Phone: +914162202722 Address: TT-613, Advanced Centre for Bio Separation Technology (CBST), Vellore Institute of Technology (VIT), Vellore, Tamil Nadu, India.
To understand the importance of arginine (Arg) in influencing electrospray ionization (ESI)-colli... more To understand the importance of arginine (Arg) in influencing electrospray ionization (ESI)-collision induced dissociation (CID) tandem mass spectrometry (MS/MS) behavior of peptides of lengths >~25 amino acid residues (a.a.r.), we chose carbamidomethyl Insulin B-chain (30 a.a.r.), glucagon (29 a.a.r.) and melittin (26 a.a.r.) as the models for this investigation. Also, two smaller peptides: Angiotensin II (8 a.a.r.) and Bradykinin (9 a.a.r.) were studied for better comprehension of the interplay between the influence of Arg and peptide's length. The motivation to study such longer peptides stems from middle-down proteomics, a recently emerging field encompassing investigations on proteolytic peptides longer than~25 a.a.r. CID MS/MS data of two different cases have been compared: (1) standard model peptides vs. chemically modified peptides, wherein sidechain guanidine group of Arg residues in these model peptides are selectively modified by 1,2-cyclohexanedione and phenylglyoxal; (2) standard model peptides vs. mutated model peptides, in which Arg residues in these model peptides are substituted by alanine (Ala) residues (Arg / Ala). Different types of stoichiometric products were obtained due to this chemical modification and each type of arginine-modified product was subjected to ESI CID MS/MS. The Arg / Ala mutated model peptides chosen for this study are: [R22A]-Insulin B-chain, [R17A & R18A]-Glucagon, [R22A & R24A]-Melittin and a shorter peptide: [R2A]-Angiotensin II. All experiments were performed in a quadrupole time-of-flight hybrid mass spectrometer, whereby CID (hexapole collision cell) was done by following fixed collision energy (CE) as well as ramped CE. Analysis of CID MS/MS spectra revealed that the sequence coverage of Arg / Ala mutated peptides was higher than the chemically modified peptides as well as their respective standard (unmodified) peptides. Examination of all the MS/MS data alluded that Arg has a greater influence on the CID MS/MS behavior of longer peptides, viz., lengths > 25 a.a.r., than the smaller peptides.
Many of the earlier studies involving the effect of isoniazid (INH) treatment have solely focused... more Many of the earlier studies involving the effect of isoniazid (INH) treatment have solely focused on the fatty acyl (FA) category of Mycobacterium tuberculosis (MTB) lipids. This motivated us with the major interest to examine the impact of INH on various other categories of MTB lipids. Towards this, we chose to interpret our mass spectral data (LC-ESI-MS) by a standalone software, MS-LAMP, in which " Mtb LipidDB " was integrated. Analysis by MS-LAMP revealed that INH treatment can alter the composition of " glycerolipids (GLs) " and " glycerophospholipids (GPLs) " categories of MTB lipids, in addition to the variations to FA category. Interpretation by " MycoMass " database yielded similar results as that of Mtb LipidDB, except that significant alterations to polyketides (PKs) category also were observed. Probing biosynthetic pathways of certain key lipids belonging to any of GLs, GPLs, and PKs categories can be attractive target(s) for drug discovery or can be useful to identify means to overcome drug resistance or to obtain insights into the causal factors of virulence. To the best of our knowledge, this is the first report hinting at the influence of INH on GLs, GPLs, and PKs of MTB.
Objectives: The purpose of the study is to characterize antimycobacterial phytoconstituent from e... more Objectives: The purpose of the study is to characterize antimycobacterial phytoconstituent from ethyl acetate extract of dried bulbs of Allium sativum Linn. (Alliaceae) and elucidating the probable mode of action of the bioactive molecule. Methods: Serial extraction, Mycobacterium tuberculosis assay by agar well diffusion method, minimal inhibitory concentration by microplate alamar blue assay, Fourier transform infrared (FT-IR), nuclear magnetic resonance (NMR) spectroscopy, liquid chromatography (LC)-electrospray ionization (ESI)-mass spectrometry (MS)/MS, cell leakage assay, scanning electron microscopy (SEM), inhibition property of linear alkylbenzene sulfonate (LAS) in the presence of rifampicin on M. tuberculosis were performed. Results: Ethyl acetate extract displayed significant inhibition properties against M. tuberculosis H37Ra (MTCC 300). Subsequently, the bioactivity-guided fractionation was employed to purify the phytochemical. Analysis of FT-IR, LC-MS (ESI), 1 H, and 13 C-NMR spectrum revealed that the bioactive phytochemicals are the variants of LAS, with C 12-alkyl being predominant, and the minimum inhibitory concentration was found to be 5.56 μg/ml. Morphological examination by SEM and cell leakage assay indicated that these molecules change the membrane fluidity. Conclusion: The results thus suggest the possibility of using low concentrations of LAS to effect changes in membrane fluidity, thereby enhancing the efficacy of antibiotic treatment.
Although extensive literature exists on antioxidant properties of medicinal plants, very few stud... more Although extensive literature exists on antioxidant properties of medicinal plants, very few studies have focused on their polyphenolic composition. Here, preliminary phytochemical screening and total flavonoid content in extracts of seeds of Myristica fragrans and leaves of Cordyline terminalis have been investigated. Screening of potential flavonoid-subclasses was done by LC-ESI-MS, whose data were interpreted using database of Lipid Metabolites and Pathways Strategy Consortium. 'Flavones and Flavonols' and 'Anthocyanidins' appear to be more abundant in C. terminalis than in M. fragrans. Higher content of isoflavonoids, chalcones and dihydrochalcones and some isoprenoids were observed in M. fragrans than in C. terminalis. The strategy and results of this study will help to choose appropriate standards prior to their identification and confirmation in natural extracts. These results may also be useful to assess antioxidant activity of only the subclass alone and accordingly appropriate subclasses can be selected to prepare formulations having nutraceutical applications.
The genome of Helicobacter pylori is rich in restriction-modification (RM) systems. Approximately... more The genome of Helicobacter pylori is rich in restriction-modification (RM) systems. Approximately 4% of the genome codes for components of RM systems. hpyAVIBM, which codes for a phase-variable C(5) cytosine methyltransferase (MTase) from H. pylori, lacks a cognate restriction enzyme. Over-expression of M.HpyAVIB in Escherichia coli enhances the rate of mutations. However, when the catalytically inactive F9N or C82W mutants of M.HpyAVIB were expressed in E. coli, mutations were not observed. The M.HpyAVIB gene itself was mutated to give rise to different variants of the MTase. M.HpyAVIB variants were purified and differences in kinetic properties and specificity were observed. Intriguingly, purified MTase variants showed relaxed substrate specificity. Homologues of hpyAVIBM homologues amplified and sequenced from different clinical isolates showed similar variations in sequence. Thus, hpyAVIBM presents an interesting example of allelic variations in H. pylori where changes in the nu...
