Imaging of Proteins in Tissue Samples Using Nanospray Desorption Electrospray Ionization Mass Spectrometry

Chemical maps of tissue samples provide important information on biological processes therein. Recently, advances in tissue imaging have been achieved using ambient ionization techniques, such as desorption electrospray ionization mass spectrometry (DESI-MS), but such techniques have been almost exclusively confined to the mapping of lipids and metabolites. We report here the use of nanospray desorption electrospray ionization (nanoDESI) that allows us to image proteins in tissue samples in a label-free manner at atmospheric pressure with only minimum sample preparation. Multiply charged proteins with masses up to 15 kDa were successfully detected by nanoDESI using an LTQ Orbitrap mass spectrometer. In an adult mice brain section, expression of proteins including ubiquitin, β-thymosin, myelin basic protein, and hemoglobin were spatially mapped and characterized. We also determined the location of methylation on myelin basic protein. This imaging modality was further implemented to MYC-induced lymphomas. We observed an array of truncated proteins in the region where normal thymus cells were infiltrated by tumor cells, in contrast to healthy tissue

Micelles Protect Intact Metallo-supramolecular Block Copolymer Complexes from Solution to Gas Phase during Electrospray Ionization

Supramolecular diblock copolymers using metal–ligand coordination can be synthesized under ambient conditions by delicate design of the end groups of the homopolymer chains. However, mass spectrometric analysis of such metallo-supramolecular copolymers is challenging. One of the reasons is the nonpolarity of the polymer chains, making it hard to disperse the complexes in electrospray ionization (ESI)-friendly environments. The other difficulty is the noncovalent nature of such copolymers, which is easily disrupted during the ionization. Here, we demonstrate that the intact metallo-supramolecular diblock copolymers can be maintained sufficiently during the ESI process in aqueous solution within micelles. The high-resolution mass spectrometric evidence revealed that the surfactant molecules effectively protect the noncovalent binding of the complexes into gaseous ions. Intriguingly, surfactant molecules were sufficiently detached away from the copolymer complexes, giving unambiguous mass spectra that were predominated by intact diblock copolymers. This ESI-based approach allowed us to investigate the relative bond strengths of metal-to-ligand complexation using collision-induced dissociation (CID) in the ion trap mass spectrometry. Conformational features and collision cross sections of the copolymers were thus obtained using subsequent ion mobility spectrometry mass spectrometry (IMS-MS). Remarkable environment-dependent conformations of the denoted diblock copolymers were found using this mass spectrometric platform

Characterization of Carotenoid Aggregates by Steady-State Optical Spectroscopy

The carotenoids have low-lying triplet excited states and can self-assemble in some solvents to form weakly or strongly coupled aggregates. These qualities make carotenoid aggregates useful for studies of singlet fission, where an outstanding goal is the correlation of interchromophoric coupling to the dynamics and yield of triplet excited states from a parent singlet excited state. Three aggregates of zeaxanthin, two weakly coupled and one strongly coupled, are characterized by steady-state spectroscopic methods including temperature-dependent absorption, fluorescence, and resonance Raman spectroscopy. The absorption spectra for each type of aggregate are distinct; however, an analysis of band positions reveals some important shared characteristics and suggests that the strongly coupled H-aggregate contains a subpopulation of weakly coupled constituents. Temperature-dependent absorption spectroscopy indicates that one of the weakly coupled aggregates can be converted to the other upon heating. The emission spectra of the three aggregates have similar profiles that are overall red-shifted by more than 1000 cm^–1 relative to the monomer. The emission quantum yields of the aggregates are 5 to 30 times less than that of the monomer, with the lowest yield for the strongly coupled aggregate. The vibrational spectra of the chromophores support only slight perturbations from the structure of solvated monomers. Our findings support the conclusion that all three aggregates are best characterized as H-aggregates, in agreement with a prior theoretical study of lutein aggregates

Quantification of Endogenous Cholesterol in Human Serum on Paper Using Direct Analysis in Real Time Mass Spectrometry

Blood testing for endogenous small metabolites to determine physiological and biochemical states is routine for laboratory analysis. Here we demonstrate that by combining the commercial direct analysis in real time (DART) ion source with an ion trap mass spectrometer, native cholesterol in its free alcohol form is readily detected from a few hundred nanoliters of human serum loaded onto chromatography paper. Deuterium-labeled cholesterol was used as the internal standard to obtain the absolute quantity of the endogenous cholesterol. The amount of the cholesterol measured by this paper-loaded DART mass spectrometry (pDART-MS) is statistically comparable with that obtained by using commercially available fluorometric-enzymatic assay and liquid chromatography/mass spectrometry. Furthermore, sera from 21 participants at three different time points in an ultramarathon were collected to obtain their cholesterol levels. The test requires only very minimal sample preparation, and the concentrations of cholesterol in each sample were acquired within a minute

