Project description:Suppressing the background interferences and enhancing the analyte signals are long-term goals in high performance electrospray ionization mass spectrometry (ESI-MS) analyses. We observed that performing electrospray in the presence of a concentrated acetonitrile atmosphere suppresses background interferences and enhances peptide signals. An enclosed nanoESI source was utilized to provide a stable atmosphere of concentrated acetonitrile vapor for high performance ESI-MS analyses. The median MS signal intensity increased by 5 times for a set of 23 BSA tryptic peptides in direct nanoESI-MS analysis. Further, the number of reproducibly and precisely quantified peptides could be improved 67% in six replicate label-free quantitative proteome analyses by this strategy.
Project description:Water electrospray is small charged droplets generated by a laboratory-designed prototype module. Extensive investigations have explored its physical properties and identified a potential non-chemical biocide against air-borne pathogens. However, since humans can inhale water electrospray particles through breath, humans' safety and potential toxicity warrant investigation. Hence, to offer insights into the effect of water electrospray on humans, we analyzed the immunopathological response to water electrospray using a nasal challenge mouse model. For this, we accessed the fundamental immune responses following the water electrospray intranasal challenge by comparing its effect with sodium hypochlorite, a biocidal agent with known toxicity. Our results indicate that water electrospray did not induce pathological immune reactions: challenged mice did not exhibit body weight loss and increased inflammatory cytokine production. Furthermore, histopathological analysis revealed that water electrospray did not damage the lung tissue, whereas sodium hypochlorite–treated mice showed significant lung tissue damage with signs of neutrophils and eosinophils infiltration. Finally, transcriptomic analysis on lung tissue confirmed the absence of pathological immune response in water electrospray-treated mice compared with sodium hypochlorite–treated mice. Together, we provide evidence that water electrospray is a safe technology for its use in disinfecting air-borne pathogens with little or no effect on immune system activation at the preclinical level.
Project description:We evaluated a number of compounds as LC additives to modify the electrospray response and found that ethylene glycol was capable of enhancing nESI response by a factor of twofold on average. This result was comparable to DMSO.
Project description:Shotgun direct infusion mass spectrometry (DI-MS) enables high-throughput proteomics. Yet, its performance critically depends on nanospray emitter quality. We present here precision-engineered nanospray emitters with an optimized 4-degree emitter tip angle, designed for sensitive, reproducible, user friendly, and compatible across mass spectrometry platforms that demonstrated enhanced ionization efficiency and spray stability, showing a 21-fold improvement in protein identification rates compared to commercial designs. Using both digested HeLa cell protein extracts and plasma samples from Alzheimer's disease (AD) patients, and controls, where the 14 most high-abundant proteins were depleted, we performed a comprehensive DI-MS protein identification and precise quantification using both Orbitrap Exploris 480 (OE480) and Orbitrap Astral mass spectrometers. We consistently identified and quantified over 160 proteins in AD plasma samples in 4 min DI-MS runs, with superior reproducibility (CV < 15%) and sensitivity on the Orbitrap Astral. Our analysis revealed several unreported disease-associated protein signatures, while confirming known AD biomarkers, with C5, C4BPA, and FAM135B as the protein combination with the highest potential as AD plasma biomarkers for the detection of the disease. The enhanced performance of these standardized engineered nanospray emitters over the established DI-MS workflow establishes a robust and scalable platform for high-throughput clinical proteomics.
Project description:Shotgun direct infusion mass spectrometry (DI-MS) enables high-throughput proteomics. Yet, its performance critically depends on nanospray emitter quality. We present here precision-engineered nanospray emitters with an optimized 4-degree emitter tip angle, designed for sensitive, reproducible, user friendly, and compatible across mass spectrometry platforms that demonstrated enhanced ionization efficiency and spray stability, showing a 21-fold improvement in protein identification rates compared to commercial designs. Using both digested HeLa cell protein extracts and plasma samples from Alzheimer's disease (AD) patients and controls, where the 14 most high-abundant proteins were depleted, we performed a comprehensive DI-MS protein identification and precise quantification using both Orbitrap Exploris 480 (OE480) and Orbitrap Astral mass spectrometers. We consistently identified and quantified over 160 proteins in AD plasma samples in 4 min DI-MS runs, with superior reproducibility (CV < 15%) and sensitivity on the Orbitrap Astral. Our analysis revealed several unreported disease-associated protein signatures, while confirming known AD biomarkers, with C5, C4BPA, and FAM135B as the protein combination with the highest potential as AD plasma biomarkers for the detection of the disease. The enhanced performance of these standardized engineered nanospray emitters over the established DI-MS workflow establishes a robust and scalable platform for high-throughput clinical proteomics.