Several arginine modification studies on enzymes have mostly been done by phenylglyoxal (PG) or 1... more Several arginine modification studies on enzymes have mostly been done by phenylglyoxal (PG) or 1,2-cyclohexanedione (CHD) as the modifying reagents. It has been found that one molecule or two molecules of PG or one CHD molecule covalently modify sidechain guanidine of arginine of the enzymes. To seek clearer insights into stoichiometry of this reaction, herein, we decided to investigate some model amino acids that include L-arginine (L-Arg) and two model peptides (an octapeptide and a 30 amino acids long peptide). Reactions were conducted at room temperature (RT: ~25 0 C), involving 'equimolar concentrations' of reactants in seven different mediums: five buffers (all at basic pH, 7.4-8.4) and two solvents. The two solvents are: (1) water (H 2 O) and (2) mixture of acetonitrile and water (ACN:H 2 O, 1:1, v/v). Progress of every reaction was monitored as a function of time by liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS). With PG, L-Arg forms 1:1 adduct ([L-Arg+PG]; m/z 309), 1:2 adduct ([L-Arg+2PG]; m/z 443) and water condensed products of respective 1:1 and 1:2 adducts (m/z 291 & m/z 425) in all six mediums, except in borate, where only uncondensed 1:1 adduct ([L-Arg+PG]; m/z 309) was observed. However, with CHD, L-Arg yielded 1:1 adduct (m/z 287), 1:2 adduct (m/z 399) and corresponding water condensed products (m/z 269 & m/z 381) in borate. Interestingly, in H 2 O and in ACN:H 2 O (1:1) too, L-Arg undergoes modification. This is the first LC-ESI-MS study illustrating modification of L-Arg by phenylglyoxal and 1,2-cyclohexanedione. Molecular structures are proposed for every observed modified product, depicting stoichiometry and mode of binding by PG or CHD onto guanidine moiety of L-Arg.
Dicyclohexylphosphinic acid (DcyHPA) and some of its metal complexes with lanthanides (La, Ce, Gd... more Dicyclohexylphosphinic acid (DcyHPA) and some of its metal complexes with lanthanides (La, Ce, Gd, Sm and Nd) and actinides (U and Th) were prepared and subsequently the structural composition was elucidated. The single crystal XRD data and theoretical analysis revealed that the ligand (DcyHPA) exists as a dimer in solid state. DcyHPA showed dual nature for the extraction of U(VI), Th(IV) and Am(III) from nitric acid medium i.e. at lower acidity, the ligand behaves as an acidic extractant and extracted the metal through cation exchange mechanism while solvation mechanism played a major role for extraction at higher acidity. Additionally, density functional theory (DFT) calculations were demonstrated to understand the electronic structure of thorium-DcyHPA metal complex formed during the extraction process.
In this study, we have probed the influence of reversal of peptide bond directionality in S pepti... more In this study, we have probed the influence of reversal of peptide bond directionality in S peptide vs Retro S (RS) peptide on the characteristics of collision induced dissociation (CID) tandem mass spectrometry (MS/MS) under electrospray ionization (ESI) conditions. S peptide: KETAAAKFERQHMDSS, which corresponds to residues (1–16) of bovine pancreatic ribonuclease A (RNase A) and RS peptide: SSDMHQREFKAAATEK were taken as models. CID was carried out within a linear trap quadrupole (LTQ) on the doubly protonated ([M+2H]2+) precursor ions (m/z 918.44) of the two peptides at different collision energies (CEs) and the product ion analysis was by high resolution mass analyzer, orbitrap. The degree of fragmentation – ‘η’ of each of the fifteen peptide bonds of the peptide molecular ions from each peptide was determined by estimating the relative abundance of product ions (b- and y-ions) with reference to precursor ions, at every CE. The greater fragility of RS peptide than S peptide was evident from determinations of CE50 and CE* (the minimum collision energy, at which, the precursor ion population is 50% and 0% of the initial populations, respectively). The values of CE50 were 23.6 and 22.6 and the values of CE* were 30 and 28 for S and RS peptides, respectively. In view of the previously determined conformational propensity of S peptide to be more structured than RS peptide (Pal-Bhowmick et al. [31]), our data suggest that the solution structures of these peptides may be preserved also in the gas phase. This augurs well for the application of high resolution CID MS/MS to probe conformational properties of peptides in gas phase.
In an attempt to understand the response of the molecule to electrospray ionization (ESI), a new ... more In an attempt to understand the response of the molecule to electrospray ionization (ESI), a new parameter called ‘molecular electrospray ionization index (MESII)’ is defined. Selected reaction monitoring (SRM) data acquired on an ESI tandem quadrupole mass spectrometer is utilized for calculating MESII, which we denote as εSRM: εSRM= - log (SRM intensity/Population of ions or molecules in-solution). Population of ions or molecules in-solution is estimated using molecular mass of the respective compound and Avogadro’s constant. Simvastatin acid (SVA), lovastatin (LV) and simvastatin (SV) are chosen as model compounds. SRM experiments in positive ion mode were performed on singly protonated ions ([M+H]+) of SVA, LV and SV. In negative ion mode, only SVA was investigated by SRM, using singly deprotonated ion ([M-H]-) as precursor ion. Thus estimated MESII values in positive ion mode are: ε+ SRM (SVA)=7.4288, ε+ SRM (LV)=7.4541 and ε+ SRM (SV)=8.6833 and in negative ion mode, ε- SRM (SVA)=7.2253. This newly defined index not only gives an idea about ionization potential (i.e., degree of ionization), but can also be an indicator of limit of detection (LOD) of an analyte. When utilizing SRM data recorded from different type of instruments for a compound, the variations that may arise in MESII values could help in understanding the influence of different instrument configurations/methods on the degree of ionization of that compound. Matrix effects on the extent of analyte’s ionization too can be understood from the differences in the MESII values. Further, it may be possible to utilize MESII for quantitation as well.
Escherichia coli RNA polymerase is a multi-subunit enzyme containing α(2)ββ'ωσ, which transcribes... more Escherichia coli RNA polymerase is a multi-subunit enzyme containing α(2)ββ'ωσ, which transcribes DNA template to intermediate RNA product in a sequence specific manner. Although most of the subunits are essential for its function, the smallest subunit ω (average molecular mass ∼ 10,105 Da) can be deleted without affecting bacterial growth. Creating a mutant of the ω subunit can aid in improving the understanding of its role. Sequencing of rpoZ gene that codes for ω subunit from a mutant variant suggested a substitution mutation at position 60 of the protein: asparagine (N) → aspartic acid (D). This mutation was verified at the protein level by following a typical mass spectrometry (MS) based bottom-up proteomic approach. Characterization of in-gel trypsin digested samples by reverse phase liquid chromatography (LC) coupled to electrospray ionization (ESI)-tandem mass spectrometry (MS/MS) enabled in ascertaining this mutation. Electron transfer dissociation (ETD) of triply charged [(M + 3H)(3+)] tryptic peptides (residues [53-67]), EIEEGLINNQILDVR from wild-type and EIEEGLIDNQILDVR from mutant, facilitated in unambiguously determining the site of mutation at residue 60.
The crystal structures of four protected beta-amino acid residues, Boc-(S)-beta3-HAla-NHMe (1); B... more The crystal structures of four protected beta-amino acid residues, Boc-(S)-beta3-HAla-NHMe (1); Boc-(R)-beta3-HVal-NHMe (2); Boc-(S)-beta3-HPhe-NHMe (3); Boc-(S)-beta3-HPro-OH (6) and two beta-dipeptides, Boc-(R)-beta3-HVal-(R)-beta3-HVal-OMe (4); Boc-(R)-beta3-HVal-(S)-beta3-HVal-OMe (5) have been determined. Gauche conformations about the C(beta)-C(alpha) bonds (theta approximately +/-60 degrees) are observed for the beta3-HPhe residues in and all four beta3-HVal residues in the dipeptides and . Trans conformations (theta is approximately 180 degrees) are observed for beta3-HAla residues in both independent molecules in and for the beta3-HVal and beta3-HPro residues in and , respectively. In the cases of compounds , molecules associate in the crystals via intermolecular backbone hydrogen bonds leading to the formation of sheets. The polar strands formed by beta3-residues aggregate in both parallel (1,3,5) and antiparallel (2,4 fashion. Sheet formation accommodates both the trans and gauche conformations about the C(beta)-C(alpha) bonds.
