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11. Česká konference hmotnostní spektrometrie 2023 (Den 1)

Út, 11.7.2023
| Originální článek z: Česká konference hmotnostní spektrometrie (ČSHS)
V pondělí 19. června byl zahájen první den 11. České konference hmotnostní spektrometrie exkurzí v Thermo Fisher Scientific, workshopem, 13 odbornými přednáškami a posterovou sekcí.
ČSHS: 11. Česká konference hmotnostní spektrometrie 2023 (Den 1)

ČSHS: 11. Česká konference hmotnostní spektrometrie 2023 (Den 1)

Program 11. České konference hmotnostní spektrometrie 2023

👉 Kompletní program včetně abstraktů ke stažení

Program CMSC 2023

Pondělí 19. června 2023

10:00 - 11:30 Exkurze v Thermo Fisher Scientific
12:00 - 19:30 Registrace
12:30 - 12:40 Zahájení 11. CMSC

ČSHS: Zahájení 11. České konference hmotnostní spektrometrie 2023 (Michael Volný)

ČSHS: 11. Česká konference hmotnostní spektrometrie 2023 (Den 1) - Odborný program

12:40 - 13:30 Plenární přednáška I: ICP-MS: the other mass spectrometry
  • Jan Kratzer (Ústav analytické Chemie AVČR v.v.i, Brno)

The aim of this lecture is to bridge the gap between the users and specialists in the field of organic mass spectrometry and the community working in inorganic (i.e. element) mass spectrometry employing the inductively coupled plasma mass spectrometry (ICP-MS) technique. ICP-MS is nowadays the most sensitive, multi-element and universal detector that allows detection of elements, especially metals, down to the pg·ml⁻¹ level. ICP-MS instrumentation has many similarities with organic mass spectrometers but it also shares many common features with other atomic spectrometric detectors used in trace element analysis. Analogously to other MS techniques, the ICP-MS consists of: 1) ion source, 2) mass analyzer and 3) the detector. ICP torch is the ion source realized by rf-driven (27 or 40 MHz) plasma sustained in argon at atmospheric pressure. This highly energetic plasma (8000 K, 1600 W) is responsible for atomization, excitation and finally ionization of analyte. The ions produced enter the low pressure environment of MS. They are separated according their mass to charge ratio (m/z) in a single quadrupole mass analyzer. Due to its low resolution (1 amu) strategies have been developed to avoid isobaric (on mass) interferences to distinguish between atomic ion of analyte and polyatomic ions as interferents having the same m/z, e.g. ⁷⁵As⁺ (analyte) versus ⁴⁰Ar³⁵Cl⁺ (interferent). For that purpose, the ICP-MS spectrometers are equipped with collision/reaction cells while their newest generation relies on triple quadrupole (ICP-MS/MS) configuration. The ions are subsequently detected by electron multiplier. Sample introduction is an inherent part of ICP-MS analysis affecting also the detector sensitivity. Like in other spectrometric detectors employed in element analysis liquid sample nebulization is the most common approach. Sample introduction efficiency is limited by the efficiency of the nebulizer (5-10%). Alternative approaches to sample introduction include solid sampling by laser ablation (LA) or gas phase sampling by vapor generation (VG). LA-ICP-MS is suitable for hard-to-degrade samples and enables also element imaging. VG-ICP-MS relies on selective analyte conversion from liquid to gas phase improving analyte introduction efficiency and decresing the risk of interferences due to matrix separation. Analysis of (metal) nanoparticles is also feasible by single particle (sp)-ICP-MS bringing information about their concentration and size distribution. ICP-MS is not only used to determine the total element content but it can be also applied to the speciation analysis, i.e. determination of different chemical species of the same element. Since ICP-MS is a ″destructive″ detector not preserving structure information about the species due to the atomization/ionization processes in the ICP torch, separation of the species by HPLC or GC have to precede their detection with speciation information derived from retention times. Applications of ICP-MS to trace element and speciation analysis in environmental and clinical samples will be discussed. Examples from the fields of metallomics will be demonstrated where both organic and inorganic MS can be combined as HPLC detectors to receieve simultaneously information on structure (ESI-MS) and ultratrace metal content (ICP-MS) in metalloproteins.

