Paolo Benigni

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 > 400 000) 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).

Fast screening of polycyclic aromatic hydrocarbons using trapped ion mobility spectrometry – mass spectrometry

In the present paper, we showed the advantages of trapped ion mobility spectrometry coupled too mass spectrometry (TIMS-MS) combined with theoretical calculations for fast identification (millisecond timescale) of polycyclic aromatic hydrocarbons (PAH) compounds from complex mixtures. Accurate PAH collision cross sections (CCS, in nitrogen as a bath gas) are reported for the most commonly encountered PAH compounds and the ability to separate PAH geometric isomers is shown for three isobaric pairs with mobility resolution exceeding 150 (3-5 times higher than conventional IMS devices). Theoretical candidate structures (optimized at the DFT/B3LYP level) are proposed for the most commonly encountered PAH compounds showing good agreement with the experimental CCS values (<5%). The potential of TIMS-MS for the separation and identification of PAH compounds from complex mixtures without the need of lengthy pre-separation steps is illustrated for the case of a complex soil mixture.

Oversampling Selective Accumulation Trapped Ion Mobility Spectrometry Coupled to FT-ICR MS: Fundamentals and Applications

In the present paper, we describe the fundamentals and analytical advantages of Oversampling Selective Accumulation Trapped Ion Mobility Spectrometry (OSA-TIMS) when coupled to ultrahigh resolution mass analyzers (e.g., FT-ICR MS). During TIMS analysis, ion packages are spatially resolved based on their mobilities along the TIMS analyzer axis and multiple strategies can be utilized during the trapping and elution of the ion population of interest. In the case of OSA-TIMS-FT-ICR MS, the TIMS operation sequence, trapping conditions, and operations are optimized to increase the signal-to-noise and the number of points across the mobility domain, which leads to more accurate mobility and mass measurements. Experimental results show that accurate ion-neutral collision cross sections (<1%) can be measured using OSA-TIMS-FT-ICR MS with high mobility resolving powers (RIMS up to 250), high mass accuracy (<1 ppm), and ultrahigh mass resolution (RMS up to 600-1200k at m/z 400) in a single analysis. The analytical advantages of OSA-TIMS over SA-TIMS were illustrated for the analysis of structural peptide isomers (SDGRG and GRGDS [M + H](+)), conformational isomers (AT-hook peptide 3 KRGRGRPRK [M + 2H](+2)), and a complex mixture of polyaromatic hydrocarbons (PAH) from coal tar. Baseline separation of the structural peptide isomers SDGRG and GRGDS, [M + H](+), was observed, and three conformations were identified for the AT-hook peptide 3 KRGRGRPRK [M + 2H](+2) during OSA-TIMS-FT-ICR MS. A 2-fold increase in the number of molecular features and a 2-6-fold signal-to-noise increase was observed for OSA-TIMS when compared with SA-TIMS during the PAH analysis. This work provides the proof-of-principle for further application of OSA-TIMS-FT-ICR MS for the unsupervised analysis of complex mixtures based on the characterization of the conformational space and the assignment of chemical formulas in a single analysis.

Towards unsupervised polyaromatic hydrocarbons structural assignment from SA-TIMS-FTMS data.

With the advent of high resolution ion mobility analyzers and their coupling to ultrahigh resolution mass spectrometers, there is a need to further develop a theoretical workflow capable of correlating experimental accurate mass and mobility measurements with tridimensional candidate structures. In the present work, a general workflow is described for unsupervised tridimensional structural assignment based on accurate mass measurements, mobility measurements, in silico 2D-3D structure generation, and theoretical mobility calculations. In particular, the potential of this workflow will be shown for the analysis of polyaromatic hydrocarbons from Coal Tar SRM 1597a using selected accumulation - trapped ion mobility spectrometry (SA-TIMS) coupled to Fourier transform- ion cyclotron resonance mass spectrometry (FT-ICR MS). The proposed workflow can be adapted to different IMS scenarios, can utilize different collisional cross-section calculators and has the potential to include MSn and IMSn measurements for faster and more accurate tridimensional structural assignment.

Analysis of Photoirradiated Water Accommodated Fractions of Crude Oils Using Tandem TIMS and FT-ICR MS

For the first time, trapped ion mobility spectrometry (TIMS) in tandem with Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) is applied to the analysis of the low energy water accommodated fraction (WAF) of a crude oil as a function of the exposure to light. The TIMS-FT-ICR MS analysis provided, in addition to the heteroatom series identification, new insights into the WAF isomeric complexity (e.g., [m/z; chemical formula; collision cross section] data sets) for a better evaluation of the degree of chemical and structural photoinduced transformations. Inspection of the [m/z; chemical formula; collision cross section] data sets shows that the WAF composition changes as a function of the exposure to light in the first 115 h by initial photosolubilization of HC components and their photo-oxidation up to O4-5 of mainly high double bond equivalence species (DBE > 9). The addition of high resolution TIMS (resolving power of 90-220) to ultrahigh resolution FT-ICR MS (resolving power over 400k) permitted the identification of a larger number of molecular components in a single analysis (e.g., over 47k using TIMS-MS compared to 12k by MS alone), with instances of over 6-fold increase in the number of molecular features per nominal mass due to the WAF isomeric complexity. This work represents a stepping stone toward a better understanding of the WAF components and highlights the need for better experimental and theoretical approaches to characterize the WAF structural diversity.

