Lipid Profiling in Cancer Diagnosis with Hand-Held Ambient Mass Spectrometry Probes: Addressing the Late-Stage Performance Concerns

Untargeted lipid fingerprinting with hand-held ambient mass spectrometry (MS) probes without chromatographic separation has shown promise in the rapid characterization of cancers. As human cancers present significant molecular heterogeneities, careful molecular modeling and data validation strategies are required to minimize late-stage performance variations of these models across a large population. This review utilizes parallels from the pitfalls of conventional protein biomarkers in reaching bedside utility and provides recommendations for robust modeling as well as validation strategies that could enable the next logical steps in large scale assessment of the utility of ambient MS profiling for cancer diagnosis. Six recommendations are provided that range from careful initial determination of clinical added value to moving beyond just statistical associations to validate lipid involvements in disease processes mechanistically. Further guidelines for careful selection of suitable samples to capture expected and unexpected intragroup variance are provided and discussed in the context of demographic heterogeneities in the lipidome, further influenced by lifestyle factors, diet, and potential intersect with cancer lipid pathways probed in ambient mass spectrometry profiling studies

Protein stabilization by specific binding of guanidinium to a functional arginine-binding surface on an SH3 domain

Guanidinium hydrochloride (GuHCl) at low concentrations significantly stabilizes the Fyn SH3 domain. In this work, we have demonstrated that this stabilizing effect is manifested through a dramatic (five- to sixfold) decrease in the unfolding rate of the domain with the folding rate being affected minimally. This behavior contrasts to the effect of NaCl, which stabilizes this domain by accelerating the folding rate. These data imply that the stabilizing effect of GuHCl is not predominantly ionic in nature. Through NMR studies, we have identified a specific binding site for guanidinium, and we have determined a dissociation constant of 90mMfor this interaction. The guanidinium-binding site overlaps with a functionally important arginine-binding pocket on the domain surface, and we have shown that GuHCl is a specific inhibitor of the peptide-binding activity of the domain. A different SH3 domain possessing a similar arginine-binding pocket is also thermodynamically stabilized by GuHCl. These data suggest that many proteins that normally interact with arginine-containing ligands may also be able to specifically interact with guanidinium. Thus, some caution should be used when using GuHCl as a denaturant in protein folding studies. Since arginine-mediated interactions are often important in the energetics of protein–protein interactions, our observations could be relevant for the design of small molecule inhibitors of protein–protein interactions

Self-Localizing Stabilized Mega-Pixel Picoliter Arrays with Size-Exclusion Sorting Capabilities

We report on a liquid self-localizing process capable of producing Mega-pixel arrays of picoliter volumes on a 1 cm(2) area, within seconds, for high throughput sampling. The chip is based on principles of spatially varying wetting and stabilization. The key is to develop differential surface contact regions, which lead to both localization of the solution and increasing the surface adsorption energy to further pin the liquid to the surface, as highlighted by other studies. By exploiting surface roughness for enhanced wettability, we demonstrate wetting of wells with the aspect ratio of 100. The high precision of reactive ion etching (RIE) of silicon substrates allows for an extremely reproducible method of preparing the array of identical well structures and increased contact area to increase surface adsorption in the wells. "Dynamic wetting" is then readily achieved through inducing contact line instability by simply moving a drop of liquid on the top surface of the array. Liquid samples self-localize into the array pattern with the associated liquid flow leading to self-localization of suspended particles or analyte. The resulting picoliter volumes are both spatially ordered and stable for long periods of time, even for such small volumes, to permit selective measurements of the contents. This development will be particularly important in the assembly of the massive amounts of crystalline particles needed for atomically resolved structural dynamics using the latest advances in high number density electron and X-ray sources

Single-cell analysis by chemical cytometry combined with fluorescence microscopy

ABSTRACT Chemical cytometry uses capillary microseparation with highly sensitive detection for detailed chemical analyses of single cells. Here, we studied microscopy and chemical cytometry in measurements of total fluorescence from single cells. An inverted fluorescence microscope was modified so that the fluorescence intensity of a sample could be automatically measured for different vertical positions of the sample with respect to the objective lens. The capillary of a custom-made chemical cytometer was mounted in a vertical position over the microscope stage. A diluted suspension of 4T1 (mouse mammary gland tumor) cells stably expressing green fluorescent protein (GFP) was placed on a microscope slide. A single cell was positioned in the center of the field of view of the microscope. The intensity of its total GFP fluorescence was measured with the microscope detector first. The cell was then analyzed in the chemical cytometer as follows. The cell was injected into a capillary and lysed to form a homogeneous cellular lysate. The lysate was driven through the capillary by pressure and its GFP fluorescence was quantified at the output of the capillary with a laserinduced fluorescence (LIF) detector. We demonstrated for the first time that in microscopy, the maximum fluorescence signal, as well as maximum signal to noise ratio, could be obtained when the cell was in one of two extremely out-of-focus positions. We proved that in chemical cytometry, the intensity of fluorescence had no memory of cell geometry and depended solely on the amount of GFP. This feature o