ČSHS: Plenární přednáška I: ICP-MS - the other mass spectrometry (Jan Kratzer, Ústav analytické Chemie AVČR v.v.i, Brno)

13:30 - 14:30 Workshop (Pragolab)

Evosep One platforma pro klinickou proteomiku

Přístroj Evosep One je standardizovaná platforma pro klinickou proteomiku, která je navržená tak, aby měla vysokou propustnost a robustnost. Toho lze docílit díky Evotips, což jsou jednorázové kolonky/špičky, ve kterých dochází k loadování a offline odsolení vzorku. Autosampler poté vezme špičku a integruje eluci s kapalnou chromatografií. Díky tomu lze redukovat kroky při přípravě a manipulaci se vzorky.

Pragolab: Evosep One - Analýza 100 vzorků denně v metodě a na standardní koloně

K eluci a tvorbě gradientu dochází při nízkém tlaku, čímž se snižuje opotřebování mechanických součástí přístroje. Jednotlivé špičky lze po každém runu ještě využít pro částečnou eluci, což prodlužuje životnost kolony. Analyticky důležité peptidy jsou efektivně zadrženy a také je snížená cross-kontaminace.

Přístroj Evosep One nabízí pět standardních metod, které lze použít od komplexní proteomické analýzy, přes frakcionační analýzu, až po vysoce výkonnou jednorázovou analýzu. Evosep One je standardizovaným řešením s výbornou reprodukovatelností a to pro „run-to-run“, „column-to-column či „instrument-to-instrument“ metody, čímž může usnadnit práci na individuálních studiích i na různých kolaboracích mezi laboratořemi.

Pragolab: Evosep One systém pro klinickou proteomiku

ČSHS: Workshop Pragolab - 11. Česká konference hmotnostní spektrometrie 2023

14:30 - 14:50 Přestávka na kávu

14:50 - 16:10 Sekce I

  • Chairperson: Lenka Hernychová
14:50 - 15:10 Orbitrap Ascend - experience in proteomics core facility
  • Karel Harant

Our core omics facility at Biocev has been using the first generation orbitrap tribrid mass spectrometer – Orbitrap Fusion Tribrid for the last eight years. This year, we have installed the latest member of the tribrid family Orbitrap Ascend Tribrid Mass spectrometer. We were able to compare the instruments’ performance for label free and isobaric labeling analysis. In addition, we tested several new features of the Orbitrap Ascend.

Isobaric labeling is a popular alternative to the label-free approach. It allows to design experiments that are not easily possible with label free work flows. One of the major limitations is ratio compression. By providing additional specificity, MS3 fragmentation solves this problem, but the solution comes at the expense of slowing down the data acquisition. Slower acquisition then results in reduction of the protein identifications obtained. The novel real-time search functionality, which was introduced with the Orbitrap Eclipse Tribrid Mass spectrometer, removes this time penalty and makes the isobaric labeling fully competitive to the label free work-flows in quantitative proteomics.

ČSHS: Orbitrap Ascend - experience in proteomics core facility (Karel Harant)

15:10 - 15:30 The characterization of proteome and protein-tissue analysis in the samples of the cervical mucus
  • Tomáš Oždian

The cervical mucus is the viscous plug of the cervix uteri, which dramatically changes its properties during the menstrual cycle and pregnancy. At the same time, it is a body fluid originating from cervical and uterine glands, which is easily and non-invasively obtained. The main task of this talk is to characterize the proteomic composition of healthy women undergoing artificial insemination with natural or stimulated menstrual cycles. According to known peristaltic properties of the uterus, we have also tried to assess proteins to the possible tissue origin using the human protein atlas database. As a result, we have identified 4370 proteins and 621 of them were differentially expressed in the samples with natural or stimulated cycles and we have found proteins with expression specific to the testis, liver, placenta, and neural tissues. Those findings further support the hypothesis of cervical mucus as the potential source of biomarkers for future studies.

ČSHS: The characterization of proteome and protein-tissue analysis in the samples of the cervical mucus (Tomáš Oždian)

15:30 - 15:50 Paclitaxel Neuropathy: A Glycoproteomic Approach to Predictive Biomarkers in Breast Cancer
  • Adam Paulin Urminský

Paclitaxel, a potent chemotherapeutic agent, is extensively used in the treatment of breast cancer, one of the most prevalent forms of cancer worldwide. Despite its efficacy, a significant challenge in the clinical use of paclitaxel is its association with peripheral neuropathy, a severe and often debilitating side effect that can dramatically affect patients' quality of life. This neuropathy is characterized by numbness, tingling, or pain in the patient's hands and feet, limiting their daily activities and often necessitating dose reduction or treatment discontinuation.

