Nicolae Dinca

Coupling of fully automated chip-based electrospray ionization to high-capacity ion trap mass spectrometer for ganglioside analysis

NanoMate robot was coupled to a high-capacity ion trap (HCT) mass spectrometer to create a system merging automatic chip-based electrospray ionization (ESI) infusion, ultrafast ion detection, and multistage sequencing at superior sensitivity. The interface between the NanoMate and HCT mass spectrometer consists of an in-laboratory constructed mounting device that allows adjustment of the robot position with respect to the mass spectrometer inlet. The coupling was optimized for ganglioside (GG) high-throughput analysis in the negative ion mode and was implemented in clinical glycolipidomics for identification and structural characterization of anencephaly-associated species. By NanoMate HCT mass spectrometry (MS), data corroborating significant differences in GG expression in anencephalic versus age-matched normal brain tissue were collected. The feasibility of chip-based nanoESI HCT multistage collision-induced dissociation (CID MS(n)) for polysialylated GG fragmentation and isomer discrimination was tested on a GT1 (d18:1/18:0) anencephaly-associated structure. MS(2)-MS(4) obtained by accumulating scans at variable fragmentation amplitudes gave rise to the first fragmentation patterns from which the presence of GT1b structural isomer could be determined unequivocally without the need for supplementary investigation by any other analytical or biochemical methods.

Analysis of novel over- and under-sulfated glycosaminoglycan sequences by enzyme cleavage and multiple stage MS.

We report on a novel strategy for identification of specific sulfation motifs in chondroitin/dermatan sulfate (CS/DS) chain derived from decorin (Dcn), based on enzyme cleavage and multistage MS (MSn). Released CS/DS chains were digested with chondroitin B and in parallel with AC I lyases to obtain oligosaccharides of known hexuronic acid (HexA) epimerization. The depolymerized chains were separated by gel filtration, and collected di‐ and hexasaccharides were analyzed by ESI MSn. MS2 on bisulfated 4,5‐Δ‐HexAGalNAc revealed an additional sulfate ester group at 4,5‐Δ‐HexA. MS2 data provided evidence upon GlcA sulfation in Dcn due to the fact that 4,5‐Δ‐HexA derived from GlcA after chondroitin AC I lyase treatment. Hexasaccharide screening in the MS1 mode indicated direct correlation between the sulfate distribution and HexA epimerization. MSn performed on ions that, according to mass calculation, correspond to pentasulfated [4,5‐Δ‐HexAGalNAc(GlcAGalNAc)2], trisulfated [4,5‐Δ‐HexAGalNAc(GlcAGalNAc)2] with IdoA‐derived 4,5‐Δ‐HexA at the nonreducing end, tetrasulfated [4,5‐Δ‐HexAGalNAc(IdoAGalNAc)2] and monosulfated [4,5‐Δ‐HexAGalNAc(IdoAGalNAc)2] with GlcA‐derived 4,5‐Δ‐HexA at the nonreducing end rendered fragmentation patterns confirming the presence of over‐, regular, and under‐sulfated regions as well as structural motifs having both types of HexA sulfated within Dcn CS/DS.

Synthesis and structural characterization of amino-functionalized polysaccharides

A variety of carbohydrates, in particular polysaccharides can be subjected to chemical modification to obtain derivatives with amphiphilic properties, which enable biochemical or biological reactions at the polymer surface. In the present work, a polydisperse maltodextrin mixture of average molecular weight 3000 was coupled with 1,6-hexamethylenediamine (HMD) via reductive amination reaction. Resulting products were characterized by thermal analysis and positive nanoelectrospray quadrupole time-of-flight (Q-TOF) mass spectrometry (MS) and tandem mass spectrometry (MS/MS). Both thermal analysis and MS screening confirmed the formation of the HMD-polysaccharide coupling products. Moreover, HMD-linked polysaccharide chains containing 2 to 26 glucose building blocks were identified by nanoESI Q-TOF MS. MS/MS fragmentation using collision-induced dissociation (CID) at low ion acceleration energies provided strong evidence for HMD-maltodextrin linkage formation and the set of sequence ions diagnostic for the composition and structure of a HMD-linked chain containing 18 glucose residues.

