A Cytotoxic Antibody Recognizing Lacto-N-fucopentaose I (LNFP I) on Human Induced Pluripotent Stem (hiPS) Cells.

We have generated a mouse monoclonal antibody (R-17F, IgG1 subtype) specific to human induced pluripotent stem (hiPS)/embryonic stem (ES) cells by using a hiPS cell line as an antigen. Triple-color confocal immunostaining images of hiPS cells with R-17F indicated that the R-17F epitope was expressed exclusively and intensively on the cell membranes of hiPS cells and co-localized partially with those of SSEA-4 and SSEA-3. Lines of evidence suggested that the predominant part of the R-17F epitope was a glycolipid. Upon TLC blot of total lipid extracts from hiPS cells with R-17F, one major R-17F-positive band was observed at a slow migration position close to that of anti-blood group H1(O) antigen. MALDI-TOF-MS and MS(n) analyses of the purified antigen indicated that the presumptive structure of the R-17F antigen was Fuc-Hex-HexNAc-Hex-Hex-Cer. Glycan microarray analysis involving 13 different synthetic oligosaccharides indicated that R-17F bound selectively to LNFP I (Fucα1-2Galß1-3GlcNAcß1-3Galß1-4Glc). A critical role of the terminal Fucα1-2 residue was confirmed by the selective disappearance of R-17F binding to the purified antigen upon α1-2 fucosidase digestion. Most interestingly, R-17F, when added to hiPS/ES cell suspensions, exhibited potent dose-dependent cytotoxicity. The cytotoxic effect was augmented markedly upon the addition of the secondary antibody (goat anti-mouse IgG1 antibody). R-17F may be beneficial for safer regenerative medicine by eliminating residual undifferentiated hiPS cells in hiPS-derived regenerative tissues, which are considered to be a strong risk factor for carcinogenesis.

Differentiation and semiquantitative analysis of an isoaspartic acid in human alpha-Crystallin by postsource decay in a curved field reflectron.

Alpha-Crystallin, which forms a huge multimeric complex that is essential for maintaining eye lens transparency, is one of the major proteins in the lens. The protein, which exists as isoforms alphaA and alphaB, functions as a molecular chaperone to restore the original conformations of distorted constituent proteins in the lens. This function is important to maintain the transparency of the lens, because there is no protein turnover in the lens. Abnormal aggregation of constituent proteins in the lens has been reported in cataract patients, and deamidation of Asn as well as racemization and isomerization of Asp have been found in the alpha-Crystallin of these patients. While the establishment of a quick and facile analytical method is eagerly anticipated to investigate the relevance of the isomerization to pathological states such as cataracts, differentiating the isomerization states is still not performed routinely. Here, we report the differentiation and semiquantitative analysis of an isoaspartic acid (betaAsp) in human alpha-Crystallin using postsource decay on a MALDI-TOF mass spectrometer incorporating a curved field reflectron. Our reproducible results of analyzing synthetic and tryptic peptides containing betaAsp corroborated results obtained using a previously reported diagnostic ion, y(l-n+1) - 46, for the differentiation of betaAsp. The relative content of betaAsp in the peptide was successfully estimated from a unique ratio, y(l-n):y(l-n+1), corresponding to cleavages at the C- and N-termini, respectively, of the isomeric residues. The betaAsp content was consistent with measurements obtained independently by reversed phase HPLC analysis. Experiments in which neighboring amino acids adjacent to betaAsp/Asp were substituted revealed that the ratio between y(l-n) and y(l-n+1) reflected the isomerization status, while the diagnostic ion was observed only in the peptides that included an arginine residue at their C-terminus. Postsource decay experiments utilizing both the diagnostic ion and the characteristic fragment pattern could be applied to various kinds of peptides containing betaAsp.

Analysis of High-Molecular-Weight Polyrotaxanes by MALDI-TOF-MS Using 3-Aminoquinoline-Based Ionic Liquid Matrix.