Currently, the capacity to predict which patients are more likely to develop Paclitaxel-induced PN is lacking. This inability to forecast this adverse effect hampers the clinicians' ability to personalize treatment plans, potentially compromising treatment efficacy and patient quality of life. To address this critical gap, our project aims to explore the blood serum glycoproteome of patients prior to paclitaxel treatment who had acquired paclitaxel neuropathy or whose treatment had not caused these issues. Glycoproteomics, the study of changes in the glycosylation status of proteins, holds promise in uncovering potential biomarkers for disease states and responses to treatment. We hypothesize that distinct patterns of protein glycosylation could signal an elevated risk of developing peripheral neuropathy in response to paclitaxel treatment.

Following our in-depth glycoproteomic investigation, our plan is to use machine learning methods to determine whether there is a particular combination of these proteoforms that could serve as a predictive model for paclitaxel-induced peripheral neuropathy.

ČSHS: Paclitaxel Neuropathy: A Glycoproteomic Approach to Predictive Biomarkers in Breast Cancer (Adam Paulin Urminský)

15:50 - 16:10 Perturbed N-glycosylation of IgG expressed in HEK293T cells in the presence of fluorinated monosaccharide analogs determined by MS
  • Tomáš Ječmen

Different fluorinated monosaccharide analogs were shown to inhibit the biosynthesis of characteristic glycan structures without being incorporated and have the potential to become a part of the treatment of diseases such as multiple sclerosis or certain types of cancer. Here, we investigate the possibility of determining whether the cellular glycosylation perturbed by different monosaccharide fluoro derivatives can be detected on a single glycoprotein level, and the inhibitor-specific N-glycan profiles can be distinguished on the affected glycoprotein. We overexpressed His-tagged Fc fragment of human IgG in HEK293T cells cultivated in the presence of one of the seven acetylated monofluoro analogs of hexosamines or fucose at 50μM concentration and in untreated cells serving as control. The proteins were digested in solution with trypsin and analyzed by RP-ESI-qTOF MS. Ion chromatograms for 19 glycoforms differing in monosaccharide composition, which were attached to a single peptide backbone, for the unglycosylated form of this peptide and two other peptides lacking glycosylation site were extracted, the areas under the curves were calculated and compared among the samples. For five of seven fluoro saccharides tested as potential glycosylation inhibitors, we observed an unchanged overall level of glycosylation at the concentration used. This was manifested as only a minimal amount of unglycosylated form of the peptide with a glycosylation site and was also observed in the controls. Also, the relative proportions of individual glycoforms did not differ significantly when compared to the proportions of the glycoforms in the respective controls. In contrast, a 1-2 orders of magnitude more abundant non-glycosylated form of the peptide was observed for the other two analogs, whose glycoprofiles differed markedly from the controls and also between them. For the acetylated 4-fluoro analog of GalNAc, the enriched glycoforms (Hex₃₋₅HexNAc₂) suggest that the later steps of the glycosylation pathways localized in the Golgi are predominantly affected by this inhibitor. In comparison, the rate of fucosylation was dramatically decreased in the presence of acetylated 2-fluoro fucose. A slight difference in total fucosylation level was also noticed for one of the controls, but the effect was much less pronounced than in the presence of 2-fluoro fucose.

ČSHS: Perturbed N-glycosylation of IgG expressed in HEK293T cells in the presence of fluorinated monosaccharide analogs determined by MS (Tomáš Ječmen)

16:10 - 16:30 Přestávka na kávu

Sponzoři a partneři

  • Bruker Daltonics
  • Merck

16:30 - 17:50 Sekce II

  • Chairperson: Jan Fiala
16:30 - 16:50 Identification and Characterization of Compounds Using Electron Activated Dissociation on SCIEX ZenoTOF 7600 System
  • Tomáš Korba

Electron Activated Dissociation (EAD) offers additional fragmentation technique to traditional collision induced dissociation (CID). In EAD, electron kinetic energies can be tuned from 0-25 eV without the use of chemical transfer reagents. This precise tunability means EAD can be performed on a wide range of analytes, from multiply charged peptides to singly-charged small molecules (1). A novel hybrid collision cell is at the heart of the technological innovations introduced with the SCIEX ZenoTOF 7600 system.