Top-Down Atmospheric Ionization Mass Spectrometry Microscopy Combined With Proteogenomics

Mass spectrometry-based protein analysis has become an important methodology for proteogenomic mapping by providing evidence for the existence of proteins predicted at the genomic level. However, screening and identification of proteins directly on tissue samples, where histological information is preserved, remain challenging. Here we demonstrate that the ambient ionization source, nanospray desorption electrospray ionization (nanoDESI), interfaced with light microscopy allows for protein profiling directly on animal tissues at the microscopic scale. Peptide fragments for mass spectrometry analysis were obtained directly on ganglia of the medicinal leech (Hirudo medicinalis) without in-gel digestion. We found that a hypothetical protein, which is predicted by the leech genome, is highly expressed on the specialized neural cells that are uniquely found in adult sex segmental ganglia. Via this top-down analysis, a post-translational modification (PTM) of tyrosine sulfation to this neuropeptide was resolved. This three-in-one platform, including mass spectrometry, microscopy, and genome mining, provides an effective way for mappings of proteomes under the lens of a light microscope

Template-Assisted Proximity for Oligomerization of Fullerenes

Demonstrated herein is an unprecedented porous template-assisted reaction at the solid–liquid interface involving bond formation, which is typically collision-driven and occurs in the solution and gas phases. The template is a TMA (trimesic acid) monolayer with two-dimensional pores that host fullerenes, which otherwise exhibit an insignificant affinity to an undecorated graphite substrate. The confinement of C₈₄ units in the TMA pores formulates a proximity that is ideal for bond formation. The oligomerization of C₈₄ is triggered by an electric pulse via a scanning tunneling microscope tip. The spacing between C₈₄ moieties becomes 1.4 nm, which is larger than the edge-to-edge diameter of 1.1–1.2 nm of C₈₄ due to the formation of intermolecular single bonds. In addition, the characteristic mass-to-charge ratios of dimers and trimers are observed by mass spectrometry. The experimental findings shed light on the active role of spatially tailored templates in facilitating the chemical activity of guest molecules

Harvesting Highly Electronically Excited Energy to Triplet Manifolds: State-Dependent Intersystem Crossing Rate in Os(II) and Ag(I) Complexes

A series of newly synthesized Os­(II) and Ag­(I) complexes exhibit remarkable ratiometric changes of intensity for phosphorescence versus fluorescence that are excitation wavelength dependent. This phenomenon is in stark contrast to what is commonly observed in condensed phase photophysics. While the singlet to triplet intersystem crossing (ISC) for the titled complexes is anomalously slow, approaching several hundred picoseconds in the lowest electronic excited state (S₁ → T₁), higher electronic excitation leads to a much accelerated rate of ISC (10¹¹–10¹² s^–1), which is competitive with internal conversion and/or vibrational relaxation, as commonly observed in heavy transition metal complexes. The mechanism is rationalized by negligible metal d orbital contribution in the S₁ state for the titled complexes. Conversely, significant ligand-to-metal charge transfer character in higher-lying excited states greatly enhances spin–orbit coupling and hence the ISC rate. The net result is to harvest high electronically excited energy toward triplet states, enhancing the phosphorescence

Microscopy ambient ionization mass spectrometry of bat wings.

<p>Tissue samples from the wings of healthy bats and individuals with white-nose syndrome were subjected to microscopy ambient ionization mass spectrometry (A). <i>Pseudogymnoascus destructans</i> infection was confirmed by the presence of fluorescent lesions when the tissue was excited with UV light (B). A nanoDESI source was used to desorb analytes from fluorescent tissue regions for MS analysis (capillary junction making contact with the tissue surface, C).</p

Detection of siderophores on the wings of bats with white-nose syndrome via molecular networking.

<p>Molecular networking of the MS/MS data was used to determine the structural relationships between the metabolites detected from wing surfaces, cultured <i>Pseudogymnoascus destructans</i>, and commercial siderophore standards. The siderophores desferrichrome (<i>m/z</i> 710.324) and ferrichrome (<i>m/z</i> 763.230) were observed from the wings of bats with white-nose syndrome and from cultured <i>P</i>. <i>destructans</i> and formed consensus nodes with commercial standards (A,B). Triacetylfusarinine C (<i>m/z</i> 928.344) was also present on WNS wings and in the <i>P</i>. <i>destructans</i> colony (C). None of the three siderophores were detected on the wings of healthy bats. In A and C insets only the node with the <i>m/z</i> of interest and its first and second neighbors have been displayed from the overall cluster for simplicity.</p

Specimen information for the <i>Myotis</i> used in the study.

<p>Specimen information for the <i>Myotis</i> used in the study.</p