Direct detection of dithiocarbamate fungicides by SALDI/MS using porous TiC ceramic powder as a substrate.

Dithiocarbamate fungicides (DTCs) have been widely used all over the world. Some of them show toxicities, such as growth toxicity and teratogenicity. Therefore, the analysis of DTCs in environments or crops is very significant. However, their direct and individual analysis was difficult, because most of them are metal complex compounds and have macromolecular properties and a low solubility in water or organic solvents. In the conventional analytical methods for DTCs, the total amount of DTCs was obtained by the quantification of the derivatives of the ligand or by measuring the carbon disulfide formed by the decomposition of the fungicides. Surface assisted laser desorption (SALDI)/MS can detect various compounds, such as metal complexes and macromolecules, present in a nanostructured substrate. The porous titanium carbide (TiC) ceramic powder shows adsorptive properties to various substances and can be used as a substrate for SALDI/MS. In this study, a method for the individual and direct detection of dithiocarbamate pesticides by SALDI/MS using porous TiC ceramics as a substrate has been developed. The dithiocarbamate fungicide was mixed with the porous TiC powder in a mortar, and the mixture was analyzed by SALDI/MS. The deprotonated ion of the ethylene-bis-dithiocarbamate complex, mancozeb or zineb, was detected in the negative ion mode. For the dimethyldithiocarbamate complexes, ferbam and ziram, the ion of the eliminated dithiocarbamate ligand was detected in the positive ion mode. Calibration curves by the present method for Manzeb showed good linearity by using an internal standard material. Based on these results, we concluded that this method is useful for the analysis of DTCs.

Imaging Analysis of Phosphatidylcholines and Diacylglycerols Using Surface-Assisted Laser Desorption/Ionization Mass Spectrometry with Metal Film Formed by Mist Chemical Vapor Deposition.

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) has been widely used for analyses of biomolecules and industrial materials. Surface-assisted laser desorption/ionization (SALDI) is studied to complement the ionization ability for the MALDI/MS. In this study, lab-made mist chemical vapor deposition (mist CVD) system was used to produce metal films as ionization assistance materials for SALDI/MS. The system could give Ag film from inexpensive silver trifluoroacetate solution rapidly and simply under atmospheric pressure. Phosphatidylcholines could be detected high sensitively and diacylglycerols (DAGs) could not be detected in MALDI/MS. In the SALDI/MS and the MS imaging with Ag film by mist CVD, both the phosphatidylcholines and the DAGs could be detected and the localized images. In the Ag film-SALDI/MS of lipids, not only Ag-adducted ions but also Na- and K-adducted ions were detected. The Ag film formed by the mist CVD to act as an ionization-assistance material and a cationization agent in SALDI would be useful in MS imaging of biological tissue sections.

Relationship between the Rod complex and peptidoglycan structure in Escherichia coli.

Peptidoglycan for elongation in Escherichia coli is synthesized by the Rod complex, which includes RodZ. Although various mutant strains of the Rod complex have been isolated, the relationship between the activity of the Rod complex and the overall physical and chemical structures of the peptidoglycan have not been reported. We constructed a RodZ mutant, termed RMR, and analyzed the growth rate, morphology, and other characteristics of cells producing the Rod complexes containing RMR. The growth and morphology of RMR cells were abnormal, and we isolated suppressor mutants from RMR cells. Most of the suppressor mutations were found in components of the Rod complex, suggesting that these suppressor mutations increase the integrity and/or the activity of the Rod complex. We purified peptidoglycan from wild-type, RMR, and suppressor mutant cells and observed their structures in detail. We found that the peptidoglycan purified from RMR cells had many large holes and different compositions of muropeptides from those of WT cells. The Rod complex may be a determinant not only for the whole shape of peptidoglycan but also for its highly dense structure to support the mechanical strength of the cell wall.

