Tracking chemical reactions on the surface of filamentous phage using mass spectrometry

Chemical modification of phage libraries has allowed the in vitro evolution of ligands having properties not provided by natural polypeptides. The development of novel and more diverse chemical reactions on phage was hampered by the lack of analytical methods to efficiently monitor the reaction products on the more than 10 000 kDa large filamentous phage particles. Herein, we present a strategy to detect chemically modified peptides on phage based on enzymatic release of peptide from phage and mass spectrometry analysis.

Polymer nanospray interfaces for on-line electrochemically induced modifications

Tatiana Rohner

The aim of this work has been to design, and fabricate a miniaturized electrospray-type interface for mass spectrometry analysis, which allows the nebulization of solutions by applying a high potential difference between the source and the mass spectrometer. The fabrication of the nanospray emitter is based on UV-laser photoablation of polymer substrates. Thick-film channel flow electrodes can be integrated in the microfluidic system, allowing the application of a high voltage to generate electrospray from the open outlet of the microchannel. The polymer-based interface first presented is characterized by a flat edge interface. However, a V-shaped nozzle has been found to present higher stability and ease of use. This design also allows the generation of spray by applying high voltage through an electrode located in a solution reservoir, thereby inducing an electroosmotic pumping. Taking advantage of the intrinsic electrolytic behavior of electrospray, a specific electrochemically induced tagging of free cysteine residues located in proteins can be performed on-line. A p-hydroquinone buffer is used to perform a continuous infusion of protein solution. When in contact with the high-voltage microelectrode, p-hydroquinone undergoes oxidation, thereby producing protonated p-benzoquinone. The latter reacts with free cysteine residues, following a Michael-type addition. The modification of the protein can be followed thanks to MS analyses. A complete study of the mechanisms involved in the electrochemically induced tagging with hydroquinone has been carried out by cyclic voltammetry and digital simulation. The process of the tagging consists of an ECE mechanism, since the formed adduct is stabilized in the reduced state, and is thus free to be oxidized. The rate constant of the chemical reaction has been extracted, by performing cyclic voltammetry studies and modeling the whole system by digital simulations. Finite element simulations have also permitted to highlight several mechanistic aspects of the electrotagging. The nanospray interface has been modeled as a channel-flow electrode cell. After validation of the model with respect to the experimental results, kinetics and mass transport conditions have been investigated. The flow profile, as well as the kinetic rate constant, has been shown to be of great importance in the electrochemically induced tagging. The use of a sacrificial electrode as metallic ion source to generate electrospray has also been investigated. Transition metal electrodes, i.e. copper, zinc, nickel and iron, as well as silver, were used to get on-line complexation with model peptides. It has been demonstrated that the use of in-reservoir sacrificial electrode was an efficient method to generate metal ions in order to form complexes with peptides, without the addition of metallic salts. These studies have demonstrated that polymer nanospray interfaces can be advantageously used as analytical devices, together with their primary function, i.e. ionization sources for protein analyses by mass spectrometry.

EPFL2003

Phage Selection of Bicyclic Peptide Ligands and Development of a New Peptide Cyclisation Method