Polyrotaxane (PR) is a necklace-like supramolecule composed of cyclic components, such as cyclodextrin (CD), and a threading polymer capped with bulky end groups. PR exhibits peculiar mechanical properties attributed to the intermolecular cross-links with CD. Various CD molecules threaded on a linear PEG chain are often modified with chemical groups to add specific physicochemical properties. In general, the stoichiometry between CD and the PEG chain is a significant parameter that defines the unique physical properties of CD-based polyrotaxane (CD-PR). To date, mass spectrometry (MS) has been applied to investigate the molecular distribution of CD-PR, modifications of CD, and the threaded ratio of CD. However, only molecular weights (MWs) up to several 10s of kDa can be subjected to such analysis, whereas the MW of CD-PR used as industrial materials is much greater. Herein, we applied two ionic liquid matrices composed of 3-aminoquinoline and a high mass detector to analyze PRs using MALDI-TOF-MS. High to very high MW PRs in the range of 90-700 kDa were successfully analyzed using this method. The threaded ratio of CD was estimated from a single MW of CD, PEG, and PR. The ratios obtained were consistent with that obtained using 1H NMR. Furthermore, a single-stranded form of PR in γ-cyclodextrin threaded PR (γCD-PR) was clearly distinguished from a double-stranded form, which is only possible in γCD -PR because of its large host cavity.

Hierarchical Cluster and Region of Interest Analyses Based on Mass Spectrometry Imaging of Human Brain Tumours.

Imaging mass spectrometry (IMS) has been rarely used to examine specimens of human brain tumours. In the current study, high quality brain tumour samples were selected by tissue observation. Further, IMS analysis was combined with a new hierarchical cluster analysis (IMS-HCA) and region of interest analysis (IMS-ROI). IMS-HCA was successful in creating groups consisting of similar signal distribution images of glial fibrillary acidic protein (GFAP) and related multiple proteins in primary brain tumours. This clustering data suggested the relation of GFAP and these identified proteins in the brain tumorigenesis. Also, high levels of histone proteins, haemoglobin subunit α, tubulins, and GFAP were identified in a metastatic brain tumour using IMS-ROI. Our results show that IMS-HCA and IMS-ROI are promising techniques for identifying biomarkers using brain tumour samples.

Improvement of Sensitivity and Reproducibility for Imaging of Endogenous Metabolites by Matrix-Assisted Laser Desorption/Ionization-Mass Spectrometry.

Matrix-assisted laser desorption/ionization (MALDI)-mass spectrometry imaging (MSI) is a powerful technique to visualize the distributions of biomolecules without any labeling. In MALDI-MSI experiments, the choice of matrix deposition method is important for acquiring favorable MSI data with high sensitivity and high reproducibility. Generally, manual or automated spray-coating and automated sublimation methods are used, but these methods have some drawbacks with respect to detection sensitivity, spatial resolution, and data reproducibility. Herein, we present an optimized matrix deposition method of sublimation coupled with recrystallization using 9-aminoacridine (9-AA) as a matrix capable of ionizing endogenous metabolites. The matrix recrystallization process after sublimation was optimized for the solvent concentration and reaction temperature for matrix-metabolite co-crystallization. This optimized method showed excellent reproducibility and spatial resolution compared to the automatic spray-coating method. Furthermore, the recrystallization step after sublimation remarkably improved the detectability of metabolites, including amino acids, nucleotide derivatives, and lipids, compared with the conventional sublimation method. To date, there have been no other reports of 9-AA-based sublimation combined with recrystallization. The present method provides an easy, sensitive, and reproducible matrix deposition method for MALDI-MSI of endogenous metabolites. Graphical Abstract.

Metalation states versus enzyme activities of Cu, Zn-superoxide dismutase probed by electrospray ionization mass spectrometry.

