Positive Ion Mode ESI-MS Research Articles

Development and application of a sensitive phosphonium-hydrazide oligosaccharide labelling reagent in capillary electrophoresis- electrospray ionization- mass spectrometry

Glycosylation is one of the most ubiquitous post-translational modifications (PTM) of proteins. Although the ionization efficiency of native glycans is fairly low, with the assistance of chemical derivation strategies, mass spectrometry (MS) has been extensively used in glycomics because of its high sensitivity, accuracy, and speed. In this study, a novel glycan labelling reagent, (4-hydrazidebutyl) triphenylphosphonium bromide (P4HZD), with a permanent positive charge was developed. The comprehensive capabilities of P4HZD for MS analysis of oligosaccharides were evaluated in detail using maltodextrin as a standard. This labelling reagent can be used in common biological MS techniques such as matrix-assisted laser desorption ionization (MALDI) and electrospray ionization (ESI) mass spectrometry. The MS signal intensity of maltodextrin species could be enhanced up to 96-fold in MALDI-MS by labelling with P4HZD, making P4HZD favorable for MALDI-MS-based high-throughput screening of oligosaccharides. Moreover, P4HZD-labelled oligosaccharides with a degree of polymerization (DP) from 1 to 18 could be separated and analysed by capillary electrophoresis (CE) combined with positive ion mode ESI-MS. In comparison with a commercialized oligosaccharide tag, Girard's reagent P (GirP), P4HZD was more effective for enhancing the signal of oligosaccharides in the middle or higher mass range using both ESI and MALDI ion sources. Two biologics, immunoglobulin G 2 (IgG 2) and fusion protein (FP), were chosen as model complex biological samples to test the efficacy of detection and separation of oligosaccharides by MALDI-MS and CE-ESI-MS analysis with P4HZD labelling. The results indicated that P4HZD is a promising labelling reagent for the detection of oligosaccharides in complex biological samples. The tandem workflow combines the strengths of MALDI-MS and CE-ESI-MS to fulfil the analytical demands of high-throughput screening, while affording good separation.

Sequences of Acretocins, Peptaibiotics Containing the Rare 1-Aminocyclopropanecarboxylic Acid, from Acremonium crotocinigenum CBS 217.70.

Sequences of Acretocins, Peptaibiotics Containing the Rare 1-Aminocyclopropanecarboxylic Acid, from Acremonium crotocinigenum CBS 217.70.

Monohexosylceramides from Rhizopus Species Isolated from Brazilian Caatinga: Chemical Characterization and Evaluation of Their Anti-Biofilm and Antibacterial Activities.

Monohexosylceramides (CMHs) are highly conserved fungal glycosphingolipids playing a role in several cellular processes such as growth, differentiation and morphological transition. In this study, we report the isolation, purification and chemical characterization of CMHs from Rhizopus stolonifer and R. microspores. Using positive ion mode ESI-MS, two major ion species were observed at m/z 750 and m/z 766, respectively. Both ion species consisted of a glucose/galactose residue attached to a ceramide moiety containing 9-methyl-4,8-sphingadienine with an amidic linkage to a hydroxylated C16:0 fatty acid. The antimicrobial activity of CMH was evaluated against Gram positive and Gram negative bacteria using the agar diffusion assay. CMH from both Rhizopus species inhibited the growth of Bacillus terrae, Micrococcus luteus (M. luteus) and Pseudomonas stutzeri (P. stutzeri) with a MIC50 of 6.25, 6.25 and 3.13 mg/mL, respectively. The bactericidal effect was detected only for M. luteus and P. stutzeri, with MBC values of 25 and 6.25 mg/mL, respectively. Furthermore, the action of CMH on the biofilm produced by methicillin-resistant Staphylococcus aureus (MRSA) was analyzed using 12.5 and 25 mg/mL of CMH from R. microsporus. Total biofilm biomass, biofilm matrix and viability of the cells that form the biofilm structure were evaluated. CMH from R. microsporus was able to inhibit the MRSA biofilm formation in all parameters tested.

The 4-pyridylmethyl ester as a protecting group for glutamic and aspartic acids: ‘flipping’ peptide charge states for characterization by positive ion mode ESI-MS

Use of the 4-pyridylmethyl ester group for side-chain protection of glutamic acid residues in solid-phase peptide synthesis enables switching of the charge state of a peptide from negative to positive, thus making detection by positive ion mode ESI-MS possible. The pyridylmethyl ester moiety is readily removed from peptides in high yield by hydrogenation. Combining the 4-pyridylmethyl ester protecting group with benzyl ester protection reduces the number of the former needed to produce a net positive charge and allows for purification by RP HPLC. This protecting group is useful in the synthesis of highly acidic peptide sequences, which are often beset by problems with purification by standard RP HPLC and characterization by ESI-MS.

