Kamlesh Shrivas

Imaging Mass Spectrometry with Silver Nanoparticles Reveals the Distribution of Fatty Acids in Mouse Retinal Sections

A new approach to the visualization of fatty acids in mouse liver and retinal samples has been developed using silver nanoparticles (AgNPs) in nanoparticle-assisted laser desorption/ ionization imaging mass spectrometry (nano-PALDI-IMS) in negative ion mode. So far, IMS analysis has concentrated on main cell components, such as cell membrane phospholipids and cytoskeletal peptides. AgNPs modified with alkylcarboxylate and alkylamine were used for nano-PALDI-IMS to identify fatty acids, such as stearic, oleic, linoleic, arachidonic, and eicosapentaenoic acids, as well as palmitic acid, in mouse liver sections; these fatty acids are not detected using 2,5-dihydroxybenzoic acid (DHB) as a matrix. The limit of detection for the determination of palmitic acid was 50 pmol using nano-PALDI-IMS. The nano-PALDI-IMS method is successfully applied to the reconstruction of the ion images of fatty acids in mouse liver sections. We verified the detection of fatty acids in liver tissue sections of mice by analyzing standard lipid samples, which showed that fatty acids were from free fatty acids and dissociated fatty acids from lipids when irradiated with a laser. Additionally, we applied the proposed method to the identification of fatty acids in mouse retinal tissue sections, which enabled us to learn the six-zonal distribution of fatty acids in different layers of the retina. We believe that the current approach using AgNPs in nano-PALDI-IMS could lead to a new strategy to analyze basic biological mechanisms and several diseases through the distribution of fatty acids.

Applications of silver nanoparticles capped with different functional groups as the matrix and affinity probes in surface‐assisted laser desorption/ionization time‐of‐flight and atmospheric pressure matrix‐assisted laser desorption/ionization ion trap mass spectrometry for rapid analysis of sulfur drugs and biothiols in human urine

A strategy is presented for the analysis of sulfur drugs and biothiols using silver nanoparticles (AgNPs) capped with different functional groups as the matrix and affinity probes in surface-assisted laser desorption/ionization time-of-flight mass spectrometry (SALDI-TOF MS) and atmospheric pressure-matrix assisted laser desorption/ionization ion trap mass spectrometry (AP-MALDI-ITMS). Biothiols adsorbed on the surface of AgNPs through covalent bonding were subjected to ultraviolet (UV) radiation that enabled desorption and ionization due to the excellent photochemical property of NPs. The proposed method has been successfully applied for the determination of cysteine and homocysteine in human urine samples using an internal standard. The limit of detection (LOD) and limit of quantification (LOQ) for cysteine and homocysteine in urine sample are 7 and 22 nM, respectively, with a relative standard deviation (RSD) of <10%. The advantages of the present method compared with the methods reported in the literature for biothiol analysis are simplicity, rapidity and sensitivity without the need for time-consuming separation and tedious preconcentration processes. Additionally, we also found that the bare AgNPs can be directly used as the matrix in MALDI-TOF MS for the analysis of sulfur drugs without the addition of an extra proton source.

Quantum dots laser desorption/ionization MS: multifunctional CdSe quantum dots as the matrix, concentrating probes and acceleration for microwave enzymatic digestion for peptide analysis and high resolution detection of proteins in a linear MALDI-TOF MS

We report the first use of functionalized cadmium selinide quantum dots (CdSe QDs) with 11‐mercaptoundecanoic acid (MUA) as the matrix for the selective ionization of proteins with high resolution and rapid analysis of amino acids and peptides by using quantum dots laser desorption/ionization mass spectrometry (QDLDI‐MS). The mercaptocarboxylic groups of CdSe QDs have been known to be an effective affinity probe to interact with the biomolecules at low abundance level. Using these QDs as the matrix, sensitivity of the method was greatly enhanced and the LOQ of peptides was found to be 100 pM with RSD <10%. The QDLDI‐MS is capable for the selective ionization of insulin, lysozyme and myoglobin with high resolution, which is not observed with sinapic acid (SA) as the matrix. The QDLDI‐MS technique offers many advantages for the analysis of amino acids, peptides and proteins with regard to simplicity, rapidity, sensitivity and the mass spectra were generated in the presence of signal suppressors such as urea and Trition X‐100. In addition, the CdSe QDs have been successfully applied as preconcentrating probes for the analysis of digested peptides in lysozyme from chicken egg white by microwave‐assisted enzymatic digestion. This indicates that the QDs are able to absorb radiation from microwave and their ability to trap peptides from microwave‐digested lysozyme. These results demonstrate that the CdSe QDs are promising candidates for the selective ionization of the analytes with an accurate platform to the rapid screening of biomolecules.

Ionic matrix for enhanced MALDI imaging mass spectrometry for identification of phospholipids in mouse liver and cerebellum tissue sections.

