Christopher J. Thompson

Application of desorption electrospray ionization mass spectrometry imaging in breast cancer margin analysis

Significance This study is the first demonstration, to our knowledge, of the application of desorption electrospray ionization mass spectrometry imaging (DESI-MSI) for discrimination of breast cancer and delineation of tumor margins. Using DESI-MSI, it is possible to discriminate between cancerous and adjacent normal tissue on the basis of the detection and specific spatial distributions of different lipid species. This study proves the feasibility of classifying cancerous and normal breast tissues using ambient ionization MSI. It will allow the surgeon to access to this information in real time so as to make accurate intraoperative decisions quickly. It will result in improved cosmesis and decrease the need for multiple operations for margin reexcision. Distinguishing tumor from normal glandular breast tissue is an important step in breast-conserving surgery. Because this distinction can be challenging in the operative setting, up to 40% of patients require an additional operation when traditional approaches are used. Here, we present a proof-of-concept study to determine the feasibility of using desorption electrospray ionization mass spectrometry imaging (DESI-MSI) for identifying and differentiating tumor from normal breast tissue. We show that tumor margins can be identified using the spatial distributions and varying intensities of different lipids. Several fatty acids, including oleic acid, were more abundant in the cancerous tissue than in normal tissues. The cancer margins delineated by the molecular images from DESI-MSI were consistent with those margins obtained from histological staining. Our findings prove the feasibility of classifying cancerous and normal breast tissues using ambient ionization MSI. The results suggest that an MS-based method could be developed for the rapid intraoperative detection of residual cancer tissue during breast-conserving surgery.

The first pilot project of the consortium for top-down proteomics: a status report.

Pilot Project #1—the identification and characterization of human histone H4 proteoforms by top‐down MS—is the first project launched by the Consortium for Top‐Down Proteomics (CTDP) to refine and validate top‐down MS. Within the initial results from seven participating laboratories, all reported the probability‐based identification of human histone H4 (UniProt accession P62805) with expectation values ranging from 10−13 to 10−105. Regarding characterization, a total of 74 proteoforms were reported, with 21 done so unambiguously; one new PTM, K79ac, was identified. Inter‐laboratory comparison reveals aspects of the results that are consistent, such as the localization of individual PTMs and binary combinations, while other aspects are more variable, such as the accurate characterization of low‐abundance proteoforms harboring >2 PTMs. An open‐access tool and discussion of proteoform scoring are included, along with a description of general challenges that lie ahead including improved proteoform separations prior to mass spectrometric analysis, better instrumentation performance, and software development.

Phase Correction of Fourier Transform Ion Cyclotron Resonance Mass Spectra Using MatLab

FT-ICR mass spectrometry has been limited to magnitude mode for almost 40xa0years due to the data processing methods used. However, it is well known that phase correction of the data can theoretically produce an absorption-mode spectrum with a mass-resolving power that is as much as twice as high as conventional magnitude mode, and that it also improves the quality of the peak shape. Temporally dispersed frequency sweep excitation followed by a time delay before detection results in a steep quadratic variation in the signal phase with frequency. Viewing this, it is possible to find the correct phase function by performing a quadratic least squares fit, modified by iterating through phase cycles until the correct quadratic function is found. Here, we present a robust manual method to rotate these signals mathematically and generate a “phased” absorption-mode spectrum. The method can, in principle, be automated. Baseline correction is also included to eliminate the accompanying baseline drift. The resulting experimental FT-ICR absorption-mode spectra exhibit a resolving power that is at least 50% higher than that of the magnitude mode.