De novo mass spectrometric sequencing of two Conus peptides, Vi1359 and Vi1361, from the vermivor... more De novo mass spectrometric sequencing of two Conus peptides, Vi1359 and Vi1361, from the vermivorous cone snail Conus virgo, found off the southern Indian coast, is presented. The peptides, whose masses differ only by 2 Da, possess two disulfide bonds and an amidated C-terminus. Simple chemical modifications and enzymatic cleavage coupled with matrix assisted laser desorption ionization (MALDI) mass spectrometric analysis aided in establishing the sequences of Vi1359, ZCCITIPECCRI-NH(2), and Vi1361, ZCCPTMPECCRI-NH(2), which differ only at residues 4 and 6 (Z = pyroglutamic acid). The presence of the pyroglutamyl residue at the N-terminus was unambiguously identified by chemical hydrolysis of the cyclic amide, followed by esterification. The presence of Ile residues in both the peptides was confirmed from high-energy collision induced dissociation (CID) studies, using the observation of w(n)- and d(n)-ions as a diagnostic. Differential cysteine labeling, in conjunction with MALDI-MS/MS, permitted establishment of disulfide connectivity in both peptides as Cys2-Cys9 and Cys3-Cys10. The cysteine pattern clearly reveals that the peptides belong to the class of T-superfamily conotoxins, in particular the T-1 superfamily.
Distinctly different effects of two closely related contryphans have been demonstrated on voltage... more Distinctly different effects of two closely related contryphans have been demonstrated on voltage-activated Ca(2+) channels. The peptides Lo959 and Am975 were isolated from Conus loroisii, a vermivorous marine snail and Conus amadis, a molluscivore, respectively. The sequences of Lo959 and Am975 were deduced by mass spectrometric sequencing (MALDI-MS/MS) and confirmed by chemical synthesis. The sequences of Lo959, GCP(D)WDPWC-NH(2) and Am975, GCO(D)WDPWC-NH(2) (O: 4-trans-hydroxyproline: Hyp), differ only at residue 3; Pro in Lo959, Hyp in Am975, which is identical to contryphan-P, previously isolated from Conus purpurascens, a piscivore; while Lo959 is a novel peptide. Both Lo959 and Am975 undergo slow conformational interconversion under reverse-phase chromatographic conditions, a characteristic feature of all contryphans reported thus far. Electrophysiological studies performed using dorsal root ganglion neurons reveal that both peptides target high voltage-activated Ca(2+) channels. While Lo959 increases the Ca(2+) current, Am975 causes inhibition. The results establish that subtle sequence effects, which accompany post-translational modifications in Conus peptides, can have dramatic effects on target ion channels.
The software Peptide Fragment Ion Analyser (PFIA) aids in the analysis and interpretation of tand... more The software Peptide Fragment Ion Analyser (PFIA) aids in the analysis and interpretation of tandem mass spectrometric data of peptides. The software package has been designed to facilitate the analysis of product ions derived from acyclic and cyclic peptide natural products that possess unusual amino acid residues and are heavily post-translationally modified. The software consists of two programmes: (a) PFIA-I lists the amino acid compositions and their corresponding product ion types for 'a queried m/z value' (z = +1) and (b) PFIA-II displays fragmentation pattern diagram(s) and lists all sequence-specific product ion types for the protonated adduct of 'a queried sequence'. The unique feature of PFIA-II is its ability to handle cyclic peptides. The two programmes used in combination can prove helpful for deriving peak assignments in the de novo sequencing of novel peptides.
The fragmentations of [M+H]+ and [M+Na]+ adducts of neutral peptides with blocked N- and C- termi... more The fragmentations of [M+H]+ and [M+Na]+ adducts of neutral peptides with blocked N- and C- termini have been investigated using electrospray ion trap mass spectrometry. The N- termini of these synthetically designed peptides are blocked with a tertiarybutyloxycarbonyl (Boc) group, and the C- termini are esterified. These peptides do not possess side chains that are capable of complexation and hence the backbone amide units are the sole sites of protonation and metallation. The cleavage patterns of the protonated peptides are strikingly different from those of sodium ion adducts. While the loss of the N- terminal blocking group occurs quite readily in the case of MS/MS of [M+Na]+, the cleavage of the C- terminal methoxy group seems to be a facile process in the case of MS/MS of [M+H]+. Fragmentation of the protonated adducts yields only bn ions, while yn and an ions are predominantly formed from the fragmentation of sodium ion adducts. The an ions arising from the fragmentation of [M+Na]+ lack the N- terminal Boc group (and are here termed an* ions). MS/MS of [M+Na]+ species also yields bn ions of substantially lower intensities that lack the N- terminal Boc group (bn). A similar distinction between the fragmentation patterns of proton and sodium ion adducts is observed in the case of peptides possessing an N- terminal acetyl group. An example of the fragmentation of the H+ and Na+ adducts of a naturally occurring peptaibol from a Trichoderma species confirms that fragmentation of these two ionized species yields complementary information, useful in sequencing natural peptides. Inspection of the isotopic pattern of bn ions derived from [M+H]+ adducts of peptaibols provided insights into the sequences of microheterogeneous samples. This study reveals that the combined use of protonated and sodium ion adducts should prove useful in de novo sequencing of peptides, particularly of naturally occurring neutral peptides with modified N- and C- termini, for example, peptaibols.
Ten new cyclic hexadepsipeptides, six isariins and four isaridins, from the fungus Isaria have be... more Ten new cyclic hexadepsipeptides, six isariins and four isaridins, from the fungus Isaria have been identified and characterized by high-performance liquid chromatography, coupled to tandem electrospray ionization mass spectrometry (LC-ESIMS/MS). The isariins possess a â-hydroxy acid residue and five R-amino acids, while isaridins contain a â-amino acid, an R-hydroxy acid, and four R-amino acids. One- and two-dimensional NMR spectroscopy confirmed the chemical identity of some of the isariin fractions. Mass spectral fragmentation patterns of [M + H]+ ions reveal clear diagnostic fragment ions for the isariins and isaridins. Previously described cyclic depsipeptides, isarfelins from Isaria felina (Guo, Y. X.; Liu, Q. H.; Ng, T. B.; Wang H. X. Peptides 2005, 26, 2384), are now reassigned as members of the isaridin family. Examination of isaridin sequences revealed significant similarities with cyclic hexadepsipeptides such as destruxins and roseotoxins. The structure of an isariin (isariin A) investigated by NMR spectroscopy indicated the presence of a hybrid Râ C11 turn, formed by the â-hydroxy acid and glycine residues and a DLeu-LAla type II¢ â-turn. Additionally, the inhibitory effect of isariins and an isaridin on the intra-erythrocytic growth of Plasmodium falciparum is presented.
