Insights into the binding sites of sulforaphane on insulin studied by electrospray ionization mass spectrometry.

RATIONALE: Sulforaphane (SFN) is a natural isothiocyanate, known to reduce the risk of cancer and also aortic damage and diabetic cardiomyopathy induced by type 2 diabetes, etc. A more detailed knowledge on the direct interaction of SFN with insulin and its binding sites is necessary for better understanding the role of SFN on diabetes. METHODS: Liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) and in-source fragmentation experiments were performed on a Thermo Exactive orbitrap mass spectrometer. The solution of insulin and SFN was incubated and analyzed by mass spectrometry. Isotopic distribution pattern, accurate mass values and theoretical product ions were used to analyze the mass spectrometry data. The nature of binding of SFN and its binding sites with insulin were evaluated by LC/MS data. RESULTS: ESI-MS analysis of the incubated solution of insulin and SFN showed 1:1 and 1:2 complexes of [Insulin-SFN]. LC/MS analysis revealed that the [Insulin+SFN] complexes were due to covalent binding of SFN at two different sites. The in-source fragmentation experiments revealed that the SFN is binding to the NH2 groups of N-terminal amino acids of A and B chains of insulin. Further study of SFN with insulin reduced with dithiothreitol (DTT) showed exclusive modification of cysteines with SFN. CONCLUSIONS: The interaction of SFN was studied with insulin using ESI-MS. SFN is found to bind covalently with the free NH2 group of the N-terminal of the A and B chains of insulin. However, when insulin is reduced SFN preferably binds to SH groups of cysteines. Hence, the present study helps in the understanding of the binding sites of SFN on insulin.

Sulforaphane interaction with amyloid beta 1-40 peptide studied by electrospray ionization mass spectrometry.

RATIONALE: Aggregation of amyloid beta 1-40 (Aß) in the brain causes Alzheimer's disease (AD) and several small molecules are known to inhibit the aggregation process. Sulforaphane (SFN) is a natural isothiocyanate which is known to prevent various neurodegenerative processes. However, its interaction with Aß is yet to be explored. Such studies could provide new mechanistic insights for its neuroprotective properties. METHODS: Liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) and in-source fragmentation experiments were performed on an Orbitrap mass spectrometer. The solution of Aß and SFN was incubated and analyzed by mass spectrometry. Isotopic distribution patterns, accurate mass values and theoretical product ions were used to analyze the mass spectrometry data. The nature of binding of SFN and its binding sites with Aß were evaluated by LC/MS and trypsin digestion experiments. RESULTS: ESI-MS analysis of the incubated solution of Aß and SFN showed a 1:1 complex of [Aß+SFN]. LC/MS analysis revealed that the solution contains three different [Aß+SFN] complexes due to covalent binding of SFN to Aß at three different sites. The in-source fragmentation experiments revealed that SFN is binding to free NH(2) groups (N-terminal amino acid and lysines) in Aß. Trypsin digestion experiments further confirmed the SFN binding sites in Aß. CONCLUSIONS: The interaction of SFN, an anticancer agent, with Aß was studied using ESI-MS. SFN is found to bind covalently and specifically with the free NH(2) group of N-terminal aspartic acid and the ε-amino group of lysine at positions 16 and 28. Aggregation assay studies showed a lesser inclination of Aß to aggregate when SFN is present. Hence the present study helps in understanding the mechanism of the action of SFN on the Aß peptide.

Targeting insulin amyloid assembly by aminosugars and their derivatives.

The use of small carbohydrates that stabilize proteins from misfolding is important from pharmaceutical point of view. We have investigated the role of small isomeric amino sugars on the in vitro aggregation of insulin amyloid. Using mass spectrometry, we screened 6 isomeric aminosugars for their role on inhibition of insulin amyloid formation and the results were compared with transmission electron microscopy imaging. We found that three N-acetylamino sugars promote insulin fibril formation. Among three isomeric aminosugars studied, only galactosamine showed few fibrils whereas other two isomers showed enhanced fibrils. The results demonstrated here may contribute to future designing of small amine derivatised galactose sugars as amyloid inhibitors and understanding their action.

Simple yet stringent screening methodologies for evaluation of putative transformants for abiotic stress tolerance: salt and cadmium stress as a paradigm.

Rigorous and stringent screening methodologies to select transformants at both seedling and plant level under cadmium or NaCl stress were developed. At seedling level, two screening strategies were standardized. One involved germination on filter paper/agar in the presence of either CdCl2 (125 µM) or NaCl (350-450 mM) for 9 days and selection of tolerant putative transformants. The other involved germination of the seedlings on soilrite by irrigation of 450 mM NaCl. Further, at plant level, in vitro evaluation for stress tolerance involved a simple leaf senescence bioassay. Combination of the seedling and plant level screening strategies would result in the initial identification of promising transformants for further analysis.

Differentiation of underivatized diastereomeric hexosamine monosaccharides and their quantification in a mixture using the kinetic method under electrospray ionization conditions.

A simple method to differentiate underivatized diastereomeric hexosamine monosaccharides, glucosamine, galactosamine, and mannosamine is reported by applying the kinetic method using N-acetylhexosamines or naturally occurring amino acids as reference bases under electrospray ionization conditions. The observed differences to distinguish the diastereomeric hexosamines are found mainly due to the proton affinity (PA) differences between the analyte and the reference base. The PA values of the hexosamines are not available in the literature, and hence, we estimated them by the kinetic method using N-acetylhexosamines as reference bases. The determined PA values are 223.97 kcal/mol for glucosamine, 224.99 kcal/mol for mannosamine, and 224.71 kcal/mol for galactosamine. The similar PA values were also obtained by using amino acids as reference bases. We have applied the same methodology to quantify these hexosamines in a mixture following the three-point calibration method suggested in the literature.

Electrospray ionisation tandem mass spectrometric study of hydrogen-bonding interactions of some disaccharides with lysine.

The efficiency of formation of protonated heterotrimers of lysine with underivatised sugars (mono-, di-, and trisaccharides) and N-acetylglucosamine (N-AcGlc) was studied under electrospray ionisation conditions. The collision induced dissociation spectra of [Lys + sugar + NAcGlc + H](+) resulted in [Lys + NAcGlc + H](+) and [Lys + sugar + H](+) as the major product ions. Relative abundances of these two fragments reflect the extent of adduct formation of protonated lysine plus sugar, with reference to the reference compound NAcGlc. This relative abundance ratio was found to be characteristic of the sugar structure. In this way it was observed that the ability of lysine to form a protonated heterodimer with neutral sugars increases with an increase in the number of acetal oxygens. Lactose showed an anomalously high affinity for protonated Lys, possibly reflecting the axial hydroxyl at C4. The postulated involvement of the epsilon-NH(2) group of lysine in the formation of protonated heterodimers with sugars was supported by similar results of similar experiments with NH(3) in place of lysine.