Synthesis and Conformational Behaviors of Unnatural Peptides Alternating Chiral and Achiral α,α-Disubstituted α-Amino Acid Units.

The development of peptidomimetics to modulate the conformational profile of peptides has been extensively studied in the fields of biological and medicinal chemistry. However, large-scale synthesis of peptidomimetics with both an ordered sequence and a controlled secondary structure is highly challenging. In this paper, the framework of peptidomimetics has been designed to be alternating an achiral α,α-disubstituted α-amino acid unit and a chiral α-methylphenylalanine unit. The polymers are synthesized via invented Ugi reaction-based polycondensation technique. The chiral higher-order structures of the alternating peptides are evaluated mainly through circular dichroism (CD) spectroscopy. The UV-Vis and CD spectra of the polymers in three solvents are systematically measured at various temperatures. The anisotropic factors of CD (gCD ) values are calculated to know the chiroptical response. The results indicate the characteristic conformational behaviors. In a polar solvent, the hydrogen bonds between the N-H group of MePhe unit and the C=O of α,α-diphenylglycine unit outweigh the intraresidue hydrogen bonds in α,α-diphenylglycine unit, leading to the formation of a prevailing preferred-handed 310 -helical conformation. On the other hand, in a less polar solvent, the intrachain hydrogen bonds switch to intraresidue hydrogen bonds in α,α-diphenylglycine unit, which make the polymer adopting a prevailing extended planar C5 -conformation.

Comprehensive Steroid Assay with Non-Targeted Analysis Using Liquid Chromatography Ion Mobility Mass Spectrometry.

Aldosterone-producing adenomas (APAs) have different steroid profiles in serum, depending on the causative genetic mutation. Ion mobility is a separation technique for gas-phase ions based on their m/z values, shapes, and sizes. Human serum (100 µL) was purified by liquid-liquid extraction using tert-butyl methyl ether/ethyl acetate at 1/1 (v/v) and mixed with deuterium-labeled steroids as the internal standard. The separated supernatant was dried, re-dissolved in water containing 20% methanol, and injected into a liquid chromatography-ion mobility-mass spectrometer (LC/IM/MS). We established a highly sensitive assay system by separating 20 steroids based on their retention time, m/z value, and drift time. Twenty steroids were measured in the serum of patients with primary aldosteronism, essential hypertension, and healthy subjects and were clearly classified using principal component analysis. This method was also able to detect phosphatidylcholine and phosphatidylethanolamine, which were not targeted. LC/IM/MS has a high selectivity for known compounds and has the potential to provide information on unknown compounds. This analytical method has the potential to elucidate the pathogenesis of APA and identify unknown steroids that could serve as biomarkers for APA with different genetic mutations.

Rapidity and Precision of Steroid Hormone Measurement.

Steroids are present in all animals and plants, from mammals to prokaryotes. In the medical field, steroids are commonly classified as glucocorticoids, mineralocorticoids, and gonadal steroid hormones. Monitoring of hormones is useful in clinical and research fields for the assessment of physiological changes associated with aging, disease risk, and the diagnostic and therapeutic effects of various diseases. Since the discovery and isolation of steroid hormones, measurement methods for steroid hormones in biological samples have advanced substantially. Although immunoassays (IAs) are widely used in daily practice, mass spectrometry (MS)-based methods have been reported to be more specific. Steroid hormone measurement based on MS is desirable in clinical practice; however, there are several drawbacks, including the purchase and maintenance costs of the MS instrument and the need for specialized training of technicians. In this review, we discuss IA- and MS-based methods currently in use and briefly present the history of steroid hormone measurement. In addition, we describe recent advances in IA- and MS-based methods and future applications and considerations.

Highly Durable Lubricity of Photo-Cross-Linked Zwitterionic Polymer Brushes Supported by Poly(ether ether ketone) Substrate.