In the past, QTOF mass spectrometers have suffered from duty cycle losses. This was mainly due to the mating of the continuous beam coming from the quadrupole ion path with time-of-flight (TOF) analysis, a pulsed, discontinuous measurement technique. High capacity ion trap just after the CID collision cell (Q2) and before the pusher region of the TOF, with optimized release of ions from this trap, allows the duty cycle losses to be mitigated leading to MS/MS sensitivity gains of 4-20 fold (2).

The increase in MS/MS sensitivity, the high speed of acquisition (133Hz) and the choice of fragmentation regimes enables improvements in data quality and depth on analysis in proteomics, metabolomics and lipidomics workflows. Protein IDs from cell lines exceeds 5000 protein groups with up to 95% of these reliably quantitated (CV <20%) at minimal protein loads. Lipids can be fully characterized at an LC time scale (around 30ms EAD reaction time) including their lipid class, acyl group structure, and the location of double bond(s) (3).

Examples of EAD utilization presented in the talk include distinguishing of isomer and isobar small molecule compounds and middle down characterization of proteins in single analysis.

ČSHS: Identification and Characterization of Compounds Using Electron Activated Dissociation on SCIEX ZenoTOF 7600 System (Tomáš Korba)

16:50 - 17:10 Lipids and bone dismorphology in insig1 gene-deficient mice
  • Karel Chalupský

Rare diseases encompass a diverse range of medical conditions that affect a small number of individuals within a population. One intriguing aspect of rare diseases is their association with lipids, which are essential molecules involved in various biological processes. Lipids play a crucial role in cellular structure, energy storage, and signal transmission. In some cases, rare diseases result from abnormalities or dysfunction in lipid metabolism, and these conditions lead to the accumulation or depletion of lipids in various tissues, causing a wide array of symptoms.

One specific protein involved in the regulation of lipid metabolism and intracellular cholesterol synthesis is INSIG1 (Insulin-induced gene 1). To investigate the effects of reduced INSIG1 function, we utilized a strain of mice with a deletion of the Insig1 gene (Insig1 KO) and looked primarily on their liver lipidome, together with other phenotyping tests.

Our analysis of hepatic lipidome revealed an accumulation of cholesterolesters and triacylglycerols in the livers of Insig1 KO mice. Additionally, we observed alterations in metabolites of vitamin D in their plasma. Furthermore, Insig1 KO mice exhibited craniofacial bone development and finger malformation.

The Insig1 KO phenotype is an example of far-reaching consequence of alterations of lipid spectra. Through our study, we demonstrated the biological significance of cholesterol metabolism and its connection to bone abnormalities in a model of Insig1 deficiency. These findings shed light on the intricate relationship between lipid metabolism, cholesterol homeostasis, and rare diseases, contributing to a better understanding of the underlying mechanisms and future diagnostics.

ČSHS: Lipids and bone dismorphology in insig1 gene-deficient mice (Karel Chalupský)

17:10 - 17:30 Novel desorption-ionization ms affinity enzyme-linked assay for Clostridium bacteria family toxin activity detection
  • Josef Dvořák

Clostridium difficile is a dangerous human pathogen causing inflammation, diarrhea, or life-threatening pseudomembranous colitis. Toxin B produced by the bacterium catalyzes the transfer of glucose from UDPglucose to a threonine 37 of Ras homolog family member A protein (RhoA). The modification by glucose inactivates RhoA GTPase activity and leads to the disruption of the intestine cytoskeleton. The novel approach for C. difficile detection is based on in-situ enrichment of glucosylated intact RhoA using functionalized chips prepared by ambient ion soft-landing and MALDI-ToF measurement. Affinity proteinmodified chips were used prior to the enzymatic reaction for binding the affinity-tagged recombinant RhoA protein. The reaction buffer containing different concentration levels of recombinant Toxin B was applied on the affinity MALDI plate and let incubate. The effectivity of the enzymatic reaction was monitored after matrix application by Autoflex speed MALDI-ToF mass spectrometer operated in linear positive mode. Data were searched for a mass shift of 162 Da corresponding to glucose modification of the RhoA. The intact RhoA was observed by a linear MALDI-ToF as a singly charged ion at m/z 25 000 and as a doubly charged ion at m/z 12 500. The glucosylated form of the RhoA was observed at m/z 25 162 and at m/z 12 581, resp. The glucose-modified RhoA was observed after in-situ enrichment using affinity protein chips at the lowest concentration of 2.4 ng/mL of toxin B. Changes in the workflow and several modifications of the reaction buffer allowed to keep RhoA protein intact and possible to detect by MALDI-ToF after transferring this method to a real-life complex human stool isolate sample.