International Journal of Mass Spectrometry, Mar 2014
Mechanisms of collision induced dissociation (CID) under the conditions of electrospray ionizatio... more Mechanisms of collision induced dissociation (CID) under the conditions of electrospray ionization mass spectrometry (ESI-MS) of cyclic-di-adenosine mono phosphate (c-di-AMP) and cyclic-di-guanosine mono phosphate (c-di-GMP) are described. The CID mass spectrum of doubly protonated ([M+2H] 2+ ) c-di-AMP differs from that of singly protonated ([M+H] + ) and the mechanisms of dissociation of these two ionic forms are proposed. CID of singly deprotonated form ([M−H] − ) of c-di-AMP was also carried out and its fragmentation mechanism is delineated. Ring-opening step seems to be imperative and foremost prior to subsequent dissociation events, during the fragmentation of both cationic and anionic forms of c-di-AMP. In the case of CID of [M+H]+ of c-di-GMP, the abundance of fragment ions indicate that ring-opening mediated fragmentation may not be a favored pathway, while loss of guanine may be relatively more preferred. Interestingly, fragmentation of [M−H] − of c-di-GMP appears to be very similar to that of c-di-AMP, which involves ring-opening step, suggesting that nitrogen base does not influence CID pathways of [M−H] − species. Understanding such dissociation mechanisms will prove useful for discovery and validation of novel hybrid cyclic dinucleotides and their analogues. Also, such comprehensions will be helpful in distinguishing different isobaric molecules possessing varied molecular structures.
Cyclic di-AMP is a recently discovered signaling molecule which regulates various aspects of bact... more Cyclic di-AMP is a recently discovered signaling molecule which regulates various aspects of bacterial physiology and virulence. Here we report the characterization of c-di-AMP synthesizing and hydrolyzing proteins from Mycobacterium tuberculosis. Recombinant Rv3586 (MtbDisA) can synthesize c-di-AMP from ATP through the diadenylate cyclase activity. Detailed biochemical characterization of the protein revealed that the diadenylate cyclase (DAC) activity is allosterically regulated by ATP. We have identified the intermediates of the DAC reaction and propose a two-step synthesis of c-di-AMP from ATP/ADP. MtbDisA also possesses ATPase activity which is suppressed in the presence of the DAC activity. Investigations by liquid chromatography -electrospray ionization mass spectrometry have detected multimeric forms of c-di-AMP which have implications for the regulation of c-di-AMP cellular concentration and various pathways regulated by the dinucleotide. We have identified Rv2837c (MtbPDE) to have c-di-AMP specific phosphodiesterase activity. It hydrolyzes c-di-AMP to 5'-AMP in two steps. First, it linearizes c-di-AMP into pApA which is further hydrolyzed to 5'-AMP. MtbPDE is novel compared to c-di-AMP specific phosphodiesterase, YybT (or GdpP) in being a soluble protein and hydrolyzing c-di-AMP to 5'-AMP. Our results suggest that the cellular concentration of c-di-AMP can be regulated by ATP concentration as well as the hydrolysis by MtbPDE.
Mass Spectrometry based Lipid(ome) Analyzer and Molecular Platform (MS-LAMP) is a new software ca... more Mass Spectrometry based Lipid(ome) Analyzer and Molecular Platform (MS-LAMP) is a new software capable of aiding in interpreting electrospray ionization (ESI) and/or matrix-assisted laser desorption/ionization (MALDI) mass spectrometric data of lipids. The graphical user interface (GUI) of this standalone programme is built using Perl::Tk. Two databases have been developed and constituted within MS-LAMP, on the basis of Mycobacterium tuberculosis (M. tb) lipid database (www.mrl.colostate.edu) and that of Lipid Metabolites and Pathways Strategy Consortium (LIPID MAPS; www.lipidmaps.org).
To understand the importance of arginine (Arg) in influencing electrospray ionization (ESI)-colli... more To understand the importance of arginine (Arg) in influencing electrospray ionization (ESI)-collision induced dissociation (CID) tandem mass spectrometry (MS/MS) behavior of peptides of lengths >~25 amino acid residues (a.a.r.), we chose carbamidomethyl Insulin B-chain (30 a.a.r.), glucagon (29 a.a.r.) and melittin (26 a.a.r.) as the models for this investigation. Also, two smaller peptides: Angiotensin II (8 a.a.r.) and Bradykinin (9 a.a.r.) were studied for better comprehension of the interplay between the influence of Arg and peptide's length. The motivation to study such longer peptides stems from middle-down proteomics, a recently emerging field encompassing investigations on proteolytic peptides longer than~25 a.a.r. CID MS/MS data of two different cases have been compared: (1) standard model peptides vs. chemically modified peptides, wherein sidechain guanidine group of Arg residues in these model peptides are selectively modified by 1,2-cyclohexanedione and phenylglyoxal; (2) standard model peptides vs. mutated model peptides, in which Arg residues in these model peptides are substituted by alanine (Ala) residues (Arg / Ala). Different types of stoichiometric products were obtained due to this chemical modification and each type of arginine-modified product was subjected to ESI CID MS/MS. The Arg / Ala mutated model peptides chosen for this study are: [R22A]-Insulin B-chain, [R17A & R18A]-Glucagon, [R22A & R24A]-Melittin and a shorter peptide: [R2A]-Angiotensin II. All experiments were performed in a quadrupole time-of-flight hybrid mass spectrometer, whereby CID (hexapole collision cell) was done by following fixed collision energy (CE) as well as ramped CE. Analysis of CID MS/MS spectra revealed that the sequence coverage of Arg / Ala mutated peptides was higher than the chemically modified peptides as well as their respective standard (unmodified) peptides. Examination of all the MS/MS data alluded that Arg has a greater influence on the CID MS/MS behavior of longer peptides, viz., lengths > 25 a.a.r., than the smaller peptides.
Many of the earlier studies involving the effect of isoniazid (INH) treatment have solely focused... more Many of the earlier studies involving the effect of isoniazid (INH) treatment have solely focused on the fatty acyl (FA) category of Mycobacterium tuberculosis (MTB) lipids. This motivated us with the major interest to examine the impact of INH on various other categories of MTB lipids. Towards this, we chose to interpret our mass spectral data (LC-ESI-MS) by a standalone software, MS-LAMP, in which " Mtb LipidDB " was integrated. Analysis by MS-LAMP revealed that INH treatment can alter the composition of " glycerolipids (GLs) " and " glycerophospholipids (GPLs) " categories of MTB lipids, in addition to the variations to FA category. Interpretation by " MycoMass " database yielded similar results as that of Mtb LipidDB, except that significant alterations to polyketides (PKs) category also were observed. Probing biosynthetic pathways of certain key lipids belonging to any of GLs, GPLs, and PKs categories can be attractive target(s) for drug discovery or can be useful to identify means to overcome drug resistance or to obtain insights into the causal factors of virulence. To the best of our knowledge, this is the first report hinting at the influence of INH on GLs, GPLs, and PKs of MTB.
Objectives: The purpose of the study is to characterize antimycobacterial phytoconstituent from e... more Objectives: The purpose of the study is to characterize antimycobacterial phytoconstituent from ethyl acetate extract of dried bulbs of Allium sativum Linn. (Alliaceae) and elucidating the probable mode of action of the bioactive molecule. Methods: Serial extraction, Mycobacterium tuberculosis assay by agar well diffusion method, minimal inhibitory concentration by microplate alamar blue assay, Fourier transform infrared (FT-IR), nuclear magnetic resonance (NMR) spectroscopy, liquid chromatography (LC)-electrospray ionization (ESI)-mass spectrometry (MS)/MS, cell leakage assay, scanning electron microscopy (SEM), inhibition property of linear alkylbenzene sulfonate (LAS) in the presence of rifampicin on M. tuberculosis were performed. Results: Ethyl acetate extract displayed significant inhibition properties against M. tuberculosis H37Ra (MTCC 300). Subsequently, the bioactivity-guided fractionation was employed to purify the phytochemical. Analysis of FT-IR, LC-MS (ESI), 1 H, and 13 C-NMR spectrum revealed that the bioactive phytochemicals are the variants of LAS, with C 12-alkyl being predominant, and the minimum inhibitory concentration was found to be 5.56 μg/ml. Morphological examination by SEM and cell leakage assay indicated that these molecules change the membrane fluidity. Conclusion: The results thus suggest the possibility of using low concentrations of LAS to effect changes in membrane fluidity, thereby enhancing the efficacy of antibiotic treatment.