Sensitive analysis of metal cations in positive ion mode electrospray ionization mass spectrometry using commercial chelating agents and cationic ion-pairing reagents

Metals play a very important role in many scientific and environmental fields, and thus their detection and analysis is of great necessity. A simple and very sensitive method has been developed herein for the detection of metals in positive ion mode ESI-MS. Metal ions are positively charged, and as such they can potentially be detected in positive ion mode ESI-MS; however, their small mass-to-charge (m/z) ratio makes them fall in the low-mass region of the mass spectrum, which has the largest background noise. Therefore, their detection can become extremely difficult. A better and well-known way to detect metals by ESI-MS is by chelating them with complexation agents. In this study eleven different metals, Fe(II), Fe(III), Mg(II), Cu(II), Ru(III), Co(II), Ca(II), Ni(II), Mn(II), Sn(II), and Ag(I), were paired with two commercially available chelating agents: ethylenediaminetetraacetic acid (EDTA) and ethylenediaminedisuccinic acid (EDDS). Since negative ion mode ESI-MS has many disadvantages compared to positive ion mode ESI-MS, it would be very beneficial if these negatively charged complex ions could be detected in the positive mode. Such a method is described in this paper and it is shown to achieve much lower sensitivities. Each of the negatively charged metal complexes is paired with six cationic ion-pairing reagents. The new positively charged ternary complexes are then analyzed by ESI-MS in the positive single ion monitoring (SIM) and single reaction monitoring (SRM) modes. The results clearly revealed that the presence of the cationic reagents significantly improved the sensitivity for these analytes, often by several orders of magnitude. This novel method developed herein for the detection of metals improved the limits of detection (LODs) significantly when compared to negative ion mode ESI-MS and shows great potential in future trace studies of these and many other species.

LC–DAD–ESI-MS-MS for Characterization and Quantitative Analysis of Diterpenoids from Coleus forskohlii

Qualitative characterization and quantitative analysis of labdane diterpenoids from Coleus forskohlii have been achieved by liquid chromatography hyphenated with photodiode-array detection and tandem electrospray ionization mass spectrometry (LC–DAD–ESI-MS n ). By use of this method, thirteen forskolin-type diterpenoids were identified in the crude extract on the basis of their fragmentation mechanisms. Fragmentation rules were deduced from nine forskolin-type standards by ESI-MS in positive-ion mode. It was found that fragmentation behavior varied with the position and number of the substituents on the skeleton; this could be used for convenient identification of this type of compound. Six marker diterpenoids were also quantified, and the quality of both the cultivated and wild plants was evaluated.

Sensitivity of positive ion mode electrospray ionization mass spectrometry (ESI MS) in the analysis of purine bases in ESI MS and on-line electrochemistry ESI MS (EC/ESI MS)

A cone-shaped MS inlet and on-line electrochemistry (EC) were used to enhance the ionization efficiency in electrospray ionization mass spectrometry (ESI MS) of purine bases. A pathway of positive ion mode ESI may involve oxidation of purine bases, guanine, adenine, xanthine and hypoxanthine, by 1e −, 1H + processes. The electrospray process generates dimers of purine bases that are detected in ESI MS as protonated ions, except for xanthine, for which a protonated radical dimer is detected. Thus electrochemical oxidation of purine bases during ESI may generate reactive radicals that can subsequently dimerize. Dimer formation is facilitated in ESI MS when the carrier solution pH is high. The positive ion mode ESI MS ionization is consistent with the reactivity of the bases toward oxidation. Furthermore, the formation of the protonated ions, and Na + and K + adducts of the bases, expected in positive ion ESI MS, are observed. In addition, unusual H-bonding of purine bases guanine and xanthine is confirmed by ESI MS. Application of low EC voltage to the on-line EC cell in EC/ESI MS improves the sensitivity and correlates with the decrease of the intensity of the dimers, possibly as a result of their further oxidation.

Relative Quantification of Carboxylic Acid Metabolites by Liquid Chromatography−Mass Spectrometry Using Isotopic Variants of Cholamine

Labeling reagents that differ only in their isotopic composition offer a powerful approach to achieve relative quantification between samples by ESI-MS. Heavy and light isotopic forms of cholamine, which contain a positively charged quaternary ammonium group, were synthesized and tested as new labeling reagents for the relative quantification of carboxylic acid-containing metabolites, specifically fatty acids. The positive charge on cholamine ensures that the labeled product is also positively charged under all LC-MS conditions, regardless of mobile-phase pH. This leads to high ionization efficiency and correspondingly high detection sensitivity, demonstrated here for the analysis of fatty acids in positive ion mode ESI-MS after reversed-phase separation under acidic conditions. Good accuracy and precision were obtained by mixing heavy- and light-labeled hydrolyzed egg lipid extracts in different known ratios. The relative quantification results for 10 observed fatty acids had an average absolute error of 4.6% and an average coefficient of variation (CV) of 2.6%. The labeling strategy yielded a median CV of 6% when employed for fatty acid analysis of eggs from chickens fed various dietary supplements.