For improving lubricity, the formation of zwitterionic polymer brushes on material surfaces is one of the most promising approaches. In this study, the photoreactive zwitterionic monomer 2-[2-(methacryloyloxy)ethyldimethylanmmonium] ethyl benzophenoxy phosphate (MBPP) was synthesized to improve the stability of zwitterionic polymer brushes. Although MBPP contains a benzophenone moiety in this molecule, it is water-soluble because of the zwitterionic linker. As a substrate, poly(ether ether ketone) (PEEK) was selected because it has recently been used to replace metals in orthopedic implants. Furthermore, PEEK is photosensitive, and UV graft polymerization of (meth)acrylic monomers on the surface can be performed without using any photoinitiators. Aqueous solutions containing various molar ratios of 2-(methacryloyloxy)ethyl phosphorylcholine (MPC) and MBPP were prepared, and the PEEK specimens were immersed in these solutions. UV light was used to irradiate the solutions for 180 min, and the formation of grafting layers of zwitterionic polymers on PEEK specimens was confirmed using contact angle measurement and X-ray photoelectron spectroscopy. The surface friction of PEEK was effectively reduced via the photopolymerization of zwitterionic monomers. However, the surface lubricity of poly(MPC) (PMPC)-grafted surface deteriorated during continuous friction because of the removal of PMPC from the surface. Nevertheless, the stability of polymer brushes was effectively improved by adding only 0.5-0.75 mol % of MBPP in the monomer solution. Moreover, the reduction of wear on the surface was determined using confocal laser microscopy. The excellent lubrication phenomenon was attributed to preserving the hydration state of grafted polymers under compressive stress. Moreover, bacterial adhesion on substrates was tested and observed on a neat PEEK and scratched regions of uncross-linked PMPC-grafted PEEK. Note that bacterial adhesion was completely suppressed on the surface of PEEK modified with cross-linked PMPC brushes with MBPP. Thus, we conclude that the surface modification of PEEK with MPC and MBPP can provide ideal surface properties for orthopedic devices.

Novel approach to enhance sensitivity in surface-assisted laser desorption/ionization mass spectrometry imaging using deposited organic-inorganic hybrid matrices.

Sample pretreatment is key to obtaining good data in matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI). Although sublimation is one of the best methods for obtaining homogenously fine organic matrix crystals, its sensitivity can be low due to the lack of a solvent extraction effect. We investigated the effect of incorporating a thin film of metal formed by zirconium (Zr) sputtering into the sublimation process for MALDI matrix deposition for improving the detection sensitivity in mouse liver tissue sections treated with olanzapine. The matrix-enhanced surface-assisted laser desorption/ionization (ME-SALDI) method, where a matrix was formed by sputtering Zr to form a thin nanoparticle layer before depositing MALDI organic matrix comprising α-cyano-4-hydroxycinnamic acid (CHCA) by sublimation, resulted in a significant improvement in sensitivity, with the ion intensity of olanzapine being about 1800 times that observed using the MALDI method, comprising CHCA sublimation alone. When Zr sputtering was performed after CHCA deposition, however, no such enhancement in sensitivity was observed. The enhanced sensitivity due to Zr sputtering was also observed when the CHCA solution was applied by spraying, being about twice as high as that observed by CHCA spraying alone. In addition, the detection sensitivity of these various pretreatment methods was similar for endogenous glutathione. Given that sample preparation using the ME-SALDI-MSI method, which combines Zr sputtering with the sublimation method for depositing an organic matrix, does not involve a solvent, delocalization problems such as migration of analytes observed after matrix spraying and washing with aqueous solutions as sample pretreatment are not expected. Therefore, ME-Zr-SALDI-MSI is a novel sample pretreatment method that can improve the sensitivity of analytes while maintaining high spatial resolution in MALDI-MSI.

Photochemical Reactions of Aminonaphthols Caused by Laser Desorption/Ionization.