Although extensive literature exists on antioxidant properties of medicinal plants, very few stud... more Although extensive literature exists on antioxidant properties of medicinal plants, very few studies have focused on their polyphenolic composition. Here, preliminary phytochemical screening and total flavonoid content in extracts of seeds of Myristica fragrans and leaves of Cordyline terminalis have been investigated. Screening of potential flavonoid-subclasses was done by LC-ESI-MS, whose data were interpreted using database of Lipid Metabolites and Pathways Strategy Consortium. 'Flavones and Flavonols' and 'Anthocyanidins' appear to be more abundant in C. terminalis than in M. fragrans. Higher content of isoflavonoids, chalcones and dihydrochalcones and some isoprenoids were observed in M. fragrans than in C. terminalis. The strategy and results of this study will help to choose appropriate standards prior to their identification and confirmation in natural extracts. These results may also be useful to assess antioxidant activity of only the subclass alone and accordingly appropriate subclasses can be selected to prepare formulations having nutraceutical applications.
The genome of Helicobacter pylori is rich in restriction-modification (RM) systems. Approximately... more The genome of Helicobacter pylori is rich in restriction-modification (RM) systems. Approximately 4% of the genome codes for components of RM systems. hpyAVIBM, which codes for a phase-variable C(5) cytosine methyltransferase (MTase) from H. pylori, lacks a cognate restriction enzyme. Over-expression of M.HpyAVIB in Escherichia coli enhances the rate of mutations. However, when the catalytically inactive F9N or C82W mutants of M.HpyAVIB were expressed in E. coli, mutations were not observed. The M.HpyAVIB gene itself was mutated to give rise to different variants of the MTase. M.HpyAVIB variants were purified and differences in kinetic properties and specificity were observed. Intriguingly, purified MTase variants showed relaxed substrate specificity. Homologues of hpyAVIBM homologues amplified and sequenced from different clinical isolates showed similar variations in sequence. Thus, hpyAVIBM presents an interesting example of allelic variations in H. pylori where changes in the nu...
Several arginine modification studies on enzymes have mostly been done by phenylglyoxal (PG) or 1... more Several arginine modification studies on enzymes have mostly been done by phenylglyoxal (PG) or 1,2-cyclohexanedione (CHD) as the modifying reagents. It has been found that one molecule or two molecules of PG or one CHD molecule covalently modify sidechain guanidine of arginine of the enzymes. To seek clearer insights into stoichiometry of this reaction, herein, we decided to investigate some model amino acids that include L-arginine (L-Arg) and two model peptides (an octapeptide and a 30 amino acids long peptide). Reactions were conducted at room temperature (RT: ~25 0 C), involving 'equimolar concentrations' of reactants in seven different mediums: five buffers (all at basic pH, 7.4-8.4) and two solvents. The two solvents are: (1) water (H 2 O) and (2) mixture of acetonitrile and water (ACN:H 2 O, 1:1, v/v). Progress of every reaction was monitored as a function of time by liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS). With PG, L-Arg forms 1:1 adduct ([L-Arg+PG]; m/z 309), 1:2 adduct ([L-Arg+2PG]; m/z 443) and water condensed products of respective 1:1 and 1:2 adducts (m/z 291 & m/z 425) in all six mediums, except in borate, where only uncondensed 1:1 adduct ([L-Arg+PG]; m/z 309) was observed. However, with CHD, L-Arg yielded 1:1 adduct (m/z 287), 1:2 adduct (m/z 399) and corresponding water condensed products (m/z 269 & m/z 381) in borate. Interestingly, in H 2 O and in ACN:H 2 O (1:1) too, L-Arg undergoes modification. This is the first LC-ESI-MS study illustrating modification of L-Arg by phenylglyoxal and 1,2-cyclohexanedione. Molecular structures are proposed for every observed modified product, depicting stoichiometry and mode of binding by PG or CHD onto guanidine moiety of L-Arg.
Dicyclohexylphosphinic acid (DcyHPA) and some of its metal complexes with lanthanides (La, Ce, Gd... more Dicyclohexylphosphinic acid (DcyHPA) and some of its metal complexes with lanthanides (La, Ce, Gd, Sm and Nd) and actinides (U and Th) were prepared and subsequently the structural composition was elucidated. The single crystal XRD data and theoretical analysis revealed that the ligand (DcyHPA) exists as a dimer in solid state. DcyHPA showed dual nature for the extraction of U(VI), Th(IV) and Am(III) from nitric acid medium i.e. at lower acidity, the ligand behaves as an acidic extractant and extracted the metal through cation exchange mechanism while solvation mechanism played a major role for extraction at higher acidity. Additionally, density functional theory (DFT) calculations were demonstrated to understand the electronic structure of thorium-DcyHPA metal complex formed during the extraction process.
In this study, we have probed the influence of reversal of peptide bond directionality in S pepti... more In this study, we have probed the influence of reversal of peptide bond directionality in S peptide vs Retro S (RS) peptide on the characteristics of collision induced dissociation (CID) tandem mass spectrometry (MS/MS) under electrospray ionization (ESI) conditions. S peptide: KETAAAKFERQHMDSS, which corresponds to residues (1–16) of bovine pancreatic ribonuclease A (RNase A) and RS peptide: SSDMHQREFKAAATEK were taken as models. CID was carried out within a linear trap quadrupole (LTQ) on the doubly protonated ([M+2H]2+) precursor ions (m/z 918.44) of the two peptides at different collision energies (CEs) and the product ion analysis was by high resolution mass analyzer, orbitrap. The degree of fragmentation – ‘η’ of each of the fifteen peptide bonds of the peptide molecular ions from each peptide was determined by estimating the relative abundance of product ions (b- and y-ions) with reference to precursor ions, at every CE. The greater fragility of RS peptide than S peptide was evident from determinations of CE50 and CE* (the minimum collision energy, at which, the precursor ion population is 50% and 0% of the initial populations, respectively). The values of CE50 were 23.6 and 22.6 and the values of CE* were 30 and 28 for S and RS peptides, respectively. In view of the previously determined conformational propensity of S peptide to be more structured than RS peptide (Pal-Bhowmick et al. [31]), our data suggest that the solution structures of these peptides may be preserved also in the gas phase. This augurs well for the application of high resolution CID MS/MS to probe conformational properties of peptides in gas phase.
