Conference CEEPC/IPM/CMSC 2022 (Day 1)
CSMS: Conference CEEPC/IPM/CMSC 2022 (Day 1)
Program CEEPC/IPM/CMSC 2022
CSMS: Conference CEEPC/IPM/CMSC 2022 (Suresh Jivan Gadher)
Thursday September 29, 2022
8:30 - 10:00 Registration
10:00 - 10:10 Opening of the 16th CEEPC, 8th IPM and 10th CMSC
10:10 - 11:00 Plenary lecture I: New insights through Single-Cell Proteomics (Chairperson: Petr Novák)
- Karl Mechtler
The analysis of single cell proteomes has recently become a viable complement to transcriptomics and genomics studies. Proteins are the main driver of cellular functionality and mRNA levels are often an unreliable proxy of such. Therefore, the global analysis of the proteome is essential to study cellular identities. Both multiplexed and label-free mass spectrometry-based approaches with single cell resolution have lately attributed surprising heterogeneity to believed homogenous cell populations. Even though specialized experimental designs and instrumentation have demonstrated remarkable advances, the efficient sample preparation of single cells still lacks behind.
Here, we introduce the proteoCHIP, optimized for multiplexed single cell proteomics sample preparation at surprising sensitivity and throughput. Sample processing using the cellenONE® robot, allows to reduce final sample volumes to low nanoliters submerged in a hexadecane layer simultaneously eliminating error prone manual sample handling and overcoming evaporation. This results in around 1,500 protein groups per analytical run at remarkable reporter ion signal to noise while reducing or eliminating the carrier proteome. We identified close to 2,600 proteins across 170 multiplexed single cells from two highly similar human cell types. This dedicated loss-less workflow allows to distinguish in vitro co-differentiated cell types of self-organizing cardiac organoids based on indicative markers across 150 single cells. In-depth characterization revealed enhanced cellular motility of endothelial cells and acute myocardium sarcomere organization in cardiomyocytes.
In addition, we evolved a robust and sensitive one-pot label free single cell workflow. By working in standard 384 well plates and compatibility with both, cell sorting in the cellenONE® or using alternatives like a FACS device the need for specialized equipment is obsolete making this workflow easy to use, cheap and accessible to a broader community. By keeping the sample in a hydrated state during proteolytic digestion and addition of DMSO for storage we improved recovery of hydrophobic peptides and boosted ID numbers to more than 1000 proteins from a single cell without match between runs. In conclusion, our versatile, and semi-automated sample preparation workflows have not only proven to be easily adoptable but are also sufficiently sensitive to drive biological applications of single cell proteomics.
11:00 - 11:20 Coffee break
Sponsors
- Bruker Daltonics
- Amedis
- Merck
- Beckman coulter
- Pragolab
- Waters Corporation
CMSC: Sponsors: CEEPC/IPM/CMSC 2022
11:20 - 13:00 Session I - Method Development
- Chairperson: Katarina Davalieva
11:20 - 11:40 Identification of potential cell-surface targets and druggable enzymes in human pheochromocytoma and paraganglioma using classical and membrane-targeting proteomic approaches
- Ondřej Vít
Integral membrane proteins (IMPs) represent optimal drug targets but are under-represented in standard proteomic analyses due to their amphipathy, lack of trypsin cleavage sites, and low expression levels. To identify tumor-upregualted IMPs and druggable enzymes in rare neuroendocrine tumors – pheochromocytoma and paraganglioma (PPGL), we combined three approaches: 1) hpTC method, where the identification of IMPs is based on their hydrophobic alpha-helices, isolated by proteolytic shaving and re-cleaved with CNBr; 2) two glycocapture methods - lectin entrapment on ultrafilters (N-glyco-FASP), and solid-phase enrichment with hydrazide chemistry (SPEG); and 3) the classical detergent-trypsin approach. We focused mainly on the high-risk tumors belonging to the so-called cluster 1, characterized by mutations in genes related to citric acid cycle and hypoxia, such as SDHB, VHL, and EPAS1). The classical trypsin-based proteomic approach pointed us toward upregulated soluble druggable enzymes (autotaxin, SHMT2, and Arginase 2) and upregulated cell surface IMPs (including CD146 and CD171). The glycopeptide enrichment approach provided additional potential cell surface targets (CD39), and the hpTC method provided additional IMP targets (e.g., anoctamin-1). The above-mentioned IMPs and soluble enzymes have all been previously shown to be upregulated in several human cancers and to affect tumor progression directly. Their marked upregulation was confirmed by specific antibodies. Together, this makes these molecules promising candidates for drug targets and/or proteins enabling sensitive tumor imaging.