The formation of monomeric and dimeric ions of seven different aminonaphthols (ANLs) has been studied by using laser desorption/ionization (LDI) with a nitrogen laser. The positive-ion data of all the ANLs merely showed molecular ion M(·+) without protonated molecule [M+H](+), while 1-amino-2-naphthol (1,2-ANL) and 2-amino-1-naphthol (2,1-ANL) showed an intense dimeric ion [2 M-2H2O+H](+). The negative-ion data showed deprotonated molecule [M-H](-) in common, while the spectra of 1,2-ANL, 2,1-ANL and 8-amino-2-naphthol (8,2-ANL) accompanied an intense peak corresponding to negative molecular ion M(·-) and the 8,2-ANL and 4-amino-1-naphthol (4,1-ANL) accompanied dehydrogenated anion [M-2H](·-). The formation of monomeric ions was discussed from the standpoints of thermochemical properties such as ionization energy, gas-phase acidity, electron affinity, and bond dissociation energy. The formation of dimeric ions [2 M-2H2O+H](+) observed in the 1,2-ANL and 2,1-ANL could be explained by the radical combination in the amino groups. An isomer 5-amino-1-naphthol (1-ANL) did not give any dimeric ions in the both positive- and negative-ion spectra. The influence of laser fluence upon the appearance of the monomeric ions such as M(·+), [M+H](+), [M-H](-) and [M-2H](·-) of the 5,1-ANL has been examined.

Direct Imaging Mass Spectrometry of Plant Leaves Using Surface-assisted Laser Desorption/Ionization with Sputter-deposited Platinum Film.

Plant leaves administered with systemic insecticides as agricultural chemicals were analyzed using imaging mass spectrometry (IMS). Matrix-assisted laser desorption/ionization (MALDI) is inadequate for the detection of insecticides on leaves because of the charge-up effect that occurs on the non-conductive surface of the leaves. In this study, surface-assisted laser desorption/ionization with a sputter-deposited platinum film (Pt-SALDI) was used for direct analysis of chemicals in plant leaves. Sputter-deposited platinum (Pt) films were prepared on leaves administered with the insecticides. A sputter-deposited Pt film with porous structure was used as the matrix for Pt-SALDI. Acephate and acetamiprid contained in the insecticides on the leaves could be detected using Pt-SALDI-MS, but these chemical components could not be adequately detected using MALDI-MS because of the charge-up effect. Enhancement of ion yields for the insecticides was achieved using Pt-SALDI, accompanied by prevention of the charge-up effect by the conductive Pt film. The movement of systemic insecticides in plants could be observed clearly using Pt-SALDI-IMS. The distribution and movement of components of systemic insecticides on leaves could be analyzed directly using Pt-SALDI-IMS. Additionally, changes in the properties of the chemicals with time, as an indicator of the permeability of the insecticides, could be evaluated.

High-Confidence Sequencing of Phosphopeptides by Electron Transfer Dissociation Mass Spectrometry Using Dinuclear Zinc(II) Complex.

Phosphorylation is the most abundant protein modification, and tandem mass spectrometry (MS2) with electron transfer dissociation (ETD) has proven to be a promising method for phosphoproteomic applications owing to its ability to determine phosphorylation sites on proteins. However, low precursor charge states hinder the ability to obtain useful information through peptide sequencing by ETD, and the presence of acidic phosphate groups contributes to a low charge state of peptide ions. In the present report, we used a dinuclear zinc complex, (Zn2L)3+ (L = alkoxide form of 1,3-bis[bis(pyridin-2-ylmethyl)amino]propan-2-ol) for electrospray ionization (ESI), followed by ETD-MS2 analysis. Since (Zn2L)3+ selectively bound to phosphopeptide with addition of a positive charge per phosphate group, the use of (Zn2L)3+ for ESI improved the ionization yield of phosphopeptides in phosphoprotein digest. Additionally, an increase in the charge state of phosphopeptides were observed by addition of (Zn2L)3+, facilitating phosphopeptide sequencing by ETD-MS2. Since the binding between (Zn2L)3+ and the phosphate group was retained during the ETD process, a comparison between the ETD mass spectra obtained using two dinuclear zinc complex derivatives containing different zinc isotopes, namely (64Zn2L)3+ and (68Zn2L)3+, provided information about the number of phosphate groups in each fragment ion, allowing the phosphorylation site to be unambiguously determined. The details of the fragmentation processes of the (Zn2L)3+-phosphopeptide complex were investigated using a density functional theory calculation. As in the case of protonated peptides, ETD induced peptide backbone dissociation in the (Zn2L)3+-phosphopeptide complex proceeded through an aminoketyl radical intermediate.