In an attempt to understand the response of the molecule to electrospray ionization (ESI), a new ... more In an attempt to understand the response of the molecule to electrospray ionization (ESI), a new parameter called ‘molecular electrospray ionization index (MESII)’ is defined. Selected reaction monitoring (SRM) data acquired on an ESI tandem quadrupole mass spectrometer is utilized for calculating MESII, which we denote as εSRM: εSRM= - log (SRM intensity/Population of ions or molecules in-solution). Population of ions or molecules in-solution is estimated using molecular mass of the respective compound and Avogadro’s constant. Simvastatin acid (SVA), lovastatin (LV) and simvastatin (SV) are chosen as model compounds. SRM experiments in positive ion mode were performed on singly protonated ions ([M+H]+) of SVA, LV and SV. In negative ion mode, only SVA was investigated by SRM, using singly deprotonated ion ([M-H]-) as precursor ion. Thus estimated MESII values in positive ion mode are: ε+ SRM (SVA)=7.4288, ε+ SRM (LV)=7.4541 and ε+ SRM (SV)=8.6833 and in negative ion mode, ε- SRM (SVA)=7.2253. This newly defined index not only gives an idea about ionization potential (i.e., degree of ionization), but can also be an indicator of limit of detection (LOD) of an analyte. When utilizing SRM data recorded from different type of instruments for a compound, the variations that may arise in MESII values could help in understanding the influence of different instrument configurations/methods on the degree of ionization of that compound. Matrix effects on the extent of analyte’s ionization too can be understood from the differences in the MESII values. Further, it may be possible to utilize MESII for quantitation as well.
Escherichia coli RNA polymerase is a multi-subunit enzyme containing α(2)ββ'ωσ, which transcribes... more Escherichia coli RNA polymerase is a multi-subunit enzyme containing α(2)ββ'ωσ, which transcribes DNA template to intermediate RNA product in a sequence specific manner. Although most of the subunits are essential for its function, the smallest subunit ω (average molecular mass ∼ 10,105 Da) can be deleted without affecting bacterial growth. Creating a mutant of the ω subunit can aid in improving the understanding of its role. Sequencing of rpoZ gene that codes for ω subunit from a mutant variant suggested a substitution mutation at position 60 of the protein: asparagine (N) → aspartic acid (D). This mutation was verified at the protein level by following a typical mass spectrometry (MS) based bottom-up proteomic approach. Characterization of in-gel trypsin digested samples by reverse phase liquid chromatography (LC) coupled to electrospray ionization (ESI)-tandem mass spectrometry (MS/MS) enabled in ascertaining this mutation. Electron transfer dissociation (ETD) of triply charged [(M + 3H)(3+)] tryptic peptides (residues [53-67]), EIEEGLINNQILDVR from wild-type and EIEEGLIDNQILDVR from mutant, facilitated in unambiguously determining the site of mutation at residue 60.
The crystal structures of four protected beta-amino acid residues, Boc-(S)-beta3-HAla-NHMe (1); B... more The crystal structures of four protected beta-amino acid residues, Boc-(S)-beta3-HAla-NHMe (1); Boc-(R)-beta3-HVal-NHMe (2); Boc-(S)-beta3-HPhe-NHMe (3); Boc-(S)-beta3-HPro-OH (6) and two beta-dipeptides, Boc-(R)-beta3-HVal-(R)-beta3-HVal-OMe (4); Boc-(R)-beta3-HVal-(S)-beta3-HVal-OMe (5) have been determined. Gauche conformations about the C(beta)-C(alpha) bonds (theta approximately +/-60 degrees) are observed for the beta3-HPhe residues in and all four beta3-HVal residues in the dipeptides and . Trans conformations (theta is approximately 180 degrees) are observed for beta3-HAla residues in both independent molecules in and for the beta3-HVal and beta3-HPro residues in and , respectively. In the cases of compounds , molecules associate in the crystals via intermolecular backbone hydrogen bonds leading to the formation of sheets. The polar strands formed by beta3-residues aggregate in both parallel (1,3,5) and antiparallel (2,4 fashion. Sheet formation accommodates both the trans and gauche conformations about the C(beta)-C(alpha) bonds.
De novo mass spectrometric sequencing of two Conus peptides, Vi1359 and Vi1361, from the vermivor... more De novo mass spectrometric sequencing of two Conus peptides, Vi1359 and Vi1361, from the vermivorous cone snail Conus virgo, found off the southern Indian coast, is presented. The peptides, whose masses differ only by 2 Da, possess two disulfide bonds and an amidated C-terminus. Simple chemical modifications and enzymatic cleavage coupled with matrix assisted laser desorption ionization (MALDI) mass spectrometric analysis aided in establishing the sequences of Vi1359, ZCCITIPECCRI-NH(2), and Vi1361, ZCCPTMPECCRI-NH(2), which differ only at residues 4 and 6 (Z = pyroglutamic acid). The presence of the pyroglutamyl residue at the N-terminus was unambiguously identified by chemical hydrolysis of the cyclic amide, followed by esterification. The presence of Ile residues in both the peptides was confirmed from high-energy collision induced dissociation (CID) studies, using the observation of w(n)- and d(n)-ions as a diagnostic. Differential cysteine labeling, in conjunction with MALDI-MS/MS, permitted establishment of disulfide connectivity in both peptides as Cys2-Cys9 and Cys3-Cys10. The cysteine pattern clearly reveals that the peptides belong to the class of T-superfamily conotoxins, in particular the T-1 superfamily.
Distinctly different effects of two closely related contryphans have been demonstrated on voltage... more Distinctly different effects of two closely related contryphans have been demonstrated on voltage-activated Ca(2+) channels. The peptides Lo959 and Am975 were isolated from Conus loroisii, a vermivorous marine snail and Conus amadis, a molluscivore, respectively. The sequences of Lo959 and Am975 were deduced by mass spectrometric sequencing (MALDI-MS/MS) and confirmed by chemical synthesis. The sequences of Lo959, GCP(D)WDPWC-NH(2) and Am975, GCO(D)WDPWC-NH(2) (O: 4-trans-hydroxyproline: Hyp), differ only at residue 3; Pro in Lo959, Hyp in Am975, which is identical to contryphan-P, previously isolated from Conus purpurascens, a piscivore; while Lo959 is a novel peptide. Both Lo959 and Am975 undergo slow conformational interconversion under reverse-phase chromatographic conditions, a characteristic feature of all contryphans reported thus far. Electrophysiological studies performed using dorsal root ganglion neurons reveal that both peptides target high voltage-activated Ca(2+) channels. While Lo959 increases the Ca(2+) current, Am975 causes inhibition. The results establish that subtle sequence effects, which accompany post-translational modifications in Conus peptides, can have dramatic effects on target ion channels.
The software Peptide Fragment Ion Analyser (PFIA) aids in the analysis and interpretation of tand... more The software Peptide Fragment Ion Analyser (PFIA) aids in the analysis and interpretation of tandem mass spectrometric data of peptides. The software package has been designed to facilitate the analysis of product ions derived from acyclic and cyclic peptide natural products that possess unusual amino acid residues and are heavily post-translationally modified. The software consists of two programmes: (a) PFIA-I lists the amino acid compositions and their corresponding product ion types for 'a queried m/z value' (z = +1) and (b) PFIA-II displays fragmentation pattern diagram(s) and lists all sequence-specific product ion types for the protonated adduct of 'a queried sequence'. The unique feature of PFIA-II is its ability to handle cyclic peptides. The two programmes used in combination can prove helpful for deriving peak assignments in the de novo sequencing of novel peptides.
