key enzymes in controlling cellular oxidative stress that catalyze reduction of glutathionyl protein disulfide bonds. Thus, protein glutathionylation is closely associated with oxidative stress.
Keywords: Glutathionyl hemoglobin; Oxidative stress; Diabetes mellitus; Uremia; Liquid chromatography/mass spectrometry; High performance liquid chromatography; Matrix-assisted laser desorption ionization-time of flight mass spectrometry;

Serum protein profile of rheumatoid arthritis treated with anti-TNF therapy (infliximab) by Tohru Takeuchi; Toyofumi Nakanishi; Yoko Tabushi; Ayu Hata; Takeshi Shoda; Takuya Kotani; Akira Shimizu; Takayuki Takubo; Shigeki Makino; Toshiaki Hanafusa (66-70).
We analyzed the changes in the serum protein profile by infliximab using two-dimensional gel electrophoresis and mass spectrometry. More than 50 gel spots were seen to increase or decrease in correlation with clinical improvements of RA. The spots corresponding to CRP, C3, and Apo J showed reduced staining intensity, while the spots corresponding to Apo A-I, RBP, and transthyretin were enhanced. The protein profile of RA patients treated with infliximab was mostly similar to that of normal healthy controls except for several protein spots. This suggested that infliximab normalized the serum protein profile of RA patients, leading to modification in the serum lipid profile and antioxidant status in RA.
Keywords: Tumor necrosis factor; Infliximab; Rheumatoid arthritis; Matrix assisted laser desorption ionization/time-of-flight mass spectrometry; Electrophoresis;

Application of a metabolomic method combining one-dimensional and two-dimensional gas chromatography-time-of-flight/mass spectrometry to metabolic phenotyping of natural variants in rice by Miyako Kusano; Atsushi Fukushima; Makoto Kobayashi; Naomi Hayashi; Pär Jonsson; Thomas Moritz; Kaworu Ebana; Kazuki Saito (71-79).
We have developed a comprehensive method combining analytical techniques of one-dimensional (1D) and two-dimensional (GC × GC) gas chromatography-time-of-flight (TOF)–mass spectrometry. This method was applied to the metabolic phenotyping of natural variants in rice for the 68 world rice core collection (WRC) and two other varieties. Ten metabolites were selected as metabolite representatives, and the selected ion current of each metabolite peak obtained from both techniques were statistically compared. Our method of combining 1D- and GC × GC-TOF/MS is useful for the metabolic phenotyping of natural variants in rice for further studies in breeding programs.
Keywords: Rice core collection; GC × GC; Two-dimensional chromatography; Gas chromatography; Metabolic phenotyping;

ESI–MS/MS study of acylcarnitine profiles in urine from patients with organic acidemias and fatty acid oxidation disorders by Hironori Kobayashi; Yuki Hasegawa; Mitsuru Endo; Jamiyan Purevsuren; Seiji Yamaguchi (80-87).
Acylcarnitines in urine from 45 patients with organic acidemias and fatty acid oxidation disorders were evaluated using ESI–MS/MS. The urinary acylcarnitine profiles in organic acidemias, SCAD deficiency and MCAD deficiency were compatible with blood acylcarnitine profiles, and abnormalities in urinary acylcarnitine profiles in these conditions were enhanced following carnitine loading. Urinary acylcarnitine profiles were not helpful for characterization of long-chain fatty acid disorders, but a combination of urine and blood acylcarnitine analysis was useful for differential diagnosis of carnitine deficit.
Keywords: Urine; Acylcarnitine; Newborn screening; Electrospray ionization tandem mass spectrometry; Organic acidemia; Fatty acid oxidation disorder; Screening; ESI–MS/MS; Carnitine deficiency; Pivoxil; Glutaric; Aciduria type 1; GA1;

Analysis of bile acid glutathione thioesters by liquid chromatography/electrospray ionization–tandem mass spectrometry by Kuniko Mitamura; Mitsuru Sogabe; Hironori Sakanashi; Saai Watanabe; Toshihiro Sakai; Yoshihiro Yamaguchi; Tateaki Wakamiya; Shigeo Ikegawa (88-97).
The formation of thioester-linked glutathione (GSH) conjugates of bile acids (BAs) is presumed to occur via trans-acylation reactions between GSH and reactive acyl-linked metabolites of BAs. The present study examines the chemical reactivity of cholyl-adenylate and cholyl-CoA thioester, acyl-linked metabolites of cholic acid (CA), with GSH to form CA-GSH conjugate in vitro. The authentic specimen of CA-GSH was synthesized along with GSH conjugates of four common BAs found in the human body. Their structures were confirmed by proton-nuclear magnetic resonance spectroscopy and electrospray ionization (ESI)–tandem mass spectrometry in positive- and negative-ion modes. Incubation of cholyl-adenylate or cholyl-CoA thioester with GSH was carried out at pH 7.5 and 37 °C for 30 min, with analysis of the reaction mixture by liquid chromatography/ESI–tandem mass spectrometry, where CA-GSH was detected on the product ion mass chromatograms monitored with stable and abundant dehydrated positive-ion [M  + H―H2O]+ at m/z 680.3 and fragmented negative-ion [GSH―H] at m/z 306.0, and was definitely identified by CID spectra by comparison with those of the authentic sample. The results show that both cholyl-adenylate and cholyl-CoA thioester are able to acylate GSH in vitro.
Keywords: Bile acid; Glutathione; Glutathione conjugate; Liquid chromatography; Electrospray ionization; Mass spectrometry; LC–MS; Metabolite;