Nanoparticle-assisted laser desorption/ionization using sinapic acid-modified iron oxide nanoparticles for mass spectrometry analysis.

Iron oxide-based nanoparticles (NP) were covalently modified with sinapic acid (SA) through a condensation reaction to assist the ionization of both large and small molecules. The morphology of SA-modified NPs (SA-NP) was characterized by transmission electron microscopy (TEM), and the modification of the NP surface with SA was confirmed using ultraviolet (UV) and infrared (IR) spectroscopy. The number of SA molecules was estimated to be 6 per NP. SA-NP-assisted laser desorption/ionization was carried out on small molecules, such as pesticides and plant hormones, and large molecules, such as peptides and proteins. A peptide fragment from degraded proteins was detected more efficiently compared with conventional methods.

Simultaneous detection of phosphatidylcholines and glycerolipids using matrix-enhanced surface-assisted laser desorption/ionization-mass spectrometry with sputter-deposited platinum film.

Matrix-assisted laser desorption/ionisation (MALDI) imaging mass spectrometry (IMS) allows for the simultaneous detection and imaging of several molecules in brain tissue. However, the detection of glycerolipids such as diacylglycerol (DAG) and triacylglycerol (TAG) in brain tissues is hindered in MALDI-IMS because of the ion suppression effect from excessive ion yields of phosphatidylcholine (PC). In this study, we describe an approach that employs a homogeneously deposited metal nanoparticle layer (or film) for the detection of glycerolipids in rat brain tissue sections using IMS. Surface-assisted laser desorption/ionisation IMS with sputter-deposited Pt film (Pt-SALDI-IMS) for lipid analysis was performed as a solvent-free and organic matrix-free method. Pt-SALDI produced a homogenous layer of nanoparticles over the surface of the rat brain tissue section. Highly selective detection of lipids was possible by MALDI-IMS and Pt-SALDI-IMS; MALDI-IMS detected the dominant ion peak of PC in the tissue section, and there were no ion peaks representing glycerolipids such as DAG and TAG. In contrast, Pt-SALDI-IMS allowed the detection of these glycerolipids, but not PC. Therefore, using a hybrid method combining MALDI and Pt-SALDI (i.e., matrix-enhanced [ME]-Pt-SALDI-IMS), we achieved the simultaneous detection of PC, PE and DAG in rat brain tissue sections, and the sensitivity for the detection of these molecules was better than that of MALDI-IMS or Pt-SALDI alone. The present simple ME-Pt-SALDI approach for the simultaneous detection of PC and DAG using two matrices (sputter-deposited Pt film and DHB matrix) would be useful in imaging analyses of biological tissue sections.

Influence of hydrophobicity on positive- and negative-ion yields of peptides in electrospray ionization mass spectrometry.