The fragmentations of [M+H]+ and [M+Na]+ adducts of neutral peptides with blocked N- and C- termi... more The fragmentations of [M+H]+ and [M+Na]+ adducts of neutral peptides with blocked N- and C- termini have been investigated using electrospray ion trap mass spectrometry. The N- termini of these synthetically designed peptides are blocked with a tertiarybutyloxycarbonyl (Boc) group, and the C- termini are esterified. These peptides do not possess side chains that are capable of complexation and hence the backbone amide units are the sole sites of protonation and metallation. The cleavage patterns of the protonated peptides are strikingly different from those of sodium ion adducts. While the loss of the N- terminal blocking group occurs quite readily in the case of MS/MS of [M+Na]+, the cleavage of the C- terminal methoxy group seems to be a facile process in the case of MS/MS of [M+H]+. Fragmentation of the protonated adducts yields only bn ions, while yn and an ions are predominantly formed from the fragmentation of sodium ion adducts. The an ions arising from the fragmentation of [M+Na]+ lack the N- terminal Boc group (and are here termed an* ions). MS/MS of [M+Na]+ species also yields bn ions of substantially lower intensities that lack the N- terminal Boc group (bn). A similar distinction between the fragmentation patterns of proton and sodium ion adducts is observed in the case of peptides possessing an N- terminal acetyl group. An example of the fragmentation of the H+ and Na+ adducts of a naturally occurring peptaibol from a Trichoderma species confirms that fragmentation of these two ionized species yields complementary information, useful in sequencing natural peptides. Inspection of the isotopic pattern of bn ions derived from [M+H]+ adducts of peptaibols provided insights into the sequences of microheterogeneous samples. This study reveals that the combined use of protonated and sodium ion adducts should prove useful in de novo sequencing of peptides, particularly of naturally occurring neutral peptides with modified N- and C- termini, for example, peptaibols.
Ten new cyclic hexadepsipeptides, six isariins and four isaridins, from the fungus Isaria have be... more Ten new cyclic hexadepsipeptides, six isariins and four isaridins, from the fungus Isaria have been identified and characterized by high-performance liquid chromatography, coupled to tandem electrospray ionization mass spectrometry (LC-ESIMS/MS). The isariins possess a â-hydroxy acid residue and five R-amino acids, while isaridins contain a â-amino acid, an R-hydroxy acid, and four R-amino acids. One- and two-dimensional NMR spectroscopy confirmed the chemical identity of some of the isariin fractions. Mass spectral fragmentation patterns of [M + H]+ ions reveal clear diagnostic fragment ions for the isariins and isaridins. Previously described cyclic depsipeptides, isarfelins from Isaria felina (Guo, Y. X.; Liu, Q. H.; Ng, T. B.; Wang H. X. Peptides 2005, 26, 2384), are now reassigned as members of the isaridin family. Examination of isaridin sequences revealed significant similarities with cyclic hexadepsipeptides such as destruxins and roseotoxins. The structure of an isariin (isariin A) investigated by NMR spectroscopy indicated the presence of a hybrid Râ C11 turn, formed by the â-hydroxy acid and glycine residues and a DLeu-LAla type II¢ â-turn. Additionally, the inhibitory effect of isariins and an isaridin on the intra-erythrocytic growth of Plasmodium falciparum is presented.
International Journal of Mass Spectrometry, Mar 2014
Mechanisms of collision induced dissociation (CID) under the conditions of electrospray ionizatio... more Mechanisms of collision induced dissociation (CID) under the conditions of electrospray ionization mass spectrometry (ESI-MS) of cyclic-di-adenosine mono phosphate (c-di-AMP) and cyclic-di-guanosine mono phosphate (c-di-GMP) are described. The CID mass spectrum of doubly protonated ([M+2H] 2+ ) c-di-AMP differs from that of singly protonated ([M+H] + ) and the mechanisms of dissociation of these two ionic forms are proposed. CID of singly deprotonated form ([M−H] − ) of c-di-AMP was also carried out and its fragmentation mechanism is delineated. Ring-opening step seems to be imperative and foremost prior to subsequent dissociation events, during the fragmentation of both cationic and anionic forms of c-di-AMP. In the case of CID of [M+H]+ of c-di-GMP, the abundance of fragment ions indicate that ring-opening mediated fragmentation may not be a favored pathway, while loss of guanine may be relatively more preferred. Interestingly, fragmentation of [M−H] − of c-di-GMP appears to be very similar to that of c-di-AMP, which involves ring-opening step, suggesting that nitrogen base does not influence CID pathways of [M−H] − species. Understanding such dissociation mechanisms will prove useful for discovery and validation of novel hybrid cyclic dinucleotides and their analogues. Also, such comprehensions will be helpful in distinguishing different isobaric molecules possessing varied molecular structures.
Cyclic di-AMP is a recently discovered signaling molecule which regulates various aspects of bact... more Cyclic di-AMP is a recently discovered signaling molecule which regulates various aspects of bacterial physiology and virulence. Here we report the characterization of c-di-AMP synthesizing and hydrolyzing proteins from Mycobacterium tuberculosis. Recombinant Rv3586 (MtbDisA) can synthesize c-di-AMP from ATP through the diadenylate cyclase activity. Detailed biochemical characterization of the protein revealed that the diadenylate cyclase (DAC) activity is allosterically regulated by ATP. We have identified the intermediates of the DAC reaction and propose a two-step synthesis of c-di-AMP from ATP/ADP. MtbDisA also possesses ATPase activity which is suppressed in the presence of the DAC activity. Investigations by liquid chromatography -electrospray ionization mass spectrometry have detected multimeric forms of c-di-AMP which have implications for the regulation of c-di-AMP cellular concentration and various pathways regulated by the dinucleotide. We have identified Rv2837c (MtbPDE) to have c-di-AMP specific phosphodiesterase activity. It hydrolyzes c-di-AMP to 5'-AMP in two steps. First, it linearizes c-di-AMP into pApA which is further hydrolyzed to 5'-AMP. MtbPDE is novel compared to c-di-AMP specific phosphodiesterase, YybT (or GdpP) in being a soluble protein and hydrolyzing c-di-AMP to 5'-AMP. Our results suggest that the cellular concentration of c-di-AMP can be regulated by ATP concentration as well as the hydrolysis by MtbPDE.
Mass Spectrometry based Lipid(ome) Analyzer and Molecular Platform (MS-LAMP) is a new software ca... more Mass Spectrometry based Lipid(ome) Analyzer and Molecular Platform (MS-LAMP) is a new software capable of aiding in interpreting electrospray ionization (ESI) and/or matrix-assisted laser desorption/ionization (MALDI) mass spectrometric data of lipids. The graphical user interface (GUI) of this standalone programme is built using Perl::Tk. Two databases have been developed and constituted within MS-LAMP, on the basis of Mycobacterium tuberculosis (M. tb) lipid database (www.mrl.colostate.edu) and that of Lipid Metabolites and Pathways Strategy Consortium (LIPID MAPS; www.lipidmaps.org).
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Papers by Sabareesh V
‘molecular electrospray ionization index (MESII)’ is defined. Selected reaction monitoring (SRM) data acquired on an
ESI tandem quadrupole mass spectrometer is utilized for calculating MESII, which we denote as εSRM:
εSRM= - log (SRM intensity/Population of ions or molecules in-solution).
Population of ions or molecules in-solution is estimated using molecular mass of the respective compound and
Avogadro’s constant. Simvastatin acid (SVA), lovastatin (LV) and simvastatin (SV) are chosen as model compounds.
