Mass Spectrometry Based Analysis and Bioanalysis

MS (mass spectrometry) has been developed as an important analytical tool to study large molecules including polymer macromolecules and proteins. Its instrumentation technique including ionization methods, mass analyzer and detector systems has been greatly improved from last century to present. Bio-molecules and organic macromolecules tend to be fragile and fragment when they are ionized by conventional ionization methods. ESI (electrospray ionization) and MALDI (matrix assisted laser desorption/ionization) are two relatively tender ionization methods and suitable for the ionization of big molecules. The study on mechanism surround these two ionization methods is a hot issue. For MALDI, one wants to figure out what exactly happens when the laser irradiates the analyte, where in-plume reaction occurs. As an example, the photo-triggered reaction can induce the fragmentation of peptides or proteins. Proteolysis is a key step for proteomic study especially bottom-up proteomic strategy. Porous materials have been developed in last decades. The catalysis of porous materials to enzymatic reaction is a hot issue, and has been demonstrated in much published works. But the mechanism of how different porous materials affect the reaction is not completely clear yet. We compared several different porous material influences for the reaction efficiencies. A kinetic model was proposed to explain and foresee the proteolysis. The model was verified by integrated CE (capillary electrophoresis)/MS detection system, where a custom-designed CE-MS interface was utilized to collect the fraction of digests for off-line MALDI TOF (time-of-flight) MS analysis. From an instrumental point, we then introduce Al-foil as disposable and once-used MALDI-target substitute, by which the sample preparation for MALDI TOF MS analysis is greatly simplified. The regeneration procedure of typical target in each run and attrition of target-plate can be neglected after holding this Al-foil on a commercially available target plate. This substrate provides comparable detection limits (LOD) of protein and digests (peptides) as the typical stainless steel MALDI target plate. The pliability of Al-foil makes it easy to be printed. While nano-scale semiconductor materials such as TiO2 have been widely used for dye-sensitized solar cell study. Following the MALDI target holder study, TiO2 nanoparticles were prepared as optimized ink and fabricated on the Al-foil via screen- and rotogravure- printing procedures. The woven mesh and engraved image carrier can be designed and controlled for different print pattern. The given apparatus here can provide two different TiO2 functionalized Al-foil. The functionalized foil then was then taken for the analysis of matrix free LDI (laser desorption ionization) and the study of protein phosphorylation, which is one of the most common and important protein post-translational modifications that occur in animal cells. Since the typical organic matrix interference always makes noise signals in low mass/charge on a mass spectrum, especially below 1000 or 700 Th, matrix free LDI is introduced to study the low mass molecules or small molecules. In this context, besides TiO2 nanoparticles, we also investigate CdSe semiconductor quantum dots, and electrochemically polymerized film of an aromatic diamine for the LDI analysis. A standard peptide was applied for the feasibility study of LDI based on these substrates. Semiconductor quantum dots are also hot target molecules for the dye-sensitized solar cell. By comparing the photocurrents on different quantum dots fabricated electrodes, the electron transfer process inside was simulated to understand the kinetics, which is also expected to guide the LDI study including the mechanism involved.

Miniaturized analytical systems for mass spectrometry-based protein studies

Mélanie Abonnenc

Current proteomic strategies depend strongly on the development of analytical methodologies and instrumentation. In parallel to the development of mass spectrometry (MS) - based proteomic workflows, microfluidic devices emerged in this field as a flexible tool for rapid and sensitive protein studies. In this context, the present work focuses on the development of miniaturized analytical systems for protein studies, especially by electrospray ionization mass spectrometric detection. Several approaches have been proposed to complement the mass spectrometric analysis of tryptic peptides using chemical tagging to isolate specific subclasses of proteins by affinity baits or for quantitation purposes. Optimization of a chemical tagging reaction in a sandwich mixer-reactor that consists of mixing two solutions providing the reactants in a channel, one central laminar flow being sandwiched between two outer flow solutions, was first investigated by finite-element method. The numerical simulations have highlighted the importance of positioning the reactant presenting the lowest diffusion coefficient close to the sidewall, as well as decreasing the outer flow velocities, to enhance a chemical reaction because of the parabolic flow profile across the microchannel. This optimization is even more relevant for consecutive reactions. As infusion-based electrospray microchips are known to present great advantages in terms of sensitivity compared to standard ionization source, an ESI emitter microchip based on polymer photo-ablation was designed. First, the hyphenation of the chip in a LC-MS workflow was successfully achieved and evaluated by comparison with a standard pneumatically-assisted ESI source. To perform enhanced on-chip post-column derivatization of peptides, a substitute design to the sandwich mixer-reactor was explored to improve reaction in a microchip before ESI – MS analysis. The electrospray micromixer chip includes a passive mixing unit to perturbate the flow along the microchannel. The mixing efficiency was demonstrated by fluorescence imaging, and on-chip chemical derivatization of peptides and kinetic studies before mass spectrometry were achieved. The on-line LC-MS derivatization of cysteinyl peptides was shown to provide a more confident and accurate protein identification by adding information on the peptidic sequence. Within the development of electrospray microchips for mass spectrometry, many efforts have been put forward in the hyphenation of functional units. Immobilization of functionalized stationary phase in microfluidic systems is widely used to achieve protein or peptide isolation, sample cleaning, separation or reaction. In this context, magnetic beads have proven to be quite useful compared to other methods, such as immobilized packed beads or monoliths, because of their high and reversible magnetization, and various surface functionalization. A polymer microchip including a magnetic track array was designed to focus the magnetic field generated by permanent magnets towards precise locations along the microchannel. A multi-plug magnetic bead capture was obtained and a significant increase of bead capture efficiency was demonstrated both numerically and experimentally, which is beneficial for affinity separation applications, as example. The research in microfluidic front-end devices for mass spectrometry is actually oriented to the development of multi-spray interfaces for high-throughput analysis. An electrospray microchip with a multi-track array was developed. The capability of the device to screen alternatively up to six samples and to perform relative quantification was assessed. Soft ionization techniques have revolutionized the analysis of proteins by mass spectrometry. In contrast with the major content of the thesis dealing with microfluidic systems for protein analysis, the last chapter presents a novel ambient ionization method, so-called membrane-desorption electrospray ionization (M-DESI). The principle is similar to the desorption electrospray ionization (DESI) method. But, in the M-DESI approach the sample is desorbed directly from a mesh membrane positioned vertically and in-axis between the ESI emitter and the mass spectrometer inlet. Analysis of peptides and proteins was demonstrated as a proof-of-concept, offering great perspectives for detection after separation on membrane.