Dramatic acceleration of protein folding by stabilization of a nonnative backbone conformation

Through a mutagenic investigation of Gly-48, a highly conserved position in the Src homology 3 domain, we have discovered a series of amino acid substitutions that are highly destabilizing, yet dramatically accelerate protein folding, some up to 10-fold compared with the wild-type rate. The unique folding properties of these mutants allowed for accurate measurement of their folding and unfolding rates in water with no denaturant by using an NMR spin relaxation dispersion technique. A strong correlation was found between β-sheet propensity and the folding rates of the Gly-48 mutants, even though Gly-48 lies in an unusual non-β-strand backbone conformation in the native state. This finding indicates that the accelerated folding rates of the Gly-48 mutants are the result of stabilization of a nonnative β-strand conformation in the transition-state structure at this position, thus providing the first, to our knowledge, experimentally elucidated example of a mechanism by which folding can occur fastest through a nonnative conformation. We also demonstrate that residues that are most stabilizing in the transition-state structure are most destabilizing in the native state, and also cause the greatest reductions in in vitro functional activity. These data indicate that the unusual native conformation of the Gly-48 position is important for function, and that evolutionary selection for function can result in a domain that folds at a rate far below the maximum possible

Contrast Agent Mass Spectrometry Imaging Reveals Tumor Heterogeneity

Mapping intratumoral heterogeneity such as vasculature and margins is important during intraoperative applications. Desorption electrospray ionization mass spectrometry (DESI-MS) has demonstrated potential for intraoperative tumor imaging using validated MS profiles. The clinical translation of DESI-MS into a universal label-free imaging technique thus requires access to MS profiles characteristic to tumors and healthy tissues. Here, we developed contrast agent mass spectrometry imaging (CA-MSI) that utilizes a magnetic resonance imaging (MRI) contrast agent targeted to disease sites, as a label, to reveal tumor heterogeneity in the absence of known MS profiles. Human breast cancer tumors grown in mice were subjected to CA-MSI using Gadoteridol revealing tumor margins and vasculature from the localization of [Gadoteridol+K]⁺ and [Gadoteridol+Na]⁺ adducts, respectively. The localization of the [Gadoteridol+K]⁺ adduct as revealed through DESI-MS complements the <i>in vivo</i> MRI results. DESI-MS imaging is therefore possible for tumors for which no characteristic MS profiles are established. Further DESI-MS imaging of the flux of the contrast agent through mouse kidneys was performed indicating secretion of the intact label

Gas-Phase FRET Efficiency Measurements To Probe the Conformation of Mass-Selected Proteins

Electrospray ionization and mass spectrometry have revolutionized the chemical analysis of biological molecules, including proteins. However, the correspondence between a protein's native structure and its structure in the mass spectrometer (where it is gaseous) remains unclear. Here, we show that fluorescence (Förster) resonance energy transfer (FRET) measurements combined with mass spectrometry provides intramolecular distance constraints in gaseous, ionized proteins. Using an experimental setup which combines trapping mass spectrometry and laser-induced fluorescence spectroscopy, the structure of a fluorescently labeled mutant variant of the protein GB1 was probed as a function of charge state. Steady-state fluorescence emission spectra and time-resolved donor fluorescence measurements of mass-selected GB1 show a marked decrease in the FRET efficiency with increasing number of charges on the gaseous protein, which suggests a Coulombically driven unfolding and expansion of its structure. This lies in stark contrast to the pH stability of GB1 in solution. Comparison with solution-phase single-molecule FRET measurements show lower FRET efficiency for all charge states of the gaseous protein examined, indicating that the ensemble of conformations present in the gas phase is, on average, more expanded than the native form. These results represent the first FRET measurements on a mass-selected protein and illustrate the utility of FRET for obtaining a new kind of structural information for large, desolvated biomolecules