Standard-Free Quantification of Dicarboxylic Acids: Case Studies with Salt-Rich Effluents and Serum

The present study evaluates the ionization efficiency (IE) of linear and branched C2–C14 dicarboxylic acids (DCAs) by electrospray ionization (ESI) under different conditions. The influence of the concentration of organic modifier (MeOH); mobile phase additive; and its concentration, pH, and DCA structure on IE values is studied using flow injection analysis. The IE values of DCAs increase with the increase of MeOH concentration but also decrease with an increase of pH. The former is due to the increase in solvent evaporation rates; the latter is caused by an ion-pairing between the diacid and the cation (ammonium), which is confirmed by the study with different amines. The investigation of DCA ionization in the presence of different acidic mobile phase additives showed that a significant improvement in the (−)ESI responses of analytes was achieved in the presence of weak hydrophobic carboxylic acids, such as butyric or propanoic acid. Conversely, the use of strong carboxylic acids, such as trichloroacetic acid, was found to cause signal suppression. The results of the IE studies were used to develop the liquid chromatography–high-resolution mass spectrometry (LC-HRMS) method that provided instrumental limits of detection in the range from 6 to 180 pg. Furthermore, upon applying the nonparametric Gaussian process, a model for the prediction of IE values was developed, which contains the number of carbons in the molecule and MeOH concentration as model parameters. As a case study, dicarboxylic acids are quantified in salt-rich effluent and blood serum samples using the developed LC-HRMS method.

New ionization and tagging methods for mass spectrometry

Xiaoqin Zhong

Mass spectrometry (MS) is an essential detection tool in bioanalytical chemistry owing to its exceptional selectivity and sensitivity, paired with rapid analyte identification and quantification. In this thesis, two classical MS and a newly developed ionization method are employed, including matrix-assisted laser desorption/ionization (MALDI) MS, electrospray ionization (ESI) MS and electrostatic spray ionization (ESTASI) MS. To meet the emerging challenges in bioanalytical chemistry, from disease diagnosis to drug development, new MS-based analytical methods ought to be developed with respect to high sensitivity, throughput, speed, as well as sample consumption and experimental simplification. For these purposes, this thesis presents four analytical strategies combined with MS detection for improved analytical performance in different research fields. Two classical analytical tools, thin layer chromatography (TLC) and 384-well plate, were coupled with ESTASI-MS respectively. In ESTASI-TLC-MS, ESTASI was applied to extract and identify a wide range of molecules of different polarities and chemical structures from both hydrophilic or hydrophobic silica plates with high sensitivity and minimal sample consumption in the femtomole range. In 384-well plate ESTASI-MS, the commercial 384-well plate could work as a container and an emitter for sample spray ionization, without any liquid delivery system or any additional interface. This approach provides fast and high throughput analyses for the large batches of reactions, such as enzyme assay and drug metabolism. Tyrosinase-catalyzed tyrosine oxidation in the presence or absence of inhibitors and cytochrome P450-catalyzed metabolic reactions of two drugs were studied respectively. To improve analysis sensitivity, two strategies, mass barcoded gold nanoparticles (Mb-AuNPs) for MALDI-MS signal amplification and microfabricated on-chip spyhole (Ø 10-12 ¿m) emitter for nanoelectrospray (spyhole-nanoESI), were developed, leading to a low sample consumption and high analysis efficiency. Mb-AuNPs combined with magnetic separation were applied for multiplex cow¿s milk allergy diagnosis in a component-resolved manner. IgE antibodies (Abs) could be extracted from a patient¿s blood serum by the formation of a sandwich structure between allergenic proteins-coated Mb-AuNPs and anti-human IgE Abs-functionalized magnetic beads (MBs). Detection of Mb-AuNPs by MALDI-MS provides a limit of detection (LOD) down to picograms per milliliter level for specific IgE Abs from only 1 ¿L of patient¿s blood serum. To take advantages of microfluidics in low sample consumption and easy integration, a novel interface of spyhole-nanoESI was designed for coupling microfluidics with MS and showed an improved sensitivity than standard ESI. This disposable microchip coupled with MS/MS shows potential application in cancer diagnosis by the successful detection of a small cell lung cancer biomarker in 1 ¿L of human serum at the extensive stage, without any complicated sample preparation steps.

EPFL2017

Identification of Isomeric Biomolecules by Infrared Spectroscopy of Solvent-Tagged Ions

Andrei Zviagin

The difference in functionality of many isomeric biomolecules requires their analytical identification for life science studies. We present a universal approach for quantitative identification of different small- to medium-sized isomeric biomolecules that can be brought to the gas phase from solution by electrospray ionization (ESI). The method involves infrared (IR) fragment cold ion spectroscopy of analyte molecules that are incompletely desolvated by soft ESI. The use of solvent molecules as natural tags removes a need for adding to solutions any special compounds, which may interfere with liquid chromatography or mass spectrometric measurements. The tested peptides and especially monosaccharides and lipids exhibit highly isomerspecific IR fragment spectra of such noncovalent complexes, which were produced from water, methanol, acetonitrile, and 2butanol solutions. The relative concentrations in solution mixtures of, for instance, two isomeric dipeptides can be quantified with the accuracy of 1.6% and 2.9% for the acquisition time of 25 min and, potentially, 5 s, respectively; for three isomeric phosphooctapeptides, the accuracy becomes 4.1% and 11% for 17 min and, potentially, 10 s measurements, respectively.

AMER CHEMICAL SOC2022

In-Spray Supercharging of Peptides and Proteins in Electrospray Ionization Mass Spectrometry

Luca Fornelli, Hubert Girault, Yu Lu, Sasa Miladinovic, Krzysztof Marek Piech, Yury Tsybin

Enhanced charging, or supercharging, of analytes in electrospray ionization mass spectrometry (ESI MS) facilitates high resolution MS by reducing an ion mass-to-charge (m/z) ratio, increasing tandem mass spectrometry (MS/MS) efficiency. ESI MS supercharging is usually achieved by adding a supercharging reagent to the electrospray solution. Addition of these supercharging reagents to the mobile phase in liquid chromatography (LC)-MS/MS increases the average charge of enzymatically derived peptides and improves peptide and protein identification in large-scale bottom-up proteomics applications but disrupts chromatographic separation. Here, we demonstrate the average charge state of selected peptides and proteins increases by introducing the supercharging reagents directly into the ESI Taylor cone (in-spray supercharging) using a dual-sprayer ESI microchip. The results are comparable to those obtained by the addition of supercharging reagents directly into the analyte solution or LC mobile phase. Therefore, supercharging reaction can be accomplished on a time-scale of ion liberation from a droplet in the ESI ion source.

2012

New ionization and tagging methods for mass spectrometry

Xiaoqin Zhong

EPFL2017