ČSHS: Novel desorption-ionization ms affinity enzyme-linked assay for Clostridium bacteria family toxin activity detection (Josef Dvořák)

17:30 - 17:50 Investigation of human Hsc70 oligomerization
  • Aleksandr Melikov

Heat shock cognate protein 70 (Hsc70) is a 71 kDa chaperone protein belonging to the ubiquitous family of heat shock proteins 70 (Hsp70). The representatives of this protein family are considered as molecular machines with ATPase activity facilitating correct folding of spatial protein structure, both in normal and stressful conditions (hypoxia, heat shock, pH fluctuations etc.). In addition, Hsc70 was identified as an uncoating enzyme for triskelion meshwork on the surface of clathrin-coated vesicles. Among other roles, Hsc70 prevents protein aggregation and assists polypeptide maturation, it facilitates the protein transport into organelles, such as endoplasmic reticulum and mitochondria. It is involved in targeting proteins for lysosomal degradation and in many others important cellular processes related to protein homeostasis. Therefore, the regulation of Hsc70 and other Hsp70 proteins is believed to be highly important, especially in a context of cellular stress.

Based on the experimental observation, the mechanism of inactivation through oligomerization was hypothesized. The dimer and trimer species of Hsp70 proteins were identified both in case of prokaryotic and eukaryotic homologs. It was also speculated that Hsp40 cofactors promote oligomerization to even higher-order oligomers. This and other possible oligomerization models of wild type HSC70 and the subset of HSC70 mutants were investigated by cross-linking mass spectrometry. The distance constraints imposed by different cross-linker lengths allowed to build structural models of Hsc70 oligomeric species. To decipher between intra and intermolecular crosslinks, the studied protein and its mutants were produced in 15N-labeled form as well.

ČSHS: Investigation of human Hsc70 oligomerization (Aleksandr Melikov)

17:50 - 18:20 Krátká sdělení I

A Comprehensive Tool for State-of-the-Art Pre-processing Analysis in Metabolomics included in updated App: COVAIN v2.0.0
  • Jana Schwarzerova

In mass spectrometry (MS)-based metabolomics data, effective data preprocessing plays a crucial role in enabling advanced analysis and facilitating meaningful insights for comparative studies. With this objective in mind, our primary focus was on enhancing and implementing a cutting-edge preprocessing method in our newly developed application, COVAIN v2.0.0, for advanced metabolomics data processing.

Our goal was to provide users with a streamlined and user-friendly environment that allows for intuitive data processing, offering various options to generate comprehensive results for final assessment. The preprocessing section alone encompasses a wide range of functionalities, including different imputation methods for handling missing values such as zero, half minimum (HM), mean, median, random forest (RF), singular value decomposition (SVD), k-nearest neighbors (kNN), and quantile regression imputation of left-censored data (QRILC).

Furthermore, subsequent preprocessing steps include outlier adjustment, where default options are provided to reduce outliers based on the winsorization transformation technique. Lastly, the transformation step offers multiple normalization options, including log(), ln(), and Z-score transformations. Additionally, this section incorporates the calculation of the ANOVA test for group selection, along with the ability to adjust the p-value.

Overall, our application, COVAIN v2.0.0, strives to deliver an advanced and user-friendly solution for metabolomics data processing, ensuring robust preprocessing techniques and a comprehensive suite of analysis tools.

Insights into the transport dynamics of prokaryotic chloride/proton antiporter
  • Jasmína Portašiková

Chloride channel family includes transmembrane channels and transporters, which are present in both eukaryotic and prokaryotic organisms. They are involved in many cellular processes and their mutation can cause serious illnesses. To treat these diseases, description of their transport mechanism is needed. Bacterial chloride transporter CLC-ec1 from E. coli is often used as model protein to study protein family of chloride channels. It functions as an antiporter of a single proton for two chloride anions. During transport of ions, the protein undergoes conformational changes that convert inward and outward-facing conformation. Outward-facing state is induced by protons, when Glu residues which are involved in ion transport are protonated. This state can be mimicked by a QQQ mutant, in which three key Glu residues are mutated to Gln residues. So far, CLC-ec1 transport has been studied by X-ray crystallography that provided detailed but static images. Here we used to hydrogen/deuterium exchange mass spectrometry to extend the recent findings and provide more detailed insight into the transport dynamics of this protein. Full-length wild-type and QQQ CLC-ec1 were over-expressed in bacteria, isolated via detergent solubilization and purified by affinity chromatography and gel filtration. Next, HDX-MS experiment was conducted at different conditions. First, we compared WT and QQQ proteins to address the role of protonation. Both proteins were followed at four pH values spanning pH range 4.4-7.4. The deuteration rate of WT protein at pH 4.4 did not completely match the deuteration of the QQQ protein, we decided to run an experiment at even lower pH 3. These data are highlighting the stepwise protonation and the associated structural changes across the ion transport path.