Absorption-mode : the next generation of Fourier transform mass spectra

The Fourier transform spectrum can be presented in the absorption-mode (commonly used in FT-NMR), magnitude-mode (FT-ICR), and power-mode (engineering applications). As is routinely used in FT-NMR, it is well-known that the absorption-mode display gives a much narrower peak shape which greatly improves the spectrum; recently, the successful solution of the phase equation allowed broadband phase correction which makes it possible to apply the absorption-mode routinely in FT-ICR. With the empirical evidence provided herein, it has been confirmed that in addition to the improvement on resolving power, compared to the conventional magnitude-mode, the new absorption-mode improves the signal-to-noise ratio (S/N) of a spectrum by 1.4-fold and can improve the mass accuracy up to 2-fold with no extra cost in instrumentation. Therefore, it is worthwhile to apply and promote absorption-mode in routine FT-ICR experiments.

Targeted High-Resolution Ion Mobility Separation Coupled to Ultrahigh-Resolution Mass Spectrometry of Endocrine Disruptors in Complex Mixtures

Traditional separation and detection of targeted compounds from complex mixtures from environmental matrices requires the use of lengthy prefractionation steps and high-resolution mass analyzers due to the large number of chemical components and their large structural diversity (highly isomeric). In the present work, selected accumulation trapped ion mobility spectrometry (SA-TIMS) is coupled to Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) for direct separation and characterization of targeted endocrine-disrupting compounds (EDC) from a complex environmental matrix in a single analysis. In particular, targeted identification based on high-resolution mobility (R ∼ 70-120) and ultrahigh-resolution mass measurements (R > 400u202f000) of seven commonly targeted EDC and their isobars (e.g., bisphenol A, (Z)- and (E)-diethylstilbestrol, hexestrol, estrone, α-estradiol, and 17-ethynylestradiol) is shown from a complex mixture of water-soluble organic matter (e.g., Suwannee River Fulvic Acid Standard II) complemented with reference standard measurements and theoretical calculations (<3% error).

Variation of the Fourier transform mass spectra phase function with experimental parameters.

It has been known for almost 40 years that phase correction of Fourier transform ion cyclotron resonance (FTICR) data can generate an absorption-mode spectrum with much improved peak shape compared to the conventional magnitude-mode. However, research on phasing has been slow due to the complexity of the phase-wrapping problem. Recently, the method for phasing a broadband FTICR spectrum has been solved in the MS community which will surely resurrect this old topic. This paper provides a discussion on the data processing procedure of phase correction and features of the phase function based on both a mathematical treatment and experimental data. Finally, it is shown that the same phase function can be optimized by adding correction factors and can be applied from one experiment to another with different instrument parameters, regardless of the sample measured. Thus, in the vast majority of cases, the phase function needs to be calculated just once, whenever the instrument is calibrated.

Coculture of Marine Invertebrate-Associated Bacteria and Interdisciplinary Technologies Enable Biosynthesis and Discovery of a New Antibiotic, Keyicin

Advances in genomics and metabolomics have made clear in recent years that microbial biosynthetic capacities on Earth far exceed previous expectations. This is attributable, in part, to the realization that most microbial natural product (NP) producers harbor biosynthetic machineries not readily amenable to classical laboratory fermentation conditions. Such cryptic or dormant biosynthetic gene clusters (BGCs) encode for a vast assortment of potentially new antibiotics and, as such, have become extremely attractive targets for activation under controlled laboratory conditions. We report here that coculturing of a Rhodococcus sp. and a Micromonospora sp. affords keyicin, a new and otherwise unattainable bis-nitroglycosylated anthracycline whose mechanism of action (MOA) appears to deviate from those of other anthracyclines. The structure of keyicin was elucidated using high resolution MS and NMR technologies, as well as detailed molecular modeling studies. Sequencing of the keyicin BGC (within the Micromonospora genome) enabled both structural and genomic comparisons to other anthracycline-producing systems informing efforts to characterize keyicin. The new NP was found to be selectively active against Gram-positive bacteria including both Rhodococcus sp. and Mycobacterium sp. E. coli-based chemical genomics studies revealed that keyicins MOA, in contrast to many other anthracyclines, does not invoke nucleic acid damage.