Cu, Zn-superoxide dismutase (SOD-1), an enzyme that catalyzes the disproportionation reaction of superoxide to produce oxygen and hydrogen peroxide, thereby protecting cells from oxidative stress, is a homodimer that coordinates one copper and one zinc ion per monomer. Cu (2+) and Zn (2+) ions play important roles in enzyme activity and structural stability, respectively. In addition, dimer formation is also essential for fulfilling the function of SOD-1. We here report on the reconstitution and enzyme activities of several metalation states of SOD-1 (Cu 4-, Cu 3Zn-, and Cu 2Zn 2-homodimers). Each metalation state of the reconstituted SOD-1 could be unambiguously differentiated by electrospray ionization mass spectrometry, the metal ions of which had been completely replaced by 99 atom % (63)Cu and (68)Zn stable isotopes. It was found that (1) the Cu 4-dimer possessed 84% of the activity of the native enzyme, (2) the Cu-site resisted being coordinated with Zn (2+) ions while the Zn-site could be bound with Cu (2+) ions, and (3) the simultaneous addition of the Cu (2+) and Zn (2+) ions to generate a fully metalated form produced the multiply metalated SOD-1 (Cu 4-, Cu 3Zn-, and Cu 2Zn 2-dimers), which were clearly distinguishable from one another by the use of the stable isotopes, while the sequential addition of Zn (2+) followed by the Cu (2+) ion predominantly produced a Cu 2Zn 2-dimer comparable to the native enzyme.

Region of Interest analysis using mass spectrometry imaging of mitochondrial and sarcomeric proteins in acute cardiac infarction tissue.

Matrix-assisted laser desorption ionization image mass spectrometry (MALDI-IMS) has been developed for the identification of peptides in various tissues. The MALDI-IMS signal distribution patterns and quantification of the signal intensities of the regions of interest (ROI) with healthy regions were compared for identification of the disease specific biomarkers. We performed a new ROI analysis using the conventional t-test and data number independent Cohen's d-value analysis. Using these techniques, we analysed heart tissues after acute myocardial infarction (AMI). As a result, IMS signals of mitochondrial adenosine triphosphate synthase alpha subunit (ATP5A), myosin-6/7(MYH6/7), aortic actin, and the myosin light chain 3 (MYL3) were identified in the infarcted region. In particular, the signals of MYH7 are significantly greater in the infarcted region using ROI analysis. ROI analysis using MALDI-IMS may be a promising technique for the identification of biomarkers for pathological studies that involve the comparison of diseased and control areas.

Complete de novo sequencing of antimicrobial peptides in the venom of the scorpion Isometrus maculatus.

Scorpion venom contains antimicrobial peptides (AMPs) in addition to neurotoxic peptides. Recent extensive transcriptomic analysis of venom glands of various scorpion species dramatically increased the number of identified AMPs. However, identification of peptides from genetic information requires reference sequences of similar peptides, and moreover, it is often difficult to predict post-translational modifications. In this study, we searched for unknown AMPs contained in the Isometrus maculatus scorpion venom based on the structural features, such as the hydrophobic nature and the lack of disulfide bonds, which are commonly observed in the majority of scorpion AMPs. Their primary structures were determined only by a de novo sequencing method using two types of MS instruments, which induce peptide fragmentation in a different fashion. Chemical derivatization techniques were also used to facilitate Leu/Ile discrimination. As a result, total 15 AMP candidates were discovered from the I. maculatus venom. Among them, three peptides were identified as AMPs by evaluating their biological activity. Other 12 candidate peptides were structurally related to the identified AMPs, possibly generated by enzymatic cleavage of the mature peptides in the venom.

Three-dimensional fractal analysis of 99mTc-MAA SPECT images in chronic thromboembolic pulmonary hypertension for evaluation of response to balloon pulmonary angioplasty: association with pulmonary arterial pressure.