SRM experiments in positive ion mode were performed on singly protonated ions ([M+H]+) of SVA, LV and SV. In
negative ion mode, only SVA was investigated by SRM, using singly deprotonated ion ([M-H]-) as precursor ion. Thus
estimated MESII values in positive ion mode are: ε+
SRM (SVA)=7.4288, ε+
SRM (LV)=7.4541 and ε+
SRM (SV)=8.6833 and
in negative ion mode, ε-
SRM (SVA)=7.2253. This newly defined index not only gives an idea about ionization potential
(i.e., degree of ionization), but can also be an indicator of limit of detection (LOD) of an analyte. When utilizing SRM
data recorded from different type of instruments for a compound, the variations that may arise in MESII values could
help in understanding the influence of different instrument configurations/methods on the degree of ionization of that
compound. Matrix effects on the extent of analyte’s ionization too can be understood from the differences in the MESII
values. Further, it may be possible to utilize MESII for quantitation as well.
have been investigated using electrospray ion trap mass spectrometry. The N- termini of these
synthetically designed peptides are blocked with a tertiarybutyloxycarbonyl (Boc) group, and the C- termini
are esterified. These peptides do not possess side chains that are capable of complexation and hence the backbone amide units are the sole sites of protonation and metallation. The cleavage patterns of the protonated peptides are strikingly different from those of sodium ion adducts. While the loss of the N- terminal blocking group occurs quite readily in the case of MS/MS of [M+Na]+, the cleavage of the C- terminal methoxy group seems to be a facile process in the case of MS/MS of
[M+H]+. Fragmentation of the protonated adducts yields only bn ions, while yn and an ions are predominantly formed from the fragmentation of sodium ion adducts. The an ions arising from the fragmentation of [M+Na]+ lack the N- terminal Boc group (and are here termed an* ions). MS/MS of
[M+Na]+ species also yields bn ions of substantially lower intensities that lack the N- terminal Boc group (bn). A similar distinction between the fragmentation patterns of proton and sodium ion adducts is observed in the case of peptides possessing an N- terminal acetyl group. An example of the
fragmentation of the H+ and Na+ adducts of a naturally occurring peptaibol from a Trichoderma species confirms that fragmentation of these two ionized species yields complementary information, useful in sequencing natural peptides. Inspection of the isotopic pattern of bn ions derived from [M+H]+ adducts of peptaibols provided insights into the sequences of microheterogeneous samples. This study reveals that the combined use of protonated and sodium ion adducts should prove useful in de novo sequencing of peptides, particularly of naturally occurring neutral peptides with modified N- and C- termini, for example, peptaibols.
characterized by high-performance liquid chromatography, coupled to tandem electrospray ionization mass spectrometry
(LC-ESIMS/MS). The isariins possess a â-hydroxy acid residue and five R-amino acids, while isaridins contain a â-amino
acid, an R-hydroxy acid, and four R-amino acids. One- and two-dimensional NMR spectroscopy confirmed the chemical
identity of some of the isariin fractions. Mass spectral fragmentation patterns of [M + H]+ ions reveal clear diagnostic
fragment ions for the isariins and isaridins. Previously described cyclic depsipeptides, isarfelins from Isaria felina (Guo,
Y. X.; Liu, Q. H.; Ng, T. B.; Wang H. X. Peptides 2005, 26, 2384), are now reassigned as members of the isaridin
family. Examination of isaridin sequences revealed significant similarities with cyclic hexadepsipeptides such as destruxins
and roseotoxins. The structure of an isariin (isariin A) investigated by NMR spectroscopy indicated the presence of a
hybrid Râ C11 turn, formed by the â-hydroxy acid and glycine residues and a DLeu-LAla type II¢ â-turn. Additionally,
the inhibitory effect of isariins and an isaridin on the intra-erythrocytic growth of Plasmodium falciparum is presented.
‘molecular electrospray ionization index (MESII)’ is defined. Selected reaction monitoring (SRM) data acquired on an
ESI tandem quadrupole mass spectrometer is utilized for calculating MESII, which we denote as εSRM:
εSRM= - log (SRM intensity/Population of ions or molecules in-solution).
Population of ions or molecules in-solution is estimated using molecular mass of the respective compound and
Avogadro’s constant. Simvastatin acid (SVA), lovastatin (LV) and simvastatin (SV) are chosen as model compounds.
SRM experiments in positive ion mode were performed on singly protonated ions ([M+H]+) of SVA, LV and SV. In
negative ion mode, only SVA was investigated by SRM, using singly deprotonated ion ([M-H]-) as precursor ion. Thus
estimated MESII values in positive ion mode are: ε+
SRM (SVA)=7.4288, ε+
SRM (LV)=7.4541 and ε+
SRM (SV)=8.6833 and
in negative ion mode, ε-
SRM (SVA)=7.2253. This newly defined index not only gives an idea about ionization potential
(i.e., degree of ionization), but can also be an indicator of limit of detection (LOD) of an analyte. When utilizing SRM
data recorded from different type of instruments for a compound, the variations that may arise in MESII values could
help in understanding the influence of different instrument configurations/methods on the degree of ionization of that
compound. Matrix effects on the extent of analyte’s ionization too can be understood from the differences in the MESII
values. Further, it may be possible to utilize MESII for quantitation as well.
have been investigated using electrospray ion trap mass spectrometry. The N- termini of these
synthetically designed peptides are blocked with a tertiarybutyloxycarbonyl (Boc) group, and the C- termini
are esterified. These peptides do not possess side chains that are capable of complexation and hence the backbone amide units are the sole sites of protonation and metallation. The cleavage patterns of the protonated peptides are strikingly different from those of sodium ion adducts. While the loss of the N- terminal blocking group occurs quite readily in the case of MS/MS of [M+Na]+, the cleavage of the C- terminal methoxy group seems to be a facile process in the case of MS/MS of
[M+H]+. Fragmentation of the protonated adducts yields only bn ions, while yn and an ions are predominantly formed from the fragmentation of sodium ion adducts. The an ions arising from the fragmentation of [M+Na]+ lack the N- terminal Boc group (and are here termed an* ions). MS/MS of
[M+Na]+ species also yields bn ions of substantially lower intensities that lack the N- terminal Boc group (bn). A similar distinction between the fragmentation patterns of proton and sodium ion adducts is observed in the case of peptides possessing an N- terminal acetyl group. An example of the
fragmentation of the H+ and Na+ adducts of a naturally occurring peptaibol from a Trichoderma species confirms that fragmentation of these two ionized species yields complementary information, useful in sequencing natural peptides. Inspection of the isotopic pattern of bn ions derived from [M+H]+ adducts of peptaibols provided insights into the sequences of microheterogeneous samples. This study reveals that the combined use of protonated and sodium ion adducts should prove useful in de novo sequencing of peptides, particularly of naturally occurring neutral peptides with modified N- and C- termini, for example, peptaibols.
characterized by high-performance liquid chromatography, coupled to tandem electrospray ionization mass spectrometry
(LC-ESIMS/MS). The isariins possess a â-hydroxy acid residue and five R-amino acids, while isaridins contain a â-amino
acid, an R-hydroxy acid, and four R-amino acids. One- and two-dimensional NMR spectroscopy confirmed the chemical
identity of some of the isariin fractions. Mass spectral fragmentation patterns of [M + H]+ ions reveal clear diagnostic
fragment ions for the isariins and isaridins. Previously described cyclic depsipeptides, isarfelins from Isaria felina (Guo,
Y. X.; Liu, Q. H.; Ng, T. B.; Wang H. X. Peptides 2005, 26, 2384), are now reassigned as members of the isaridin
family. Examination of isaridin sequences revealed significant similarities with cyclic hexadepsipeptides such as destruxins
and roseotoxins. The structure of an isariin (isariin A) investigated by NMR spectroscopy indicated the presence of a
hybrid Râ C11 turn, formed by the â-hydroxy acid and glycine residues and a DLeu-LAla type II¢ â-turn. Additionally,
the inhibitory effect of isariins and an isaridin on the intra-erythrocytic growth of Plasmodium falciparum is presented.