11:40 - 12:00 Global SEC-PCP-SILAC mapping reveals protein complexes mediating NF-κB activation in breast cancer
- Petr Lapčík
NF-κB has essential role in immune response and is associated with lymph node metastasis of luminal A breast tumors (1). Analysis of protein interactome and its changes in response to NF-κB modulation could uncover pro-metastatic mechanisms related to NF-κB. We apply metabolic isotope labeling SILAC, size exclusion chromatography (SEC) and protein correlation profiling (PCP) (2) to construct a network of interactome rearrangement in response to NF-κB modulation in MCF-7 breast cancer cells.
We generated two co-fractionation datasets consisting of 80 fractions from SILAC-labeled and 80 fractions from label-free native MCF-7 lysates with inhibited or native NF-κB activity. LC-MS/MS analysis of SEC fractions using Orbitrap Lumos and Bruker Impact II mass spectrometers quantified 3308 and 5460 protein groups in SILAC and label-free datasets, respectively (FDR = 0.01). Interactome reconstruction using PrinCE (3) detected 7568 interactions among 1520 proteins. Co-elution of subunits of known complexes, such as ribosome, proteasome and MCM, was observed. Modulation of NF-κB was linked to interactome changes of proteins involved in immune response, cell cycle and DNA replication. NF-κB factor RELA interacted with proteins co-eluting with activators of NF-κB and these interactions were modulated by NF-κB inhibition.
Our interaction network represents a complex insight into dynamics of MCF-7 protein interactome associated with NF-κB pathway and could serve as a basis for future studies characterizing NF-κB in breast cancer.
12:00 - 12:20 Determination of changes in the milk proteome in production and storage of kefir using mass spectrometry-based omics approaches
- Haci Mehmet Kayili
Kefir is an important food source based in the Caucasus region. The beneficial effects of kefir on human health are indicated in the literature. The mechanisms underlying these critical properties of kefir have not yet been elucidated. This study aims to examine the changes in milk protein profiles during kefir production and storage by using mass spectrometry-based-omics approaches. In addition, the detection of kefir-based proteins that transfer to milk from kefir microflora was investigated within the scope of the study. First, Kefir production was carried out. During the production and storage of kefir, kefir samples were taken from the periods determined in the study, and milk proteins were extracted. Peptides were produced using Lys-C and trypsin enzymes by a classical proteomics approach. Afterward, the fractionation of the peptides was carried out. Peptide-containing samples were analyzed by nLC-QExactive-Plus mass spectrometry. The data obtained as a result of the analysis were processed with the Maxquant software, and statistical analyzes were performed. According to the results obtained, significant changes were found in the milk proteome in 3 proteins between 0-12 hours, 39 proteins between 0-24 hours, 41 proteins between 0-7 days, and finally, 42 proteins between 0-28 days. From the 24th hour, a significant change was observed in 23 proteins. In the analysis, it was determined in the study that 398 kefir-based proteins were released into the milk. It is anticipated that these results will provide valuable contributions to the literature in terms of showing the changes in the milk proteome in kefir production.
CSMS: Conference CEEPC/IPM/CMSC 2022: Determination of changes in the milk proteome in production and storage of kefir using mass spectrometry-based omics approaches (Haci Mehmet Kayili)
12:20 - 12:40 Non-Immunoaffinity Extraction of Intact Proteins from Biological Fluids and Their Analysis by Liquid Chromatography – Triple Quadrupole Mass Spectrometry
- Katarína Maráková
One of the crucial steps in quantitation of intact proteins from complex biological matrices is, except their reliable analysis, also sample preparation to achieve sufficient specificity and sensitivity. Commonly used immunoaffinity-based methods are characterized by their superior selectivity, although this can be a drawback if simultaneous analysis of multiple different proteins from a single sample is required. In our work, we developed non-immunoaffinity sample preparation based on a generally widely affordable microelution solid phase extraction for eleven model intact proteins (5.5 – 29 kDa) with various isoelectric points (4.5 - 11.3). Extracted intact proteins were analysed by reversed-phase liquid chromatography coupled with a triple quadrupole mass spectrometer operated in a multiple reaction monitoring (MRM) mode. Reversed-phase separations were performed on the Restek wide-pore Viva C4 column, as mobile phases served water and acetonitrile acidified by 0.1% difluoroacetic acid and 0.2% formic acid. The best recoveries for most of the selected proteins were obtained by using the HLB stationary phase. 1% trifluoroacetic acid and 0.2% Triton X-100 were used as efficient pretreatment reagents to release interactions between the proteins and biological matrix. Multiple sample loading was found out to be essential to obtain recoveries >65% in urine for all targeted proteins (up to 30kDa) and >50% in serum/plasma for most of the proteins. Limits of quantitation in biological matrices were in the range 2 - 1200 ng/mL, corresponding to 0.23 - 97.6 nM.