RATIONALE: The influence of hydrophobicity originating from an amino acid phenylalanine (Phe) residue on the ion yields of peptides has been empirically evaluated using positive- and negative-ion electrospray ionization (ESI) mass spectrometry. The enhancement effect of hydrophobicity was compared with that of the presence of basic and acidic residues of peptides. METHODS: In order to empirically understand the ion yields in soft ionization methods, we have divided the total ionization process into ionization efficiency of analyte molecules and the rate of desorption or vaporization of molecules. The ion yields of protonated and deprotonated molecules of peptides were evaluated. RESULTS: The presence of a Phe residue resulted in an increase in the ion yields of both the analyte ions [M + nH](n+) and [M-nH](n-). The relationship between the ion yields and hydrophobicities of peptides was evaluated using the partition coefficient measured by thin-layer chromatography (PACTLC). A peptide containing a Phe residue at its C-terminus gave a higher ion yield than when it was at the N-terminus. CONCLUSIONS: The ion yields of peptides increased with increasing hydrophobicity both in positive- and negative-ion ESI. The enhancement effect of hydrophobicity on the ion yields was higher than that of basicity and acidity of the peptides in ESI.

Two-dimensional mapping using different chromatographic separations coupled with mass spectrometry for the analysis of ginsenosides in Panax ginseng root and callus.

Two-dimensional (2D) mapping using different chromatographic separations coupled with mass spectrometry is a rapid and simple method for the analysis of a mixture using conventional liquid chromatography mass spectrometry. The 2D map could be created from two different chromatograms obtained with the same detector and different columns or separation methods. In this study, 2D mapping was applied to the analysis of components contained in Panax ginseng, and was evaluated in terms of its effectiveness in the separation of these components. The several glycosides included in Panax ginseng could not be sufficiently separated by one-dimensional chromatography with a reverse phase or a hydrophilic interaction chromatography (HILIC) column, but the components of Panax ginseng could be separated and visualized as a component pattern by 2D mapping. We showed that the components contained in the calli and their quantities were altered by the culture conditions in which the calli were grown by 2D mapping. 2D mapping is expected to be a useful method for visualizing complex component patterns found in glycosides and unknown compounds in foods.

5-Amino-1-naphthol, a novel 1,5-naphthalene derivative matrix suitable for matrix-assisted laser desorption/ionization in-source decay of phosphorylated peptides.

RATIONALE: Although matrix-assisted laser desorption/ionization in-source decay (MALDI-ISD) is an important method for post-translational modification (PTM) analysis, the conventional matrices, 2,5-dihydroxybenzoic acid (2,5-DHB) and 1,5-diaminonaphthalene (1,5-DAN), are poor in terms of the fragment ion yields of the phosphorylated peptides. The use of 5-amino-1-naphthol (5,1-ANL) as a novel matrix for ISD of phosphorylated peptides in MALDI time-of-flight mass spectrometry (TOFMS) is described here. METHODS: We have evaluated the ion yields of ISD fragments obtained from phosphorylated peptides using three 1,5-naphthalene derivatives as MALDI-ISD matrices, i.e., 5,1-ANL, 1,5-DAN and 1,5-dihydroxynaphthalene (1,5-DHN). The signal-to-noise ratio (S/N) of c'-series ions obtained from these matrices was used to estimate their suitability for MALDI-ISD of non-modified and phosphorylated peptides. RESULTS: The order of the S/N values of the ISD fragments for non-modified and phosphorylated peptides were 1,5-DAN > 5,1-ANL > 1,5-DHN and 5,1-ANL > 1,5-DHN > 1,5-DAN, respectively. CONCLUSIONS: The newly introduced matrix 5,1-ANL gave highest ion yields of ISD fragments from mono-, di-, and tetraphosphorylated peptides, while 1,5-DAN was poor in the ISD ion yields for phosphorylated peptides.

Cytotoxic prodigiosin family pigments from Pseudoalteromonas sp. 1020R isolated from the Pacific coast of Japan.

Pseudoalteromonas sp. 1020R, isolated from the Pacific coast of Japan, produces prodigiosin family pigments. Structural analysis indicated that these are prodigiosin (2-methyl-3-pentyl-prodiginine) and three other prodigiosin congeners which differ only in the lengths of the alkyl side chains. These compounds exhibited different extents of cytotoxicity against U937 leukemia cells, and cell death was accompanied by typical features of apoptosis.