EPFL2009

Microfluidic front-end technologies for protein electrospray mass spectrometry

Niels Erik Lion

In the present work, a polymer microfabricated microsprayer is characterised and tested for the mass spectrometric analysis of biomolecules, in particular in the context of protein mass spectrometry and proteomics. As the core of this work deals with analytical sciences and device development, Chapter 1 puts this work in perspective with the emergence of proteomics and more generally systems biology. In particular, major epistemological concepts that allowed the emergence of these new disciplines among life science research are reviewed. It is shown that the new paradigm of integrative biology did not emerge ex nihilo, but was preceded by major epistemological shifts in medicine, physics, and engineering. As such, the rise of systems biology appears as a new episode in the balance between reductionist and holistic approaches in the history of biology and medicine, and its biomedical promises are discussed. Chapter 2 reviews the state of the art in the hyphenation of microfluidic devices with electrospray mass spectrometry, both from a technical and applicative standpoint. Chapter 3 and 4 present the characterisation of the new polymer microspray for the analysis of peptides, proteins and glycoconjugates when coupled with various electrospray ion sources and instruments. In particular, a detailed comparison with classical pulled nanospray capillaries is performed, that established the performances in terms of sensitivity, stability and applicable ranges of flow rates and solvents. Chapter 5 introduces a functionalised polymer microspray for online sample clean-up. Basically, a hydrophobic polyvinylidene fluoride membrane is interfaced at the inlet of the polymer microspray to serve as a capture solid-phase of hydrophobic compounds; once captured, they can be cleaned up, and further eluted by the spray solution containing organic solvents. The process is further investigated in the presence of compounds usually used in protein sample preparation, such as chaotropes, reducing agents or detergents. Further microchip developments include the introduction of a dual channel microsprayer in Chapter 6, that allows to spray two solutions at the same time; given the microchip design, the two solutions are mixed only in the Taylor cone, where the electrospray is generated, and their respective flow rates can be controlled independently. This unique feature allows to analyse pure aqueous samples with integration of minimum amounts of organic sheath flow to promote desolvation and ionisation. Moreover, when proteins are analysed, it allows to measure them in their folded state, as demonstrated by their charge state distribution in the mass spectrum. Further tuning of the organic sheath flow allows to denature the protein within the Taylor cone, which can be monitored by the evolution of the protein charge state distribution in the mass spectrum. Potential of this technology for protein thermodynamic stability measurement is discussed. Lastly, Chapter 7 presents in silico evaluation of two technologies developed in the laboratory, namely Off-Gel electrophoresis for isoelectric fractionation of proteins and peptides mixtures, and online counting of cysteine residues within peptides during their analysis by electrospray mass spectrometry, to provide useful information to speed-up proteome profiling: if one wants to seek within a whole digested proteomes for peptides that are unique, measurement of their mass alone is hardly sufficient to perform useful protein identification, even with very high resolution, high mass accuracy instruments. The purpose of these simulations is to estimate how much information peptide isoelectric point and number of cysteines within each peptide provide in terms of number of unique peptides (and hence unambiguous peptide identification without MS/MS). Applicability of this strategy for high-throughput proteome profiling and its limitations are further discussed.

EPFL2006

Kinetics of Proteolytic Reactions in Nanoporous Materials

Hongyan Bi, Hubert Girault, Liang Qiao

Proteolysis with proteases preloaded within nanopores of porous material is a very fast process, where proteins can be digested in minutes compared to the conventional bulk enzyme reactions taking place over hours. To model this surprising phenomenon, a modified sequential proteolytic mechanism has been developed to simulate the kinetics of the reaction. Digestion of myoglobin was used as an example to show the high efficiency of the in-nanopore enzymatic reaction, while angiotensin 1 and ACTH (1-14) were selected as model peptides to validate the theoretical considerations. The proteolytic peptides were quantified by capillary electrophoresis and sequenced by mass spectrometry using bottom-up strategy. The simulation clearly shows that the major factor for the very fast digestion kinetics observed stems from a peptide confinement effect, where the generated peptides are trapped within a confined space for further proteolysis to the final products. On the other hand, the ingress and diffusion of the proteins into the porous cavity can accelerate or limit the first proteolytic step requiring the encounter between the substrates and enzymes. The present model can be widely applied to different enzyme catalyzed reactions for high-throughput protein profiling, and can promote the study of enzyme reactions occurring inside the cell.