Electron-induced dissociation (EID) for structure characterization of glycerophosphatidylcholine: determination of double-bond positions and localization of acyl chains

Glycerophospholipids are a highly abundant and diverse collection of biologically relevant lipids, and distinction between isomeric and isobaric species is a fundamental aspect for confident identification. The ability to confidently assign a unique structure to a glycerophospholipid of interest is dependent on determining the number and location of the points of unsaturation and assignment of acyl chain position. The use of high-energy electrons (>20u2009eV) to induce gas-phase dissociation of intact precursor ions results in diagnostic product ions for localizing double-bond positions and determining acyl chain assignment. We describe a high-resolution, tandem mass spectrometry method for structure characterization of glycerophospholipids using electron-induced dissociation (EID). Furthermore, the inclusion of nomenclature to systematically assign bond cleavage sites with acyl chain position and double-bond location enables a uniform platform for lipid identification. The EID methodology detailed here combines novel application of an electron-based dissociation technique with high-resolution mass spectrometry that facilitates a new experimental approach for lipid biomarker discovery and validation.

2-Aminoethanol Extraction as a Method for Purifying Sc3N@C80 and for Differentiating Classes of Endohedral Fullerenes on the Basis of Reactivity

Extraction with 2-aminoethanol is an inexpensive method for removing empty cage fullerenes from the soluble extract from electric-arc-generated fullerene soot that contains endohedral metallofullerenes of the type Sc3N@C2n (n = 34, 39, 40). Our method of separation exploits the fact that C60, C70, and other larger, empty cage fullerenes are more susceptible to nucleophilic attack than endohedral fullerenes and that these adducts can be readily extracted into 2-aminoethanol. This methodology has also been employed to examine the reactivity of the mixture of soluble endohedral fullerenes that result from doping graphite rods used in the Krätschmer-Huffman electric-arc generator with the oxides of Y, Lu, Dy, Tb, and Gd. For example, with Y2O3, we were able to detect by mass spectrometry several new families of endohedral fullerenes, namely Y3C108 to Y3C126, Y3C107 to Y3C125, Y4C128 to Y4C146, that resisted reactivity with 2-aminoethanol more than the empty cage fullerenes and the mono- and dimetallo fullerenes. The discovery of the family Y3C107 to Y3C125 with odd numbers of carbon atoms is remarkable, since fullerene cages must involve even numbers of carbon atoms. The newly discovered families of endohedral fullerenes with the composition M4C2n (M = Y, Lu, Dy, Tb, and Gd) are unusually resistant to reaction with 2-aminoethanol. Additionally, the individual endohedrals, Y3C112 and M3C102 (M = Lu, Dy, Tb and Gd), were remarkably less reactive toward 2-aminoethanol.

Quantification of cocaine and its adulterants (lidocaine and levamisole) using the Dragendorff reagent allied to paper spray ionization mass spectrometry

Thin layer chromatography is a simple, easy and cheap technique widely used in Brazilian forensic laboratories, being a screening test for the separation and identification of illicit drugs, such as cocaine and its adulterants. Herein, paper chromatography using the Dragendorff reagent (revealing agent) was employed to analyze cocaine and its adulterants (levamisole, lidocaine, caffeine, and phenacetin). Positive results, i.e. visualization of the orange color, were only observed for cocaine, lidocaine and levamisole. Paper spray ionization mass spectrometry (PS-MS) was applied on revealed spots for the construction of a quantification model, for which some figures of merit were determined such as linearity, limit-of-detection (LOD), limit-of-quantification (LOQ) and accuracy. The results showed that PS-MS in positive-ion ionization mode, PS(+)-MS, is an efficient technique for direct analysis of the chromatographic spots on office-type paper revealed by the Dragendorff reagent. The method presented linearity greater than 0.98, and LODs of 6.51 μg mL−1 and 13.53 μg mL−1 for cocaine and levamisole, respectively, and 0.35 mg mL−1 for lidocaine. PS(+)MS was also applied to quantify cocaine in ten street crack samples, where there was no statistically significant difference between PS(+)MS and gas chromatography with flame ionization detection (GC-FID) data at a significance level of 5%.