Detection, characterization and identification of crucifer phytoalexins using high-performance liquid chromatography with diode array detection and electrospray ionization mass spectrometry

We have analyzed 23 crucifer phytoalexins (e.g. brassinin, dioxibrassinin, cyclobrassinin, brassicanals A and C) by HPLC with diode array detection and electrospray ionization mass spectrometry (HPLC-DAD–ESI-MS) using both negative and positive ion modes. Positive ion mode ESI-MS appeared more sensitive than negative ion mode ESI-MS in detecting this group of compounds. A new HPLC separation method, new LC–MS and LC–MS 2 data and proposed fragmentation pathways, LC retention times, and UV spectra for selected compounds are reported.

A Deoxynucleotide Derivatization Methodology for Improving LC-ESI-MS Detection

We have developed a novel LC-UV-MS derivatization method for the analysis of deoxyguanosine monophosphate adducts that demonstrates enhanced signal intensities relative to underivatized analytes in positive ion mode electrospray ionization MS. Detection of DNA nucleotide adducts is normally conducted in negative ion mode, which requires basic mobile phases that make chromatographic separations difficult and reduce MS sensitivity. Utilizing coupling reagents typically employed in peptide synthesis, several different deoxyguanosine nucleotide phosphoramidates and phosphomonoesters were synthesized in high conversion yield and under mild reaction conditions. The derivatives were characterized by MS/MS and reaction conversion yields determined from the DAD-UV traces. The derivatives were evaluated for ionization efficiencies, fragmentation patterns, and reversed-phase chromatographic properties by LC/ESI-MS/MS. Overall, the hydrophobic derivatives showed increases in ionization efficiency and improved peak shape. Rank ordering of the derivatizing agents was initially established using the dGp-modified derivatives. The best derivatizing agent, hexamethyleneimine, showed a 3-4-fold signal enhancement compared to underivatized dGp and was selected for additional evaluation. A model system using the carcinogen, N-acetoxy-2-acetylaminofluorene (AAAF), was used to synthesize a N-acetyl-(2-aminofluorenyl)-guanosine 5'-monophosphate (dGpAAF) adduct, which was subsequently derivatized with hexamethyleneimine. Detection limits for dGphex and dGpAAFhex, purified by HPLC, were 10- and 3-fold higher (S/N) than their respective underivatized analogues. Practical applicability, with similar improvements in sensitivity, was established by derivatizing adducts isolated from calf thymus DNA exposed to AAAF. Our results demonstrate the utility of simple reactions for the enhanced detection of a mononucleotide in positive ion mode ESI MS and the application of this technique for the detection of dGp-DNA adducts at the low-femtomole level.

Determination of domperidone in human serum and human breast milk by high-performance liquid chromatography–electrospray mass spectrometry

A sensitive and selective method for the determination of domperidone in human breast milk and serum has been developed. The same method may be successfully applied to both matrices to a lower limit of quantitation of 0.5 ng/ml. Samples are processed by a liquid–liquid extraction, and analyzed by LC–ESI-MS in positive ion mode. There was no interference, on the domperidone quantitation, from over 30 drugs. Samples from patients, at various times post-dose, were analyzed and a large number showed significant levels of domperidone in the breast milk as well as in the serum.

Separation and identification of positively charged and neutral nucleoside adducts by capillary electrochromatography-microelectrospray mass spectrometry

Capillary electrochromatography (CEC) is shown to be capable of separating mixtures containing both positively charged and neutral styrene oxide–adenosine adducts. In a study of the mechanism of deamination of positively charged 1-(2-hydroxy-1-phenylethyl) adenosine using 18O-labeled water, possible contamination of the chromatographically purified deamination product, 1-(2-hydroxy-1-phenylethyl) inosine, with the positively charged 1-(2-hydroxy-1-phenylethyl) adenosine was observed. Because the deamination product and the presumed contamination have the same molecular weights and similar structures, CEC-microelectrospray mass spectrometry (CEC-μESI/MS) was used to confirm the presence and identity of the suspected impurity. A trace amount of the positively charged 1-(2-hydroxy-1-phenylethyl) adenosine, which could not be observed by either HPLC-UV or CEC-UV, was detected by CEC-μESI/MS. This discriminatory ability of CEC-μESI/MS is attributed to the fact that positive ion mode ESI-MS is a more sensitive detector for a positively charged compound than a UV detector, and that the combination of electroosmotic and electrophoretic flows and hydrophobic interactions with the stationary phase contributes to the separation of the positively charged compound. As a result, the positively charged compound was observed to elute much earlier and with much sharper peaks than the neutral compounds for which electroosmotic flow is the only “pumping” force for the solvent.