Enhanced electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry of long-chain polysaccharides

A novel strategy was developed to extend the application of electrospray ionization (ESI) Fourier transform ion cyclotron resonance (FTICR) mass spectrometry (MS) to the analysis of long-chain polysaccharides. High molecular weight polydisperse maltodextrins (poly-alpha(1-4) glucose) and dextrans (poly-alpha(1-6) glucose) were chosen as model compounds in the present study. Increased ionization efficiency of these mixtures in the positive ion mode was achieved upon modification of their reducing end with nitrogen-containing groups. The derivatization method is based on the formation of a new C--N bond between 1,6-hexamethylenediamine (HMD) and the reducing end of the polysaccharide, which exists in solution as an equilibrium between the hemiacetal and the open-ring aldehyde form. To achieve the chemical modification of the reducing end, two synthetic pathways were developed: (i) coupling of HMD by reductive amination and (ii) oxidation of the hemiacetal to lactone, followed by ring opening by HMD to yield the maltodextrin lactonamide of 1,6-hexanediamine (HMMD). Amino-functionalized polysaccharides were analyzed by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI FTICR-MS) in the positive ion mode by direct flow injection. The hexamethylenediamine (HMD) and maltodextrin lactonamide of 1,6-hexanediamine (HMMD) moieties provide increased proton affinities which dramatically improve the detection of the long-chain polysaccharides by FTICR-MS. The present approach allowed for identification of single components in mixtures with prominent heterogeneity in the degree of polymerization (DP), without the need for chromatographic separation prior to MS. The high mass accuracy was essential for the unambiguous characterization of the species observed in the analyzed mixtures. Furthermore, molecular components containing up to 42 glucose residues were detected, representing the largest polysaccharide chains analyzed so far by ESI FTICR-MS.

Structural identification by differential mass spectrometry as a criterion for selecting the best quantum chemical calculation of formation enthalpy for tetrachlorinated biphenyls.

RATIONALE The assignment of correct structures for isomers with similar mass spectra (e.g. polyhalogenated aromatic compounds) is not always successful when spectral libraries alone are employed or, even worse, when the compounds are not present in commercial spectral libraries. METHODS We present a computational method based on differential mass spectrometry (Diff-MS) for the validation of formation enthalpy (Δ(f)H) series calculated using quantum chemistry for the fragments produced in electron ionization (EI)-MS. The method simulates the chemical structure identification (CSI) of isomers with similar mass spectra using differential mass spectra and Δ(f)H series. The best Δ(f)H values were those from which the correct structures could be derived. RESULTS We have used six tetrachlorinated biphenyl isomers (TeCBs 44, 46, 52, 66, 74, 77). Their EI mass spectra were acquired at 70 eV and, for the principal ions, five series of Δ(f) H values were computed by the semi-empirical methods, AM1, MINDO3, MNDO, PM3, and RM1. The generation of differential mass spectra and the correlation with the Δ(f)H series for the calculation of probabilities from the list of structural assignments were carried out with the ordering algorithm (ORD) of the CSI-Diff-MS Data Analysis 3.1.1 program. CONCLUSIONS Intelligent software, used for structural elucidation based on MS and QCC, was employed to select the best values of the formation enthalpies of TeCBs. The advantages and disadvantages of the semi-empirical methods for the calculation of Δ(f)H values for different TeCB ions are critically presented. The best semi-empirical methods were RM1, AM1 and MINDO3, which can be used to calculate the Δ(f)H database necessary to identify TeCB isomers. This approach allowed the correct assignment of structures for isomers with very similar mass spectra and demonstrated the reliability of the correlation between differential mass spectra and the formation enthalpies of the fragment ions.

Chemical Structure Identification by Differential Mass Spectra

In this paper a new technique FOR mass spectra interpretation based on chemical structure identification by differential mass spectra (CSI Diff-ms) is presented. CSI Diff-ms is broadening the applicability of the mass spectrometry in the area of identification of isomer structure for analytes exhibiting similar mass spectra. By this technique are correlated differential mass spectra (diff ms) of the isomers and formation enthalpies that are calculated quantumchemical for the molecules and the formed ions in the mass spectrum for the structure identification. Here are presented types of differential spectra the theoretical justification of the correlation between the spectra ratio and formation enthalpies of the ions and three applications which allow to establish the chemical structures of some aromatic position isomers’ and of configuration (endo- si exo-) using the fragmentation spectra. The CSI Diff-ms software allows to import mass spectra, is calculating the differential mass spectra, the spectra similarity, and makes the correlation diff ms with the formation enthalpies calculated in order to obtain the structures probabilities list. The knowledge of the intimate structure of the isomers with help of this technique represents an important progress in the knowledge of the relation structure-activity or toxicity of the natural and synthetic chemical compounds.

