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

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.

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

Of gels, offgel and off with the gel

Hoang-Trang Lam

In the context of prefractionation methods for proteomics, this work deals mainly with the development of electrophoretic tools for isoelectric focusing of peptides and proteins for the analysis of biological mixtures. In the light of existing devices for isoelectric focusing (IEF), the objective has been to develop multicompartment devices, designed for the IEF of peptides and proteins. The first choice of the OFFGEL for IEF among other techniques is justified by the easy recovery of liquid fractions of peptides, of small volumes, further amenable to liquid chromatography (as a second dimension separation) or mass spectrometry analyses. The resolution is a key point to consider in the design of separation units. Finite element simulation of the isoelectric focusing of peptides by OFFGEL has allowed the design of a multicompartment OFFGEL device for high resolution separation of peptides. The numerical simulations have highlighted the importance of the mobility near pI for the IEF kinetics and the final peak shape. The calculation of the distribution of peptides mobility near pI for three proteomes has allowed concluding on the optimal width of the well to obtain best separation. This mathematical study has also illustrated the high focusing power of the OFFGEL technique as a separation tool for shotgun proteomics application. The design of a multicompartment OFFGEL device was then done, based on the results of the simulations. The reproducibility of the pH gradient was validated, the loading capacity was evaluated for proteins, and a demonstration of the high resolution separation of peptides and proteins from a complex biological mixture was performed. The OFFGEL separation was then integrated in a workflow combining chemical tagging of the cysteine residues. This approach showed that the high resolution of peptide OFFGEL and the added information on the sequence of the peptides permitted a more confident and accurate protein identification. In the context of gel-free proteomics, another separation cell has been designed, that enables performing isoelectric focusing without the need of an immobilized pH gradient (IPG) gel. The novel device has been characterized in terms of performances and has been be applied to a biological sample of Escherichia coli, showing a more rapid separation of proteins than OFFGEL IEF, thus demonstrating its potential for fast proteome prefractionation purposes. An electrochemical cell has also been designed, for the transfer of ionizable species by electrochemistry at the micro-interface of two immiscible electrolytes (µ-ITIES), supported by an IPG gel as the aqueous phase and a small drop of organic phase. This study was initially motivated by the aim of performing online extraction of proteins/peptides during IEF separation. The use of this device for the transfer of model molecules was demonstrated, opening the door to further developments concerning the electrochemical transfer of proteins.

EPFL2008

Spectroscopic studies of kinetically trapped conformations in the gas phase: the case of triply protonated bradykinin

Thomas Rizzo, Liudmila Voronina

Understanding the relation between the gas-phase structure of biological molecules and their solution-phase structure is important when attempting to use gas-phase techniques to address biologically relevant questions. Directly after electrospray ionization, molecules can be kinetically trapped in a state that retains some “memory” of its conformation in solution and is separated from the lowest-energy gas-phase structure by barriers on the potential energy surface. In order to identify and characterize kinetically trapped structures, we have explored the conformational space of triply protonated bradykinin in the gas phase by combining field-asymmetric ion mobility spectrometry (FAIMS) with cold ion spectroscopy. We isolate three distinct conformational families and characterize them by recording their UV-photofragment spectra and vibrational spectra. Annealing of the initial conformational distribution produced by electrospray reveals that one of the conformational families is kinetically trapped, while two others are stable, gas-phase structures. We compare our results to previously published results obtained using drift-tube ion mobility spectrometry (IMS) and propose a correspondence between the conformational families separated by FAIMS and those by IMS.

Royal Soc Chemistry2015

Of gels, offgel and off with the gel

Hoang-Trang Lam

EPFL2008