Jeremy Touati

Bicyclic peptides binding to targets of interest can be isolated from combinatorial libraries using a phage display-based approach. In a first project of this thesis, we aimed at generating bicyclic peptide inhibitors of matrix metalloproteinases (MMPs), a family of proteases that share high structural similarities and have been difficult to target in a specific manner. From phage-encoded combinatorial libraries, we isolated a bicyclic peptide that inhibits specifically the gelatinase MMP-2 with a Ki of 2 μM. This inhibitor was less potent than the best peptidic MMP-2 inhibitor, the linear APP-derived inhibitory peptide (Ki = 13 nM). However, due to its conformational constraint, the bicyclic peptide is expected to be significantly more stable and hence more suitable for applications in biological systems. It may be used as a research tool alternatively to the broadly applied monocyclic peptide MMP-2 inhibitor CTT which is less potent (Ki of 142 μM). If affinity matured, the bicyclic peptide MMP-2 inhibitor might even be developed into an anti-cancer therapeutic. In a second project, bicyclic peptide binders to the centriolar protein SAS-6 were generated. SAS-6 plays an important structural role in centrioles and bicyclic peptide binders are currently applied in cellular assays by the laboratory of Professor Pierre Gönczy (EPFL) to study the process of centriole formation. In a third project of this thesis, we established an enzymatic method to selectively cyclise peptides with unprotected side chains using a transglutaminase (TGase). TGases catalyse the formation of stable amide bonds between the side chains of glutamine and primary amines. They have been used extensively in biotechnological applications to cross-link peptides and proteins or to attach labels to proteins. We found that the microbial transglutaminase (MTGase) of Streptomyces mobaraensis can cyclise quantitatively peptides with an N- terminal glutamine-donor sequence and a C-terminal lysine residue in an intramolecular reaction. Experiments with minimised glutamine-donor substrates revealed that peptides with only an Ala-Leu dipeptide N-terminal to the glutamine and a randomly chosen peptide between the glutamine and lysine residues are efficiently cyclised. By combining the MTGase-based cyclisation strategy with a chemical thiol-based cyclisation reaction, we were able to generate tricyclic peptide structures. It remains to be tested if this method can be applied to cyclise combinatorial peptide libraries on the surface of phage. In a fourth and last project, we developed a method to follow qualitatively and quantitatively chemical or enzymatic modifications applied to peptides displayed on phage. In this method, peptides on phage were chemically modified, cleaved off by a protease, purified, concentrated and analysed by mass spectrometry. The method will help to assess new reactions applied to phage peptides such as the MTGase-based enzymatic cyclisation reaction.

EPFL2012

Mass Spectrometry based Analytical Methods for Proteome Analysis and Redoxomics

Liang Qiao

Proteomics is nowadays one of the most important research topics in life science for understanding many life activities from the molecular level. The objective is to find out proteins or modifications on proteins that regulate biological events, aging, diseases and so forth. Because of the complexity in the structure of proteins, the dynamic range of protein-abundances, and the differences from time-to-time and tissue-to-tissue, proteomics requires development of analytical methods with respect to resolution, sensitivity, speed, etc. In this thesis, analytical methods based on mass spectrometry are developed for proteome research and especially redox proteomics. Two major types of mass spectrometers are employed, including matrix-assisted laser desorption/ionization mass spectrometer (MALDI-MS) and electrospray ionization mass spectrometer (ESI-MS). Nano and micro metal oxide materials are used to modify the MALDI sample plate for in-situ extraction of phosphorylated peptides or for in-source photochemical reactions. The former is valuable for the study of protein posttranslational modifications. The latter results in online tagging of cysteine for counting the number of cysteine in peptides that is valuable information for the identification of cysteine-containing proteins. Functional microfluidic chips are designed as microreactor and ESI emitter for online chemical reactions followed with MS characterization. Specifically, the multi-channel microchip is applied in tyrosine nitration study. Tyrosine nitration is considered as a biomarker for radical associated diseases, such as cardiovascular, Alzheimer’s, and Parkinson’s diseases. With the microchip ESI-MS, a new pathway for tyrosine nitration induced by copper(II) is demonstrated. Considering that abnormally high concentration of copper(II) was found in the brain tissue of patient with Alzheimer’s disease, the current finding is important for understanding the mechanism of neurodegenerative diseases. An electrostatic-spray ionization (ESTASI) method is developed to generate ions from sample solutions. The ESTASI can be realized with various geometries, such as samples in a microchannel, in a capillary, in a micropipette and on a plastic plate. Comparing to ESI, voltage is not directly applied on the sample during ESTASI, thereby avoiding redox reactions during ionization. The ESTASI has been used to couple separation strategies with MS, including capillary electrophoresis and gel isoelectric focusing. These new methods can be useful in top-down proteomics and mass imaging.

EPFL2013