New Quantitative Structure-Fragmentation Relationship Strategy for Chemical Structure Identification Using the Calculated Enthalpy of Formation as a Descriptor for the Fragments Produced in Electron Ionization Mass Spectrometry: A Case Study with Tetrachlorinated Biphenyls

Differential mass spectrometry correlated with quantum chemical calculations (QCC-ΔMS) has been shown to be an efficient tool for the chemical structure identification (CSI) of isomers with similar mass spectra. For this type of analysis, we report here a new strategy based on ordering (ORD), linear correlation (LCOR) algorithms, and their coupling, to filter the most probable structures corresponding to similar mass spectra belonging to a group with dozens of isomers (e.g., tetrachlorinated biphenyls, TeCBs). This strategy quantifies and compares the values of enthalpies of formation (Δ(f)H) obtained by QCC for some isobaric ions from the electron ionization (EI)-MS mass spectra, to the corresponding relative intensities. The result of CSI is provided in the form of lists of decreasing probabilities calculated for all the position-isomeric structures using the specialized software package CSI-Diff-MS Analysis 3.1.1. The simulation of CSI with ORD, LCOR, and their coupling of six TeCBs (IUPAC no. 44, 46, 52, 66, 74, and 77) has allowed us to find the best semiempirical molecular-orbital methods for several of their common isobaric fragments. The study of algorithms and strategy for the entire group of TeCBs (42 isomers) was made with one of the optimal variants for the computation of Δ(f)H using semiempirical molecular orbital methods of HyperChem: AM1 for M(+•) and [M - 4Cl](+•) ions and RM1 for [M - Cl](+) and [M - 2Cl](+•). The analytical performance of ORD, LCOR, and their coupling resulted from the CSI simulation of an analyte of known structure, using a decreasing number of isomeric standards, s = 5, 4, 3, and 2. Compared with the results obtained by a classical library search for TeCB isomers, the novel strategies of assigning structures of isomers with very similar mass spectra based on ORD, LCOR, and their coupling were much more efficient, because they provide the correct structure at the top of the probability list. Databases used in these CSI do not contain mass spectra, as in the case of a library search, but a series of Δ(f)H values obtained by QCC. These techniques are capable of relating relative intensities to the chemical structures of analytes via Δ(f)H of ions which turns out to be a good quantitative structure-fragmentation relationship (QSFR) descriptor.

Stereochemistry Studies of Some 1,3-dioxane Derivatives by Differential Mass Spectrometry and Computational Chemistry

In this work mass spectrometry is for the first time used in the stereo-chemical study of and conformational equilibriums of the saturated 6 atom rings, namely of 1,3 dioxans: cis- and trans-2-phenyl-4-methyl-l,3-dioxane and cis- and trans-2-phenyl-5-methyl-1, 3-dioxane. The technique involved is based on the correlation of the experimental differential mass spectra (diff ms) with the enthalpies of formation obtained through quantum-chemical calculations for the main fragmentation ions from the mass spectra. The lists with the correlation probabilities were obtained using the computer program Chemical Structure Identification by Differential Mass Spectra (CSI Diff ms). The obtained results are in agreement with NMR and X-Ray studies made of these compounds. This also highlighted the preference of the aromatic substituent for the axial position, when the molecule is isolated (in vacuum).

Structural Analysis of Chondroitin Sulfate Disaccharides by Electrospray Ionization High Capacity Ion Trap Mass Spectrometry

Decorin is a small leucine-rich repeat proteoglycan linked to a single chondroitin or dermatan sulfate (CS/DS) glycosaminoglycan (GAG) chain depending on tissue expressed. Determination of molecular characteristics of CS/DS, is essential for understanding decorin functions. CS/DS analysis includes the identification of glycoforms obtained after detachment from coreprotein followed by collecting information upon epimerization, sulfate content and its distribution. For this purpose, one of the most efficient methods is electrospray ionization (ESI) tandem mass spectrometry (MS/MS). Here we present an approach for structural investigation of human skin decorin CS disaccharides based on combining a specific enzyme digestion with negative ion mode ESI MS-screening followed by fragmentation analysis by collision-induced dissociation (CID) MS/MS. The obtained results showed clearly that human decorin encompasses oversulfated CS motifs in which the site of oversulfation is at glucuronic acid. Moreover, it was possible to demonstrate that the presence of regular and/or irregular GAG domains, which play a critical biological role, can be directly documented by ion sequencing in tandem MS experiments.