12:40 - 13:00 Advancing Cyclic Ion Mobility Mass Spectrometry Methods for Studying Biomolecules: Towards the Conformational Dynamics of Mega Dalton Protein Aggregates
- Adam Pruška
Native mass spectrometry is a powerful tool for the analysis of non-covalent complexes. When coupled with high-resolution ion mobility, this technique can be used to investigate the conformational changes induced in said complexes by different solution or gas-phase conditions. In this study, we describe how a new generation high-resolution ion mobility instrument equipped with a cyclic ion mobility cell can be utilized for the analysis of large biomolecular systems, including temperature-induced protein aggregates of masses greater than 1.5 MDa, as well as a 63 kDa oligonucleotide complex. The effects of and the interplay between the voltages applied to the different components of the cyclic ion mobility spectrometry system on ion transmission and arrival time distribution were demonstrated using biomolecules covering the m/z range 2 000 to 10 000. These data were used to establish a theoretical framework for achieving the best separation on the cyclic ion mobility system. Finally, the cyclic ion mobility mass spectrometer was coupled with a temperature-controlled electrospray ionization source to investigate high-mass protein aggregation. This analysis showed that it was possible to continuously monitor the change in abundance for several conformations of MDa aggregates with increasing temperature. This work significantly increases the range of biomolecules that can be analyzed by both cyclic ion mobility and temperature-controlled electrospray ionization mass spectrometry, providing new possibilities for high-resolution ion mobility analysis.
13:00 - 15:30 Lunch + Poster session I (Odd-numbered posters)
CSMS: Conference CEEPC/IPM/CMSC 2022 Poster session I (Odd-numbered posters)
15:30 - 16:30 Company Workshop – Bruker Daltonics
The latest proteomics development at Bruker
- Gary Kruppa
Clinical (phospho)proteomics with TIMS and PASEF
- Florian Meier
16:30 - 16:50 Coffee break
CSMS: Conference CEEPC/IPM/CMSC 2022: Petr Novák and Michael Volný
16:50 - 17:30 Short Talks
- Chairperson: Petr Man
Detection of C. difficile Toxin B by biomolecule-modified chips and mass spectrometry
- Josef Dvořák
Optimized HDX-MS workflow for antibody structure monitoring by HDX-MS
- Zuzana Kalaninová
RPLC-UV and HILIC-UV characterisation of on-site produced Ramucirumab-DTPA immunoconjugate
- Denis Naplekov
Collision energy setting in proteomics and glycoproteomics: From individual species to a practical perspective
- Agnes Revesz
Novel circulating biomarkers of biventricular heart failure
- Matěj Běhounek
Comprehensive metabolomic and lipidomic study of tauopathy and Alzheimer's disease patients
- Dana Dobešová
Overlooked oligomerization process in Azurin, a model metalloprotein
- Roman Tuzhilkin
CSMS: Conference CEEPC/IPM/CMSC 2022 Day 1
17:30 - 18:00 Josef Chmelik Award
18:00 - 19:30 Guided tour to the conference dinner
19:30 - 23:00 Conference dinner and sightseeing boat trip
CSMS: Conference CEEPC/IPM/CMSC 2022 Conference dinner and sightseeing boat trip
CSMS: Conference CEEPC/IPM/CMSC 2022 Conference dinner and sightseeing boat trip
CSMS: Conference CEEPC/IPM/CMSC 2022 Conference dinner and sightseeing boat trip
