MALDI/post ionization-ion mobility mass spectrometry of noncovalent complexes of dopamine receptors' epitopes.

Protein domains involved in receptor heteromer formation are disordered and rich in the amino acids necessary for the formation of noncovalent complexes (NCX). We present mass spectral NCX data from proteins and protein receptors' epitopes obtained by combining ion mobility (IM) and MALDI. We focus on NCX involved in heteromer formation occurring between epitopes of the Dopamine D2 (D2R) and Adenosine A2A receptors (A2AR) as well as D2R and the α2 nicotinic (NR) receptor's subunit. The IM data yield information on the gas phase conformation of the singly charged NCX which are observed either directly from MALDI or as codesorbed neutrals that are subsequently postionized by a time-delayed excimer laser pulse directed onto a portion of the neutral plume created by the MALDI desorption laser. Imaging mass spectrometry of the matrix/epitope dried droplet surface shows that the acidic and basic epitopes and their NCX are found to be spatially collocated within regions as small as 25 × 50 µm(2). Subtle differences in the relative abundance of protonated and cationized NCX and epitopes are measured in spatial regions near the sodium-rich outer border of the droplet.

Cellular membrane phospholipids act as a depository for quaternary amine containing drugs thus competing with the acetylcholine/nicotinic receptor.

We previously demonstrated that ammonium- or guanidinium-phosphate interactions are key to forming noncovalent complexes (NCXs) through salt bridge formation with G-protein coupled receptors (GPCR), which are immersed in the cell membrane's lipids. The present work highlights MALDI ion mobility coupled to orthogonal time-of-flight mass spectrometry (MALDI IM oTOF MS) as a method to determine qualitative and relative quantitative affinity of drugs to form NCXs with targeted GPCRs' epitopes in a model system using, bis-quaternary amine based drugs, α- and ß- subunit epitopes of the nicotinic acetylcholine receptor' (nAChR) and phospholipids. Bis-quaternary amines proved to have a strong affinity for all nAChR epitopes and negatively charged phospholipids, even in the presence of the physiological neurotransmitter acetylcholine. Ion mobility baseline separated isobaric phosphatidyl ethanolamine and a matrix cluster, providing an accurate estimate for phospholipid counts. Overall this technique is a powerful method for screening drugs' interactions with targeted lipids and protein respectively containing quaternary amines and guanidinium moieties.

Gangliosides' analysis by MALDI-ion mobility MS.

The combination of ion mobility with matrix-assisted laser desorption/ionization allows for the rapid separation and analysis of biomolecules in complex mixtures (such as tissue sections and cellular extracts), as isobaric lipid, peptide, and oligonucleotide molecular ions are pre-separated in the mobility cell before mass analysis. In this study, MALDI-IM MS is used to analyze gangliosides, a class of complex glycosphingolipids that has different degrees of sialylation. Both GD1a and GD1b, structural isomers, were studied to see the effects on gas-phase structure depending upon the localization of the sialic acids. A total ganglioside extract from mouse brain was also analyzed to measure the effectiveness of ion mobility to separate out the different ganglioside species in a complex mixture.

Laser Desorption/Ionization Mass Spectrometric Imaging of Endogenous Lipids from Rat Brain Tissue Implanted with Silver Nanoparticles.

Mass spectrometry imaging (MSI) of tissue implanted with silver nanoparticulate (AgNP) matrix generates reproducible imaging of lipids in rodent models of disease and injury. Gas-phase production and acceleration of size-selected 8 nm AgNP is followed by controlled ion beam rastering and soft landing implantation of 500 eV AgNP into tissue. Focused 337 nm laser desorption produces high quality images for most lipid classes in rat brain tissue (in positive mode: galactoceramides, diacylglycerols, ceramides, phosphatidylcholines, cholesteryl ester, and cholesterol, and in negative ion mode: phosphatidylethanolamides, sulfatides, phosphatidylinositol, and sphingomyelins). Image reproducibility in serial sections of brain tissue is achieved within <10% tolerance by selecting argentated instead of alkali cationized ions. The imaging of brain tissues spotted with pure standards was used to demonstrate that Ag cationized ceramide and diacylglycerol ions are from intact, endogenous species. In contrast, almost all Ag cationized fatty acid ions are a result of fragmentations of numerous lipid types having the fatty acid as a subunit. Almost no argentated intact fatty acid ions come from the pure fatty acid standard on tissue. Graphical Abstract ᅟ.

MALDI-Ion Mobility Mass Spectrometry of Lipids in Negative Ion Mode.

Profiling and imaging MALDI mass spectrometry (MS) allows detection and localization of biomolecules in tissue, of which lipids are a major component. However, due to the in situ nature of this technique, complexity of tissue and need for a chemical matrix, the recorded signal is complex and can be difficult to assign. Ion mobility adds a dimension that provides coarse shape information, separating isobaric lipids, peptides, and oligonucleotides along distinct familial trend lines before mass analysis. Previous work using MALDI-ion mobility mass spectrometry to analyze and image lipids has been conducted mainly in positive ion mode, although several lipid classes ionize preferentially in negative ion mode. This work highlights recent data acquired in negative ion mode to detect glycerophosphoethanolamines (PEs), glycerophosphoserines (PSs), glycerophosphoglycerols (PGs), glycerolphosphoinositols (PIs), glycerophosphates (PAs), sulfatides (STs), and gangliosides from standard tissue extracts and directly from mouse brain tissue. In particular, this study focused on changes in ion mobility based upon lipid head groups, composition of radyl chain (# of carbons and double bonds), diacyl versus plasmalogen species, and hydroxylation of species. Finally, a MALDI-ion mobility imaging run was conducted in negative ion mode, resulting in the successful ion mapping of several lipid species.

Color-blind fluorescence detection for four-color DNA sequencing.

We present an approach called pulsed multiline excitation (PME) for measurements of multicomponent, fluorescence species and demonstrate its application in capillary electrophoresis for DNA sequencing. To fully demonstrate the advantages of PME, a fluorescent dye set has been developed whose absorption maxima span virtually the entire visible spectrum. Unlike emission wavelength-dependent approaches for identifying fluorescent species, the removal of the spectral component in PME confers a number of advantages including higher and normalized signals from all dyes present in the assay, the elimination of spectral cross-talk between dyes, and higher signal collection efficiency. Base-calling is unambiguously determined once dye mobility corrections are made. These advantages translate into significantly enhanced signal quality as illustrated in the primary DNA sequencing data and provide a means for achieving accurate base-calling at lower reagent concentrations.