Fast, ultra-trace detection of juvenile hormone III from mosquitoes using mass spectrometry.

In the present work, a new protocol for fast separation and quantification of JH III from biological samples using liquid chromatography coupled to electrospray tandem mass spectrometry is described. In particular, the proposed protocol improves existing methodologies by combining a limited number of sample preparation steps with fast LC-MS/MS detection, providing lower limits of detection and demonstrated matrix effect control, together with high inter and intraday reproducibility. A limit of detection of 8pg/mL (0.32pg on column) was achieved, representing a 15-fold gain in sensitivity with respect to previous LC-MS based protocols. The performance of the LC-MS/MS protocol is comparable to previously described JH III quantitation protocol based on fluorescence detection, with the added advantage that quantification is independent of the availability of fluorescent tags that are often unavailable or show quite diverse responses on a batch-to-batch basis. Additionally, a detailed description of the JH III fragmentation pathway is provided for the first time, based on isolation of the molecular ion and their intermediate fragments using in-source MS/MS, MS/MS(n) and FT-ICR MS/MS measurements. The JH III workflow was evaluated as a function of developmental changes, sugar feeding and farnesoic acid stimulation in mosquitoes and can be applied to the detection of other juvenile hormones.

Towards Discovery and Targeted Peptide Biomarker Detection Using nanoESI-TIMS-TOF MS

AbstractIn the present work, the potential of trapped ion mobility spectrometry coupled to TOF mass spectrometry (TIMS-TOF MS) for discovery and targeted monitoring of peptide biomarkers from human-in-mouse xenograft tumor tissue was evaluated. In particular, a TIMS-MS workflow was developed for the detection and quantification of peptide biomarkers using internal heavy analogs, taking advantage of the high mobility resolution (R = 150–250) prior to mass analysis. Five peptide biomarkers were separated, identified, and quantified using offline nanoESI-TIMS-CID-TOF MS; the results were in good agreement with measurements using a traditional LC-ESI-MS/MS proteomics workflow. The TIMS-TOF MS analysis permitted peptide biomarker detection based on accurate mobility, mass measurements, and high sequence coverage for concentrations in the 10–200 nM range, while simultaneously achieving discovery measurements of not initially targeted peptides as markers from the same proteins and, eventually, other proteins. Graphical Abstractᅟ

Increasing Analytical Separation and Duty Cycle with Nonlinear Analytical Mobility Scan Functions in TIMS-FT-ICR MS

In this work, nonlinear, stepping analytical mobility scan functions are implemented to increase the analytical separation and duty cycle during tandem Trapped Ion Mobility Spectrometry and FT-ICR MS operation. The differences between linear and stepping scan functions are described based on length of analysis, mobility scan rate, signal-to-noise, and mobility resolving power. Results showed that for the linear mobility scan function only a small fraction of the scan is sampled, resulting in the lowest duty cycle 0.5% and longest experiment times. Implementing nonlinear targeted scan functions for analysis of known mobilities resulted in increased duty cycle (0.85%) and resolving powers (R up to 300) with a 6-fold reduction in time from 30 to 5 min. For broad range characterization, a nonlinear mobility stepping scan function provided the best sensitivity, resolving power, duty cycle (4%), and points per peak. The applicability of nonlinear mobility scan functions for the analysis of complex mixtures is illustrated for the case of a direct infusion of a MCF-7 breast cancer cell digest, where isobaric peptides (e.g., DFTPAELR and TTILQSTGK) were separated in the mobility domain (RIMS: 110) and identified based on their CCS, accurate mass (RMS: 550k), and tandem MS using IRMPD in the ICR cell.

Chemical Analysis of Water-accommodated Fractions of Crude Oil Spills Using TIMS-FT-ICR MS.

Multiple chemical processes control how crude oil is incorporated into seawater and also the chemical reactions that occur overtime. Studying this system requires the careful preparation of the sample in order to accurately replicate the natural formation of the water-accommodated fraction that occurs in nature. Low-energy water-accommodated fractions (LEWAF) are carefully prepared by mixing crude oil and water at a set ratio. Aspirator bottles are then irradiated, and at set time points, the water is sampled and extracted using standard techniques. A second challenge is the representative characterization of the sample, which must take into consideration the chemical changes that occur over time. A targeted analysis of the aromatic fraction of the LEWAF can be performed using an atmospheric-pressure laser ionization source coupled to a custom-built trapped ion mobility spectrometry-Fourier transform-ion cyclotron resonance mass spectrometer (TIMS-FT-ICR MS). The TIMS-FT-ICR MS analysis provides high-resolution ion mobility and ultrahigh-resolution MS analysis, which further allow the identification of isomeric components by their collision cross-sections (CCS) and chemical formula. Results show that as the oil-water mixture is exposed to light, there is significant photo-solubilization of the surface oil into the water. Over time, the chemical transformation of the solubilized molecules takes place, with a decrease in the number of identifications of nitrogen- and sulfur-bearing species in favor of those with a greater oxygen content than were typically observed in the base oil.