MALDI-based imaging mass spectrometry revealed abnormal distribution of phospholipids in colon cancer liver metastasis by Shuichi Shimma; Yuki Sugiura; Takahiro Hayasaka; Yutaka Hoshikawa; Tetsuo Noda; Mitsutoshi Setou (98-103).
We present the results of matrix-assisted laser desorption/ionization (MALDI) imaging and direct molecular identification using tandem mass spectrometry (MS/MS) in colon cancer liver metastasis. Cancer tissue was removed from a Japanese patient and frozen immediately without any fixations. The sections were sliced to a thickness of 3 μm. The matrix for lipid ionization was 2,6-dihydroxy acetophenone. The matrix solution was applied with an airbrush into a thin uniform matrix layer on the tissue surface. After two-dimensional laser scanning, the images were reconstructed as a function of m/z from a few hundred obtained spectra. In the obtained images, the existence of molecules was represented by a pseudo-color corresponding to the signal intensity. In a feasibility study, we picked up a localized signal, m/z 725 in a cancerous area. The MS/MS result suggested that m/z 725 was sphingomyelin(16:0)+Na. Thus, we successfully show the feasibility of MALDI imaging as a tool for the analysis of pathological specimens.
Keywords: Imaging mass spectrometry; Colon cancer; Phospholipids; Sphingomyelin; Aging; Molecular imaging;

Here we report a simple, sensitive, and accurate method for detecting urinary sulfated tauro- and glyco-bile acids that uses electrospray ionization mass spectrometry. The sulfated tauro- and glycodihydroxycholic acids mainly generated [M  − 2H]2− negative ions at m/z 288.6 and m/z 263.6, respectively. These doubly charged ions appeared primarily in samples prepared from the urine of patients with cholestasis and were detected quantitatively. Cholestatic jaundice is the primary clinical sign of biliary atresia. The measurement of doubly charged negative ions, especially of sulfated taurodihydroxycholic acid (principally taurochenodeoxycholate-3-sulfate), is a useful screening modality for biliary atresia in neonates.
Keywords: Sulfated bile acids; ESIMS; Cholestasis; Biliary atresia;

Changes in urinary level and configuration ratio of d-lactic acid in patients with short bowel syndrome by Yoshito Inoue; Toshihiro Shinka; Morimasa Ohse; Miyuki Kohno; Kunio Konuma; Hiromichi Ikawa; Tomiko Kuhara (109-114).
The present study showed that the d-lactic acid configuration ratio in the urine rose earlier than that in blood or the urinary or blood d-lactic acid levels upon disease onset, and that the d-lactic acid measurement in urine is more sensitive and useful than that in blood. As this result, a prediction of a d-lactic acidosis may be possible. To simplify the procedure for detecting d-lactic acid, we first showed a correlation between the d-lactic acid configuration ratio in urine and blood, indicating urine could be used. To separate the optical isomers of lactic acid, we simplified our previous procedure. For chiral recognition, we chose O-acetyl-(−)-menthylation and analyzed the samples under GC/MS by capillary gas chromatography on a DB-5MS column. This procedure is less sensitive than the former method, but it is faster and simpler, requiring only one derivatization step. This method may be useful for predicting d-lactic acidosis in patients with short bowel syndrome.
Keywords: Short bowel syndrome; Urinary d-lactic acid; Separation of enantiomers; O-acetylated-(−)-menthyl ester;

Metamphetamine (MA) is one of the most frequently encountered abused drugs in Japan and the Triage™ immunoassay kit is often used to screen for this drug. However, immunoassay screening also gives positive results with other structurally related compounds, such as 3,4-methylenedioxymethamphetamine (MDMA), 3,4-methylenedioxyamphetamine (MDA), p-methoxyamphetamine (PMA), an ephedrine metabolite and β-phenethylamine (PEA). Therefore, it is important to develop a simple and reliable method which can determine these drugs simultaneously. This paper describes a simple method for simultaneous identification and quantification of 13 amphetamine related drugs in human whole blood. The method consists of a solid phase extraction using a new polar-enhanced Focus™ column followed by acetylation and gas chromatography–mass spectrometry in the scan mode. Tetradeuterated MA and trideuterated methylephedrine (ME) were used as internal standards. As the Focus™ column required only simple extraction steps and provided a clean extract, identification of each drug was feasible even at low concentrations. The calibration curves were linear over the concentration range from 50 to 5000 ng/ml for all drugs with correlation coefficients that exceeded 0.99. The lower limits of detection of the drugs were 5–50 ng/ml. The absolute recoveries for the drugs were 65–95% and 64–89% at concentrations of 100 and 1000 ng/ml, respectively. Accuracy and precision data were satisfactory when using 2 internal standards. The applicability of the assay was proven by the analysis of blood samples in forensic cases. This method should be most useful for confirmation of positive immunoassay results for amphetamines and related drugs.
Keywords: Amphetamines; Ephedrine; Enhanced polymer column; Gas chromatography-mass spectrometry;