The ionic matrix (IM) is considered to be versatile for matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) for the identification of a wide range of biomolecules due to its good solubility for a variety of analytes, formation of homogeneous crystals with analytes, and high vacuum stability. When these advantages are exploited, the performance of IM of α-cyano-4-hydroxycinnamic acid butylamine (CHCAB) and 2,5-dihydroxybenzoic acid butylamine (DHBB) was compared with other matrixes for the identification of phospholipids in standard mixtures and mouse liver tissue sections. The results showed that the IM of CHCAB caused higher signal intensity and allowed the detection of a number phospholipids such as phosphatidylethanolamine (PE) and phosphatidylserine (PS) in addition to detection of phosphatidylcholine (PC) on the surface of the liver tissue sample. The IM of CHCAB was also used to identify the species of lipids present in different layers of cerebellum where the greater numbers of biomolecules were detected as compared to DHB matrix. Further, the feasibility of the proposed method was extended for the analysis of tryptic digested cytochrome c for increased signal intensity and number of peptide sequences in MALDI-MS. Thus, the application of IM to MALDI-MS could be a promising tool for imaging biomolecules in tissue sections in high throughput analyses with high sensitivity.

Method for simultaneous imaging of endogenous low molecular weight metabolites in mouse brain using TiO2 nanoparticles in nanoparticle-assisted laser desorption/ionization-imaging mass spectrometry.

We report the detection of a group of endogenous low molecular weight metabolites (LMWM) in mouse brain (80-500 Da) using TiO(2) nanoparticles (NPs) in nanoparticle-assisted laser desorption/ionization-imaging mass spectrometry (Nano-PALDI-IMS) without any washing and separation step prior to MS analysis. The identification of metabolites using TiO(2) NPs was compared with a conventional organic matrix 2,5-dihydroxybenzoic acid (DHB) where signals of 179 molecules were specific to TiO(2) NPs, 4 were specific to DHB, and 21 were common to both TiO(2) NPs and DHB. The use of TiO(2) NPs enabled the detection of a higher number of LMWM as compared to DHB and gold NPs as a matrix. This approach is a simple, inexpensive, washing, and separation free for imaging and identification of LMWM in mouse brain. We believe that the biochemical information from distinct regions of the brain using a Nano-PALDI-IMS will be helpful in elucidating the imbalances linked with diseases in biomedical samples.

Multifunctional nanoparticles composite for MALDI-MS: Cd2+-doped carbon nanotubes with CdS nanoparticles as the matrix, preconcentrating and accelerating probes of microwave enzymatic digestion of peptides and proteins for direct MALDI-MS analysis.

For the first time, we utilized multifunctional nanoparticles composite (NPs composite) for matrix-assisted laser desorption/ionization mass spectrometric (MALDI-MS) analysis of peptides and proteins. Multiwalled carbon nanotubes doped with Cd(2+) ions and modified with cadmium sulfide NPs were synthesized by a chemical reduction method at room temperature. The multifunctional NPs composite applied for the analysis of peptides and microwave-digested proteins in the atmospheric pressure matrix-assisted laser desorption/ionization ion-trap and MALDI time-of-flight (TOF) mass spectrometry (MS) was successfully demonstrated. The maximum detection sensitivity for peptides in MALDI-MS was achieved by the adsorption of negatively charged peptides onto the surfaces of NP composite through electrostatic interactions. The optimal conditions of peptide mixtures were obtained at 20 min of incubation time using 1 mg of NPs composite when the pH of the sample solution was kept higher than the pI values of peptides. The potentiality of the NP composite in the preconcentration of peptides was compared with that of the individual NP by calculating the preconcentration factors (PF) and found that the NPs composite showed a 4-6 times of PF than the other NPs. In addition, the NPs composite was also applied as heat-absorbing materials for efficient microwave tryptic digestion of cytochrome c and lysozyme from milk protein in MALDI-TOF-MS analysis. We believe that the use of NPs composite technique would be an efficient and powerful preconcentrating tool for MALDI-MS for the study of proteome research.

Ultrasonication followed by single‐drop microextraction combined with GC/MS for rapid determination of organochlorine pesticides from fish

A novel, rapid and simple sample pretreatment technique termed ultrasonication followed by single-drop micro-extraction (U-SDME) has been developed and combined with GC/MS for the determination of organochlorine pesticides (OCPs) in fish. In the present work, the lengthy procedures generally used in the conventional methods like, Soxhlet extraction, supercritical fluid extraction, pressurized liquid extraction and microwave assisted solvent extraction for extraction of OCPs from fish tissues are minimized by the use of two simple extraction procedures. Firstly, OCPs from fish were extracted in organic solvent with ultrasonication and then subsequently preconcentrated by single-drop micro-extraction (SDME). Extraction parameters of ultrasonication and SDME were optimized in spiked sample solution in order to obtain efficient extraction of OCPs from fish tissues. The calibration curves for OCPs were found to be linear between 10-1000 ng/g with correlation of estimations in the range 0.990-0.994. The recoveries obtained in blank fish tissues were ranged from 82.1 to 95.3%. The LOD and RSD for determination of OCPs in fish were 0.5 ng/g and 9.4-10.0%, respectively. The proposed method was applied for the determination of bioconcentration factor in fish after exposure to different concentrations of OCPs in cultured water. The present method avoids the co-extraction of lipids, long extraction steps (>12 h) and large amount of organic solvent for the separation of OCPs. The main advantages of the present method are rapid, selective, sensitive and low cost for the determination of OCPs in fish.