Project description:Due to the characteristics of multi-walled carbon nanotubes (MWCNTs) to aggregate and bind non-specifically, attempts have been made to inhibit their aggregation and increase their dispersion stability for biomedical applications by coating with bovine serum albumin (BSA). However, BSA concentration has not been studied in detail in terms of cellular response. To investigate the surface proteins formed in 10% fetal bovine serum cell culture media depending on the BSA concentration used to stabilize MWCNTs and to correlate the cellular responses with BSA concentration, we performed nanoflow liquid chromatography–electrospray ionization–tandem mass spectrometry analysis to quantify proteins of the protein corona (PC) and of A549 cells. Results showed that increasing the concentration of pre-incubated BSA changed the composition ratio of the proteins of the PCs adsorbed on the MWCNTs and also decreased the cellular uptake of A549 cells treated with BSA-coated MWCNTs. In the proteomic analysis, proteins related to the ribosome, ribosome biogenesis in eukaryotes, and oxidative phosphorylation pathways were downregulated in the low concentration BSA-treated cell groups, while proteins related to the mRNA surveillance pathway were downregulated in the high-BSA-concentration cell groups. Moreover, circular dichroism and Fourier-transform infrared spectroscopy results showed that the α-helix structure, a secondary structure of the adsorbed proteins, increased as a function of BSA concentration. Taken together, BSA concentration affects the adsorption and secondary structure of PCs on MWCNTs, which in turns affect their intracellular behavior. Our results may provide insight for the design of modification methods to increase the biocompatibility of MWCNTs.
Project description:Desorption electrospray ionization-mass spectrometry (DESI-MS) was evaluated for the detection of proteins ranging in molecular mass from 12 to 66 kDa. Proteins were uniformly deposited on a solid surface without pretreatment and analyzed with a DESI source coupled to a quadrupole ion trap mass spectrometer. DESI-MS parameters optimized for protein detection included solvent flow rate, temperature of heated capillary tube, incident and reflection angle, sheath gas pressure, and ESI voltage. Detection limits were obtained for all protein standards, and they were found to decrease with decreasing protein molecular mass: for cytochrome c (12.3 kDa) and lysozyme (14.3 kDa) a detection limit of 4 ng/mm2 was obtained; for apomyoglobin (16.9 kDa) 20 ng/mm2; for beta-lactoglobulin B (18.2 kDa) 50 ng/mm2; and for chymotrypsinogen A (25.6 kDa) 100 ng/mm2. The DESI-MS analysis of higher molecular mass proteins such as ovalbumin (44.4 kDa) and bovine serum albumin (66.4 kDa) yielded mass spectra of low signal-to-noise ratio, making their detection and molecular weight determination difficult. In this study, DESI-MS proved to be a rapid and robust method for accurate MW determination for proteins up to 17 kDa under ambient conditions. Finally, we demonstrated the DESI-MS detection of the bacteriophage MS2 capsid protein from crude samples with minimal sample preparation.
Project description:The formation of high charge-state protein ions with nanoelectrospray ionization (nESI) from purely aqueous ammonium bicarbonate solutions at neutral pH, where the proteins have native or native-like conformations prior to ESI droplet formation, is demonstrated. This "electrothermal" supercharging method depends on the temperature of the instrument entrance capillary, the nESI spray potential, and the solution ionic strength and buffer, although other factors almost certainly contribute. Mass spectra obtained with electrothermal supercharging appear similar to those obtained from denaturing solutions where charging beyond the total number of basic sites can be achieved. For example, a 17+ ion of bovine ubiquitin was formed by nESI of a 100 mM ammonium bicarbonate, pH 7.0, solution, which is three more charges than the total number of basic amino acids plus the N-terminus. Heating of the ESI droplets in the vacuum/atmosphere interface and the concomitant denaturation of the protein in the ESI droplets prior to ion formation appears to be the primary origin of the very high charge-state ions formed from these purely aqueous, buffered solutions. nESI mass spectra resembling those obtained under traditional native or denaturing conditions can be reversibly obtained simply by toggling the spray voltage between low and high values.
Project description:We report here the development of a corona discharge (CD) initiated electrochemical (EC) electrospray ionization (ESI) technique using a standard electrospray ion source. This is a new ionization technique distinct from ESI, electrochemistry inherent to ESI, APCI, and techniques using hydroxyl radicals produced under atmospheric pressure conditions. By maximizing the observable CD at the tip of a stainless steel ESI capillary, efficient electrochemical oxidation of electrochemically active compounds is observed. For electrochemical oxidation to be observed, the ionization potential of the analyte must be lower than Fe. Ferrocene labeled compounds were chosen as the electrochemically active moiety. The electrochemical cell in the ESI source was robust, and generated ions with selectivity according to the ionization potential of the analytes and up to zeptomolar sensitivity. Our results indicate that CD initiated electrochemical ionization has the potential to become a powerful technique to increase the dynamic range, sensitivity, and selectivity of ESI experiments.
Project description:A 193-nm wavelength deep ultraviolet laser was used for ambient laser ablation electrospray ionization mass spectrometry of biological samples. A pulsed ArF excimer laser was used to ablate solid samples, and the resulting plume of the desorbed material merged with charged electrospray droplets to form ions that were detected with a quadrupole time-of-flight mass spectrometer. Solutions containing peptide and protein standards up to 66-kDa molecular weight were deposited on a metal target, dried, and analyzed. No fragmentation was observed from peptides and proteins as well as from the more easily fragmented vitamin B12 molecule. The mass spectra contained peaks from multiply charged ions that were identical to conventional electrospray. Deep UV laser ablation of tissue allowed detection of lipids from untreated tissue. The mechanism of ionization is postulated to involve absorption of laser energy by a fraction of the analyte molecules that act as a sacrificial matrix or by residual water in the sample.