Accelerating photofragmentation UV Spectroscopy–Mass spectrometry fingerprinting for quantification of isomeric peptides

Identification of isomeric biomolecules remains a challenging analytical problem. A recently developed spectroscopic method that combines UV photofragmentation and mass spectrometry for fingerprinting of cold ions (2D UV-MS), has already demonstrated its high performance in the library-based identification and quantification of different types of biomolecular isomers. The practical use of the method has been hindered by a slow rate of data acquisition, which makes the fingerprinting incompatible with high-throughput analysis and online liquid chromatography (LC) separation. Herein we demonstrate how the use of a few pre-selected wavelengths can accelerate the method by two orders of magnitude without a significant loss of accuracy. As a proof of principle, 2D UV-MS fingerprinting was coupled to online LC separation and tested for quantification of isomeric peptides containing either Asp or isoAsp residues. The relative concentrations of the peptides mixed in solution have been determined, on average, with better than 4% and 6% accuracy for resolving and non-resolving gradients of LC separation, respectively.

Practical Considerations for Improving the Productivity of Mass Spectrometry-based Proteomics

Luca Fornelli, Anton Kozhinov, Unige Anna Laskay, Kristina Srzentic, Yury Tsybin, Oxana Upir

Mass spectrometry (MS) is currently the most sensitive and selective analytical technique for routine peptide and protein structure analysis. Top-down proteomics is based on tandem mass spectrometry (MS/MS) of intact proteins, where multiply charged precursor ions are fragmented in the gas phase, typically by electron transfer or electron capture dissociation, to yield sequence-specific fragment ions. This approach is primarily used for the study of protein isoforms, including localization of post-translational modifications and identification of splice variants. Bottom-up proteomics is utilized for routine high-throughput protein identification and quantitation from complex biological samples. The proteins are first enzymatically digested into small (usually less than ca. 3 kDa) peptides, these are identified by MS or MS/MS, usually employing collisional activation techniques. To overcome the limitations of these approaches while combining their benefits, middle-down proteomics has recently emerged. Here, the proteins are digested into long (3-15 kDa) peptides via restricted proteolysis followed by the MS/MS analysis of the obtained digest. With advancements of high-resolution MS and allied techniques, routine implementation of the middle-down approach has been made possible. Herein, we present the liquid chromatography (LC)-MS/MS-based experimental design of our middle-down proteomic workflow coupled with post-LC supercharging.

Schweizerische Chemische Gesellschaft2013

Novel Approaches in Cold Ion Spectroscopy for Structural Characterization of Biomolecules in the Gas Phase

Vladimir Kopysov

Mass spectrometry is a powerful analytical technique, which has become invaluable across a wide range of scientific fields, such as "omics" sciences (e.g., proteomics), doping control and forensic sciences, drug development, environmental analysis, geologic research, etc. Originally developed over a century ago to measure elemental atomic weights and the natural abundances of isotopes, nowadays mass spectrometry is a vital tool for structural characterization of biomolecules, ranging from small metabolites to large macromolecular assemblies. This includes identifying unknown compounds, quantifying known compounds, and exploring molecular structures. However, the mass spectrometric analysis is based on measurements of the mass-to-charge ratio of ions, a quantity that is not sensitive to their three dimensional (3D) structure. Cold ion spectroscopy is another method of studying ions in the gas phase, that has demonstrated its capability to reveal their spectroscopic fingerprints, which are characteristic of the 3D structure of the ions on a fundamental level. In this thesis we present novel approaches to molecular structure identification, which are based on integration of high-resolution mass spectrometry (MS) with high-resolution ultraviolet (UV) photofragmentation spectroscopy of cold ions. The first part of this work describes the coupling of an Exactive Orbitrap-based mass spectrometer to a cold ion spectrometer and the use of this novel hybrid instrument for measuring two-dimensional (2D) UV-MS spectra of ionic species. The synergy of the two complementary techniques makes these spectra unique fingerprints of biomolecular ions. The second part presents an approach that uses mathematical analysis of these highly specific fingerprints for identification of isomeric biomolecules in mixtures. Using preliminarily recorded libraries of fingerprints, the UV-MS approach was successfully employed for quantitative identification of positional isomers of a phosphorylated peptide, diastereomers of an opioid peptide, and diastereomers of a drug molecule. We also demonstrate a library-free approach, which allows estimating the number of components present in a mixture and deriving their UV absorption and fragmentation mass spectra directly from the 2D UV-MS spectrum of the mixture. In the third part, we employ the library-free approach and the phenomenon of nonstatistical photofragmentation to identify conformers of gas-phase biomolecular ions and to recover their individual UV absorption and fragmentation mass spectra. We validate this approach with a benchmark dipeptide, Tyr-Ala, and then apply it to a decapeptide, gramicidin S, and a small drug molecule, homatropine. A careful analysis of the derived UV and mass spectra of the conformers of gramicidin S and homatropine provides some structural information that can be used for solving their 3D structures. In the last part, we demonstrate a use of 2D UV-MS spectroscopy for estimating the distances between the phenylalanine and tyrosine chromophores in four gas-phase opioid peptides. We have also calculated the 3D structures of their lowest energy conformers. We discuss these structures in the context of the estimated interchromophore distances and the pharmacological properties of the peptides.

EPFL2016

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

Novel Approaches in Cold Ion Spectroscopy for Structural Characterization of Biomolecules in the Gas Phase

Vladimir Kopysov

EPFL2016