OBJECTIVE: Balloon pulmonary angioplasty (BPA) is used for inoperable chronic thromboembolic pulmonary hypertension (CTEPH), but its effect cannot be evaluated noninvasively. We devised a noninvasive quantitative index of response to BPA using three-dimensional fractal analysis (3D-FA) of technetium-99m-macroaggregated albumin (Tc-MAA) single-photon emission computed tomography (SPECT). PATIENTS AND METHODS: Forty CTEPH patients who underwent pulmonary perfusion scintigraphy and mean pulmonary arterial pressure (mPAP) measurement by right heart catheterization before and after BPA were studied. The total uptake volume (TUV) in bilateral lungs was determined from maximum intensity projection Tc-MAA SPECT images. Fractal dimension was assessed by 3D-FA. Parameters were compared before and after BPA, and between patients with post-BPA mPAP more than 30 mmHg and less than or equal to 30 mmHg. Receiver operating characteristic analysis was carried out. RESULTS: BPA significantly improved TUV (595±204-885±214 ml, P<0.001) and reduced the laterality of uptake (238±147-135±131 ml, P<0.001). Patients with poor therapeutic response (post-BPA mPAP≥30 mmHg, n=16) showed a significantly smaller TUV increase (P=0.044) and a significantly greater post-BPA fractal dimension (P<0.001) than the low-mPAP group. Fractal dimension correlated with mPAP values before and after BPA (P=0.013 and 0.001, respectively). A post-BPA fractal dimension threshold of 2.4 distinguished between BPA success and failure with 75% sensitivity, 79% specificity, 78% accuracy, and area under the curve of 0.85. CONCLUSION: 3D-FA using Tc-MAA SPECT pulmonary perfusion scintigraphy enables a noninvasive evaluation of the response of CTEPH patients to BPA.

Convenient structural analysis of glycosphingolipids using MALDI-QIT-TOF mass spectrometry with increased laser power and cooling gas flow.

Matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight mass spectrometry (MALDI-QIT-TOF MS) was applied to the structural characterization of neutral glycosphingolipids. Lithium adduct ions of glycosphingolipids were analyzed using MALDI-QIT-TOF MS under strong conditions of increased laser power and cooling gas flow. The relative intensities of fragment ions were increased under the strong conditions, and the resulting spectra revealed the presence of oligosaccharide ions fragmented from the glycosphingolipids. Consequently, the oligosaccharide sequences of the glycosphingolipids were readily obtained. To obtain more detailed structural information, MS/MS (MS2) and MS/MS/MS (MS3) analyses were performed with selection of the lactosylceramide and ceramide ions, respectively. The resulting data were sufficient to determine the structures of both the oligosaccharide and the ceramide moiety of each glycosphingolipid. The fragmentation patterns of MS2 and MS3 for Forssman glycolipid under the strong conditions were comparable to those of MS3 and MS4 obtained under standard conditions, respectively. Thus, MALDI-QIT-TOF MS with increased laser power and cooling gas flow is a convenient method for glycosphingolipid analysis.

Analysis on heat stress-induced hyperphosphorylation of stathmin at serine 37 in Jurkat cells by means of two-dimensional gel electrophoresis and tandem mass spectrometry.

Two-dimensional gel electrophoresis (2-DE) and tandem mass spectrometry were successfully used for determination of a phosphorylation site of stathmin induced by heat stress to Jurkat cells of a human T lymphoblastic cell line. The cells were incubated for 30 min at 41 degrees C up to 45 degrees C in a serum free 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffered culture medium. The intracellular soluble proteins were separated by 2-DE, and some of the proteins increased their abundance by heat stress. Those proteins were identified to be calmodulin, protein kinase C substrate, thymosin beta-4 and F-actin capping protein beta-subunit by peptide mass fingerprinting (PMF) with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). On the contrary, protein phosphatase 2C gamma-isoform, nucleophosmin, translationally controlled tumor protein, Rho GDP-dissociation inhibitor-1, eukaryotic translation initiation factors 5A and 3A subunit 2, ubiquitin-like protein SMT 3B and chloride intracellular channel protein-1 were decreased their abundance. A protein spot of M(r) 18,000 and pI 5.9 was markedly increased at temperatures higher than 43 degrees C at which the cells were led to apoptosis. The spot was identified to be stathmin of a signal relay protein which has a function of sequestering microtubule. MALDI-quadrupole ion trap (QIT)-TOF-MS/MS and immunoblotting with a monoclonal antibody specific for a phosphorylation site of stathmin showed that the spot was a phosphorylated stathmin at serine 37 (Ser 37). The phosphorylation was suppressed by treatment of cells with olomoucine of an inhibitor specific for cyclin dependent kinase (Cdk-1). These results strongly suggest that heat stress activates Cdk-1 which phosphorylates Ser 37 on the stathmin molecule. The phosphorylation may cause the functional loss of stathmin for dynamic microtubule assembly and leads Jurkat cells to cell cycle arrest and apoptosis.