ČSHS: Posterová sekce (Den 1)

Quantitative Cross-linking Mass Spectrometry Using Data-Independent Acquisition
  • Valérie Procházková

Chemical cross-linking in combination with mass spectrometry (CXMS) has been developed into a powerful tool for mapping interaction networks and three-dimensional structures of proteins and their complexes. However, proteins are intrinsically dynamic, and they can form different conformations. Adding quantitative information to CXMS offers a unique opportunity to study flexibility and structural rearrangement of proteins. In this study, we report the benefits of utilizing data-independent acquisition and novel urea-based isotopically labeled cross-linkers.

First, we used different mixtures (9:1, 1:1 and 1:9) of MS-cleavable DSPU and its isotopically labeled analogue to modify model proteins (BSA, BCA) testing quantitative potential of our strategy. Subsequently holo and apo forms of calmodulin and myoglobin were modified by isotopically labeled and non-labeled reagents to quantify the structural rearrangement upon calcium or heme binding, respectively. Both, the cross-link formation and quantification were performed in single dataindependent experiment where tryptic peptides were measured in the broad-band mode with mass accuracy below 1 ppm and subsequently fragmented without isolating precursor ions at fixed collision energy resulting in sub-ppm accuracy for fragment ions as well. The raw data were converted into hybrid mgf file which was interpreted by MEROX search engine.

Acquired data on model proteins clearly demonstrate the potential of our quantitative cross-linking strategy. The high mass accuracy in MS1 and MS2 modes enables unambiguous identification of crosslinked peptides. Moreover, the quantitative information is not derived from the MS1 experiment only. Due to the presence of isotopically labelled reporter ions in MS/MS spectra, it is possible to improve the qualitative and quantitative aspects of quantitative cross-linking experiments. Observed changes nicely overlap with high resolution structural models and previously published data. Our results lead to an assumption that presented data-independent acquisition method can be utilized for quantitative crosslinking experiments studying structure and dynamics of proteins and protein assemblies in solution.

Fast FluoroAlkylation of Proteins (FFAP): A Novel Cross-linking Strategy for Aromatic Residues
  • Michael Karpíšek

Chemical cross-linking in combination with mass spectrometry (CXMS) has been developed into a powerful method for mapping protein structures, dynamics and interaction networks including molecular interfaces in protein-protein and protein-nucleic acid complexes. Although many cross-linkers have been developed in last two decades, majority of CXMS analyses still utilizes lysinespecific cross-linking reagents based on N-hydroxysuccinimide esters. Other cross-linking reagents have only limited use for various reasons such as low reactivity, low occurrence of targeted residues, unwanted side products etc. In this study we show new generation of cross-linkers based on recently published Fast FluoroAlkylation of Proteins (FFAP) technology that enables targeting of aromatic amino acids residues.

Cross-linking reaction is based on two step FFAP mechanism which includes activation of hypervalent iodine using metal ions or lewis acid and subsequent attack of aromatic residues by the resulting radical. Since the induction of fluoroalkyl radicals is triggered by ascorbic acid and the labeling pulse is stopped by tryptophan, it enables to perform protein labeling experiments in quench flow system in short time range (3s). The studied proteins were analyzed by bottom up approach using high resolution mass spectrometry (solariX XR 15T, Bruker Daltonics) where samples were digested by trypsin protease, separated on reverse phase column online coupled to mass spectrometer.

The results on several protein models such as horse heart myoglobin complex clearly demonstrate the potential of our novel cross-linking strategy. The cross-links of aromatic residues generated by fluoroalkyl radicals nicely correspond with protein crystal structures of studied protein and provide information not attainable by lysine-specific cross-links. Our data lead to assumption the cross-linker based on fluoroalkyl radicals could be use as fast and powerful method to study protein structure and dynamics in solution.

18:20 - 22:00 Občerstvení a posterová sekce I

ČSHS: 11. Česká konference hmotnostní spektrometrie 2023 - Program

Czech Mass Spectrometry Conferences
 

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