Oligonucleotide analysis by nanoparticle-assisted laser desorption/ionization mass spectrometry.

We analyzed oligonucleotides by nanoparticle-assisted laser desorption/ionization (nano-PALDI) mass spectrometry (MS). To this end, we prepared several kinds of nanoparticles (Cr-, Fe-, Mn-, Co-based) and optimized the nano-PALDI MS method to analyze the oligonucleotides. Iron oxide nanoparticles with diammonium hydrogen citrate were found to serve as an effective ionization-assisting reagent in MS. The mass spectra showed both [M - H](-) and [M + xMe(2+)- H](-) (Me: transition metal) peaks. The number of metal-adducted ion signals depended on the length of the oligonucleotide. This phenomenon was only observed using bivalent metal core nanoparticles, not with any other valency metal core nanoparticles. Our pilot study demonstrated that iron oxide nanoparticles could easily ionize samples such as chemical drugs and peptides as well as oligonucleotides without the aid of an oligonucleotide-specific chemical matrix (e.g., 3-hydroxypicolinic acid) used in conventional MS methods. These results suggested that iron-based nanoparticles may serve as the assisting material of ionization for genes and other biomolecules.

Influence of secondary structure on in-source decay of protein in matrix-assisted laser desorption/ionization mass spectrometry.

The susceptibility of the N-Cα bond of the peptide backbone to specific cleavage by in-source decay (ISD) in matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) was studied from the standpoint of the secondary structure of three proteins. A naphthalene derivative, 5-amino-1-naphtol (5,1-ANL), was used as the matrix. The resulting c'-ions, which originate from the cleavage at N-Cα bonds in flexible secondary structures such as turn and bend, and are free from intra-molecular hydrogen-bonded α-helix structure, gave relatively intense peaks. Furthermore, ISD spectra of the proteins showed that the N-Cα bonds of specific amino acid residues, namely Gly-Xxx, Xxx-Asp, and Xxx-Asn, were more susceptible to MALDI-ISD than other amino acid residues. This is in agreement with the observation that Gly, Asp and Asn residues usually located in turns, rather than α-helix. The results obtained indicate that protein molecules embedded into the matrix crystal in the MALDI experiments maintain their secondary structures as determined by X-ray crystallography, and that MALDI-ISD has the capability for providing information concerning the secondary structure of protein.

Roles of the C-terminal residues of calmodulin in structure and function.

Electrospray ionization mass spectrometry (ESI-MS), circular dichroism (CD), nuclear magnetic resonance (NMR) spectroscopy, flow dialysis, and bioactivity measurements were employed to investigate the roles of the C-terminal residues of calmodulin (CaM). In the present study, we prepared a series of truncated mutants of chicken CaM that lack four (CCMΔ4) to eight (CCMΔ8) residues at the C-terminal end. It was found that CCMΔ4, lacking the last four residues (M145 to K148), binds four Ca2+ ions. Further deletion gradually decreased the ability to bind the fourth Ca2+ ion, and CCMΔ8 completely lost the ability. Interestingly, both lobes of Ca2+-sturated CCMΔ5 showed instability in the conformation, although limited part in the C-lobe of Ca2+-saturated CCMΔ4 was instable. Moreover, unlike CCMΔ4, structure of the C-lobe in CCMΔ5 bound to the target displayed dissimilarity to that of CaM, suggesting that deletion of M144 changes the binding manner. Deletion of the last five residues (M144 to K148) and further truncation of the C-terminal region decreased apparent capacity for target activation. Little contribution of the last four residues including M145 was observed for structural stability, Ca2+-binding, and target activation. Although both M144 and M145 have been recognized as key residues for the function, the present data suggest that M144 is a more important residue to attain Ca2+ induced conformational change and to form a proper Ca2+-saturated conformation.