Investigation of 5-(3-Trifluoromethylbenzylidene)thiazolidine-2,4-dione as a Matrix for Analyses of Biogenic Monoamine Transmitters Using MALDI-MS.

In order to discover new matrices suitable for the analyses of low molecular-weight compounds using positive-ion mode matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry (MS), 5-(3-trifluoromethylbenzylidene)thiazolidine-2,4-dione (3-CF3-BTD) was synthesized, and its effectiveness was compared with that when commercially available α-cyano-4-hydroxycinnamic acid was used. 3-CF3-BTD was sufficiently sensitive to analyze neurotransmitters, i.e., dopamine, serotonin, histamine, and epinephrine, in amounts of several picomoles. Similar to vacuum MALDI experiments, atmospheric-pressure MALDI-MS measurements using 3-CF3-BTD as a matrix also detected dopamine.

Improvement of automated right ventricular segmentation using dual-bolus contrast media injection with 256-slice coronary CT angiography.

RATIONALE AND OBJECTIVES: To investigate the effect of dual-bolus contrast media injection (dual-CM) on the accuracy of automated right ventricular (RV) segmentation on coronary computed tomography angiography (CCTA). MATERIALS AND METHODS: A total of 104 patients with suspected and known coronary artery disease underwent 256-slice CCTA with retrospective electrocardiographic (ECG) gating. The patients were divided into 51 patients who underwent single-bolus CM injection (single-CM) and 53 patients who underwent dual-CM. The dual-CM method consisted of an initial bolus of CM followed by an injection of dilute CM with saline (CM:saline, 1:9). Three-dimensional CCTA images were automatically segmented into the RV, left ventricle (LV), and myocardium using commercially available software (Comprehensive Cardiac Analysis; Philips Medical Systems, Cleveland, OH). Prevalence and locations of segmentation errors were compared between single-CM and dual-CM. Segmentation errors were defined as segment deviation of >1 cm from the actual ventricular margin. RESULTS: Prevalence of segmentation errors was significantly lower with dual-CM than with single-CM in the diastolic phase (4/41 vs. 20/41, respectively; P < .01), and there was no difference between the two methods in the systolic phase (2/12 vs. 2/10, respectively). With dual-CM and single-CM, the locations of segmentation errors were mostly the RV wall (4/53 and 18/51, respectively) and secondly the LV wall (2/53 and 9/51, respectively). CONCLUSIONS: Dual-CM improved the accuracy of automated ventricular segmentation using diastolic data from 256-slice CCTA.

Application of high-resolution ESI and MALDI mass spectrometry to metabolite profiling of small interfering RNA duplex.