Dispersive liquid-liquid microextraction for the determination of copper in cereals and vegetable food samples using flame atomic absorption spectrometry.

Dispersive liquid-liquid microextraction (DLLME) is applied for the determination of copper in cereals and vegetable food samples using flame atomic absorption spectrometry (FAAS). The maximum extraction efficiency of copper was obtained after the optimisation of parameters such as extraction and dispersing solvents, pH, concentration of 2,9-dimethyl-1,10-phenanothroline (DPT), N-phenylbenzimidoyl thiourea (PBITU) and salt. The optimised methodology exhibited a good linearity in the range of 0.2-20 ng/mL copper with relative standard deviations percentage (RSD,%) from ±1.5% to 3.5%. The method is found to be simple and rapid for the analysis of copper in food samples with the limit of detection (LOD) and quantitation (LOQ) were 0.05 and 0.16 ng/mL, respectively. Good recoveries of copper were obtained in the range of 93.5-98.0% in food samples as well as in Certified Reference Material (99.1%). The application of the proposed method has been successfully tested for the determination of copper in cereals (maize, millet, rice, wheat, gram, lentils, kidney beans and green beans) and vegetable (potato, cauliflower, tomato, spinach, green beans, lettuce, egg plants and bitter gourd) food samples.

Single drop microextraction using silver nanoparticles as electrostatic probes for peptide analysis in atmospheric pressure matrix‐assisted laser desorption/ionization mass spectrometry and comparison with gold electrostatic probes and silver hydrophobic probes

Single drop microextraction using tetraalkylammonium bromide coated silver nanoparticles (SDME-AgNPs) prepared in toluene has been successfully applied as electrostatic affinity probes to preconcentrate peptide mixtures in biological samples prior to atmospheric pressure matrix-assisted laser desorption/ionization ion trap mass spectrometry (AP-MALDI-MS) analysis. This approach is based on the isoelectric point (pI) of peptides and surface charge of AgNPs. Using the SDME-AgNPs technique, from a peptide mixture, Met- and Leu-enkephalins (Met-enk and Leu-enk) were extracted into a droplet of toluene containing AgNPs, but not the neutral peptides (gramicidins). The best peptide extraction efficiency for SDME-AgNPs was observed with the optimized parameters: extraction time 2 min, sample agitation rate 240 rpm, and sample pH 7. The limits of detection (LODs) of the SDME-AgNPs/AP-MALDI-MS technique for Met-enk and Leu-enk peptides were 160 and 210 nM, respectively. Furthermore, the application of the technique has been shown for the analysis of peptides from a sample containing high matrix interferences such as 1% Triton X-100 and 6 M urea. Finally, this approach has been compared with the SDME-AuNPs technique and the results have clearly revealed that the SDME-AgNP affinity probe exhibits higher affinity to extract the sulfur-bearing peptide (Met-enk). We also compared this electrostatic affinity probe of AgNPs with the previously demonstrated hydrophobic affinity probe of AgNPs and found that the electrostatic probe can greatly reduce the extraction time from 1.5 h to 2 min. This is due to the fact that electrostatic attraction forces are much stronger than the hydrophobic attraction forces. Therefore, we concluded that the electrostatic affinity probe based on SDME-AgNPs coupled with AP-MALDI-MS is a high-throughput technique for the analysis of low-abundance peptides from biological samples containing complex matrices.

Oxidized multiwalled carbon nanotubes for quantitative determination of cationic surfactants in water samples using atmospheric pressure matrix-assisted laser desorption/ionization mass spectrometry

Oxidized multiwalled carbon nanotubes (O-MWCNT) are used as preconcentrating probes for the quantitative determination of cationic surfactants (CS(+)) in water samples using atmospheric pressure matrix-assisted laser desorption/ionization mass spectrometry (AP-MALDI-MS). The method is based on the electrostatic interactions of positively charged CS(+) with the negatively charged O-MWCNT for the preconcentration and isolation of analytes from sample solutions. The O-MWCNT/AP-MALDI-MS is performed for the determination of CS(+) in river- and waste-water samples. The optimum extraction efficiency of CS(+) is observed for 7 min of contact time at 1.0 mg dosage of O-MWCNT at pH 7.0. The acceptable relative recovery percentage in water sample is obtained in the range 90.5-97.8% with relative standard deviation (R.S.D.)<14.5%. The results suggested that the newly proposed method is rapid, accurate and effective and could be successfully applied for the enrichment and quantitative determination of CS(+) in water samples.