Matrix-assisted laser desorption/ionization

In mass spectrometry, matrix-assisted laser desorption/ionization (MALDI) is an ionization technique that uses a laser energy-absorbing matrix to create ions from large molecules with minimal fragmentation. It has been applied to the analysis of biomolecules (biopolymers such as DNA, proteins, peptides and carbohydrates) and various organic molecules (such as polymers, dendrimers and other macromolecules), which tend to be fragile and fragment when ionized by more conventional ionization methods. It is similar in character to electrospray ionization (ESI) in that both techniques are relatively soft (low fragmentation) ways of obtaining ions of large molecules in the gas phase, though MALDI typically produces far fewer multi-charged ions. MALDI methodology is a three-step process. First, the sample is mixed with a suitable matrix material and applied to a metal plate. Second, a pulsed laser irradiates the sample, triggering ablation and desorption of the sample and matrix material. Fi

Mass Spectrometry Based Analysis and Bioanalysis

Hongyan Bi

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.

EPFL2010

Novel Analytical Methods for Allergy-Related Studies

Mikaël Frossard

Managing and treating diseases require a deep understanding of the pathologies coupled with accurate diagnostic procedures. The constant development and improvement of bioanalytical methods reflect the need of diagnosis ameliorations with respect to accuracy, sensitivity and efficiency. The field of allergy diagnosis is a good example. Despite affecting almost a third of the population in western countries, allergies still present complex challenges for the physicians in terms of diagnosis and allergen characterization. This thesis presents novel analytical methods developed for the fast and personalized allergy component-resolved diagnosis (CRD), the allergen identification and quantification in food matrices and the characterization of allergen modifications, notably nitration. Immunomagnetic separation (IMS) was used to perform the allergy diagnosis at the molecular level by extracting the IgE antibodies from allergic patient blood sera and probing them against individual allergens and natural allergenic extracts. The fast and highly selective IMS procedure was coupled to matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) mass spectrometry (MS) to identify accurately and with high sensitivity the proteins recognized as sensitizers by the patient antibodies. The developed CRD methods were successful to diagnose cowâs milk, egg, peanut and tree nuts allergies, allowing the resolution of several complex clinical cases. Additionally, precious information was collected for the cross-reactivity and cross-sensitization in peanut and tree nuts allergies using blood sera from the PronNut clinical study. Allergen nitration is one of the potential causes for the increase in allergy prevalence observed recently and requires thorough investigation. Herein, various nitrating agents were tested and compared for in vitro allergen nitration. After modifying allergens using two of the most efficient nitrating procedures, the nitration sites were identified by bottom-up proteomics (BUP) and compared to the known epitopes, bringing useful information and novel insights to the problem of correlation between protein nitration and allergenicity. The presence of a minute amount of allergens in a food product is responsible for potential life-threatening reactions in highly allergic patients and sensitive analytical methods are therefore required in food quality assessments. A proteomic-based method was used for the quality control of several food products suspected of causing severe reactions for peanut and hazelnut-allergic patients. Peptide markers were identified in a first screening of the food extracts by BUP using a high-resolution MS instrument. A standard addition method was then used to quantify peanut and hazelnut proteins by BUP using an untargeted âshotgunâ approach.

EPFL2020

An Experimental Setup for Heterogeneous Catalysis on Atomically Defined Metal Nanostructures

Jean-Guillaume De Groot

The main objective of this PhD thesis is to link the atomic scale structure to the catalytic properties of self-assembled nanostructures. The growth and characterization of these nanostructures was carried out in an ultra-high vacuum (UHV) chamber initially designed for the study of the kinetics of epitaxial growth of thin films and nanostructures by variable temperature scanning tunneling microscopy. During this PhD thesis, we built a very sensitive and selective gas detector based on a quadrupole mass spectrometer and adapted to the geometry of this UHV system. The proper functioning of the gas detector is confirmed by temperature programmed desorptions of Xe on Ag(100) which demonstrate the very high sensitivity of the device of about

10^{-6}

~monolayer (ML). The zero-order desorption parameters obtained for the first monolayer of Xe adsorbed on Ag(100) are

E_d=0.22-0.23

~eV with a prefactor

\nu_0

between

4\times10^{12}

^{-1}

and

1 \times10^{13}

^{-1}

. An exchange process between Fe adatoms and Ag atoms from the Ag(100) surface is identified by means of temperature programmed desorption of N

_2

. Desorption spectra suggest that the exchange is complete from an annealing temperature of 140~K. Our study of the catalytic activity of metal nanostructures focuses on the ability of passivated Fe clusters to adsorb N

_2

molecules without dissociating them, which is a fundamental step in the bio-inspired heterogeneous catalysis of ammonia. The first system investigated for the growth of Fe clusters is MgO/Ag(100). Diffusion of Fe adatoms deposited on a MgO monolayer is observed from an annealing temperature of 280~K onwards. Fe clusters with an average size of

12.1\pm 0.5

~atoms are obtained on a MgO monolayer by thermal ripening of 0.072~ML of Fe deposited at 40~K. No sign of N

_2

adsorption was observed on these clusters by thermal programmed desorption investigation. The second system chosen for the growth of Fe clusters is graphene/Ir(111) obtained by chemical vapor deposition. The deposition of 0.3 and 0.4 ML of Fe on a graphene/Ir(111) sample at 50~K, followed by annealing at 300~K, generates a superlattice of Fe clusters which matches the periodicity of the moiré formed by graphene/Ir(111). The theoretically expected mean cluster size is 26 atoms for 0.3~ML of Fe, and 35 atoms for 0.4~ML of Fe. The temperature stability of the clusters obtained with 0.4~ML Fe is investigated from 300~K to 600~K. It is established that Smoluchowski ripening takes place as the annealing temperature increases and the superlattice structure has completely disappeared after annealing at 600~K. Temperature programmed desorption of molecularly adsorbed N

_2

at 50~K on a sample with 0.4~ML of Fe deposited on graphene/Ir(111) reveals 3 desorption peaks at 120~K, 92~K, and 60~K. In addition, slow dissociation of nitrogen molecules at 50~K adsorbed on the higher energy adsorption sites is demonstrated.

EPFL2021