We investigated the application of a high-resolution Orbitrap mass spectrometer equipped with an electrospray ionization (ESI) source and a matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometer to the metabolite profiling of a model small interfering RNA (siRNA) duplex TSR#34 and compared their functions and capabilities. TSR#34 duplex was incubated in human serum in vitro, and the duplex and its metabolites were then purified by ion exchange chromatography in order to remove the biological matrices. The fraction containing the siRNA duplex and its metabolites was collected and desalted and then subjected to high-performance liquid chromatography (HPLC) equipped with a reversed phase column. The siRNA and its metabolites were separated into single strands by elevated chromatographic temperature and analyzed using the ESI-Orbitrap or the MALDI-TOF mass spectrometer. Using this method, the 5' and/or 3' truncated metabolites of each strand were detected in the human serum samples. The ESI-Orbitrap mass spectrometer enabled differentiation between two possible RNA-based sequences, a monoisotopic molecular mass difference which was less than 2 Da, with an intrinsic mass resolving power. In-source decay (ISD) analysis using a MALDI-TOF mass spectrometer allowed the sequencing of the RNA metabolite with characteristic fragment ions, using 2,4-dihydroxyacetophenone (2,4-DHAP) as a matrix. The ESI-Orbitrap mass spectrometer provided the highest mass accuracy and the benefit of on-line coupling with HPLC for metabolite profiling. Meanwhile, the MALDI-TOF mass spectrometer, in combination with 2,4-DHAP, has the potential for the sequencing of RNA by ISD analysis. The combined use of these methods will be beneficial to characterize the metabolites of therapeutic siRNA compounds.

Ionic liquid matrixes optimized for MALDI-MS of sulfated/sialylated/neutral oligosaccharides and glycopeptides.

1,1,3,3-tetramethylguanidium (TMG) salt of alpha-cyano-4-hydroxycinnamic acid (CHCA) (G(2)CHCA) was reported by Tatiana et al. as a useful ionic liquid matrix (ILM) for sulfated oligosaccharides to suppress the loss of sulfate groups. However, the report mainly referred to positive ion spectra only and amounts of 10 pmol or more of the analyte were used. Herein, we demonstrated highly sensitive detection of sulfated/sialylated/neutral oligosaccharides and preferential ionization of glycopeptides by optimizing a newly synthesized ILM: TMG salt of p-coumaric acid (G(3)CA) and the existing G(2)CHCA in both positive and negative ion extraction modes. Sulfated oligosaccharides were detected with high sensitivity (e.g., 1 fmol) in both ion extraction modes, and the dissociation of sulfate groups was suppressed especially using G(3)CA. Sialylated and neutral oligosaccharides were also detected with high sensitivity (e.g., 1 fmol) with positive ion extraction while the dissociation of sialic acids was suppressed especially using G(3)CA. Additionally, glycopeptide ions were detected preferentially using the ILMs among the digest of a glycoprotein, ribonuclease B, in both ion extraction modes but particularly in the negative ion mode. As a result, the use of optimized ILMs provides an effective method for carbohydrate analysis due to the highly sensitive soft-ionization achieved in both ion extraction modes as well as the homogeneity of analyte-matrix mixtures.

Rodent alpha-chymases are elastase-like proteases.

Although the alpha-chymases of primates and dogs are known as chymotrypsin-like proteases, the enzymatic properties of rodent alpha-chymases (rat mast cell protease 5/rMCP-5 and mouse mast cell protease 5/mMCP-5) have not been fully understood. We report that recombinant rMCP-5 and mMCP-5 are elastase-like proteases, not chymotrypsin-like proteases. An enzyme assay using chromogenic peptidyl substrates showed that mast cell protease-5s (MCP-5s) have a clear preference for small aliphatic amino acids (e.g. alanine, isoleucine, valine) in the P1 site of substrates. We used site-directed mutagenesis and computer modeling approaches to define the determinant residue for the substrate specificity of mMCP-5, and found that the mutant possessing a Gly substitution of the Val at position 216 (V216G) lost elastase-like activity but acquired chymase activity, suggesting that the Val216 dominantly restricts the substrate specificity of mMCP-5. Structural models of mMCP-5 and the V216G mutant based on the crystal structures of serine proteases (rMCP-2, human cathepsin G, and human chymase) revealed the active site differences that can account for the marked differences in substrate specificity of the two enzymes between elastase and chymase. These findings suggest that rodent alpha-chymases have unique biological activity different from the chymases of other species.