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Rapid, direct analysis of cholesterol by charge labeling in reactive desorption electrospray ionization.

ABSTRACT: Direct and rapid analysis of cholesterol was accomplished in the ambient environment using reactive desorption electrospray ionization (DESI) mass spectrometry. This was achieved by electrospraying reagent solutions in the form of high velocity charged droplets at surfaces such as dried serum samples and animal tissue sections. Betaine aldehyde, incorporated into the spray solvent, reacts selectively and rapidly with the alcohol group of cholesterol by nucleophilic addition, forming a hemiacetal salt. Limits of detection for pure cholesterol and related compounds were approximately 1 ng when a solution of cholesterol of 1 microg/mL was spotted onto the surface. Quantitative analysis of free cholesterol in serum using reactive DESI was demonstrated using cholesterol-d7 as internal standard. High throughput analysis of small volumes of serum spotted onto a suitable substrate was achieved at an analysis rate of approximately 14 s per sample, with a relative standard deviation (RSD) of ca. 6%. Use of reactive DESI in the imaging mode allowed 2D spatial distributions of phospholipids and cholesterol to be recorded simultaneously in rat brain tissues.

SUBMITTER: Wu C

PROVIDER: S-EPMC2747033 | biostudies-literature | 2009 Sep

REPOSITORIES: biostudies-literature

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Rapid, direct analysis of cholesterol by charge labeling in reactive desorption electrospray ionization.

Wu Chunping C Ifa Demian R DR Manicke Nicholas E NE Cooks R Graham RG

Analytical chemistry 20090901 18

Direct and rapid analysis of cholesterol was accomplished in the ambient environment using reactive desorption electrospray ionization (DESI) mass spectrometry. This was achieved by electrospraying reagent solutions in the form of high velocity charged droplets at surfaces such as dried serum samples and animal tissue sections. Betaine aldehyde, incorporated into the spray solvent, reacts selectively and rapidly with the alcohol group of cholesterol by nucleophilic addition, forming a hemiacetal ...[more]

PMID: 19746995

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Project description:We recently reported the use of desorption electrospray ionization (DESI) as a novel interface to couple high-performance liquid chromatography (HPLC) with mass spectrometry (MS) (Chem. Commun. 2011, 47, 4171). One of the benefits of such an interface is that post-column derivatization of separated analytes can be integrated with ionization via a "reactive" DESI approach in which a derivatizing reagent is doped into the spray solvent. The reactive DESI interface allows analyte desorption/ionization from the end of the chromatographic column with prompt MS detection; a short time delay of ~20 ms was demonstrated. In this study, we extended this application by "supercharging" proteins following HPLC separation using a DESI spray solvent containing supercharging reagents, m-nitrobenzyl alcohol (m-NBA) or sulfolane. Proteins (insulin, ubiquitin, lysozyme and α-lactalbumin) eluted out of the LC column can be supercharged with the protein charge state distributions (CSDs) significantly increased (to higher charge), which would be advantageous for subsequent top-down MS analysis of proteins. Interestingly, supercharging combined with reactive DESI enhances tolerance towards trifluoroacetic acid (TFA), which is known to be a superior additive in the mobile phase for premium peptide/protein chromatographic separation but has severe signal suppression effects for conventional electrospray ionization (ESI). In comparison to electrosonic spray ionization (ESSI), a variant form of ESI, the sensitivity of protein analysis using LC/DESI-MS with the mobile phase containing TFA can be improved by up to 70-fold for lysozyme and α-lactalbumin by including m-NBA in the DESI spray solvent. Presumably, by reducing TFA dissociation in the droplet, supercharging agents lower trifluoroacetate anion concentrations and concomitantly reduce ion pairing to analyte cationic sites. The reduced ion pairing therefore decreases the TFA signal suppression effect. The supercharging capability and the reduction of TFA signal suppression suggest that LC/DESI-MS is a valuable method for protein analysis.

Project description:There has been a recent surge in applications of mass spectrometry (MS) to tissue analysis, particularly lipid-based tissue imaging using ambient ionization techniques. This recent growth highlights the need to examine the effects of sample handling, storage conditions, and experimental protocols on the quality of the data obtained. Variables such as time before freezing after organ removal, storage time at -80 °C, time stored at room temperature, heating, and freeze/thaw cycles were investigated for their effect on the data quality obtained in desorption electrospray ionization (DESI)-MS using mouse brain. In addition, analytical variables such as tissue thickness, drying times, and instrumental conditions were also examined for their impact on DESI-MS data. While no immediate changes were noted in the DESI-MS lipid profiles of the mouse brain tissue after spending 1 h at room temperature when compared to being frozen immediately following removal, minor changes were noted between the tissue samples after 7 months of storage at -80 °C. In tissue sections stored at room temperature, degradation was noted in 24 h by the appearance of fatty acid dimers, which are indicative of high fatty acid concentrations, while in contrast, those sections stored at -80 °C for 7 months showed no significant degradation. Tissue sections were also subjected to up to six freeze/thaw cycles and showed increasing degradation following each cycle. In addition, tissue pieces were subjected to 50 °C temperatures and analyzed at specific time points. In as little as 2 h, degradation was observed in the form of increased fatty acid dimer formation, indicating that enzymatic processes forming free fatty acids were still active in the tissue. We have associated these dimers with high concentrations of free fatty acids present in the tissue during DESI-MS experiments. Analytical variables such as tissue thickness and time left to dry under nitrogen were also investigated, with no change in the resulting profiles at thickness from 10 to 25 μm and with optimal signal obtained after just 20 min in the dessicator. Experimental conditions such as source parameters, spray solvents, and sample surfaces are all shown to impact the quality of the data. Inter-section (relative standard deviation (%RSD), 0.44-7.2%) and intra-sample (%RSD, 4.0-8.0%) reproducibility data show the high quality information DESI-MS provides. Overall, the many variables investigated here showed DESI-MS to be a robust technique, with sample storage conditions having the most effect on the data obtained, and with unacceptable sample degradation occurring during room temperature storage.

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Rapid, direct analysis of cholesterol by charge labeling in reactive desorption electrospray ionization.

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Rapid, direct analysis of cholesterol by charge labeling in reactive desorption electrospray ionization.

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