Introducing BIOCEV OMICS Mass Spectrometry Core Facility
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About us
Our laboratory is equipped with high-end mass spectrometry instruments, which offers services in field of PROTEOMICS and METABOLOMICS. All our services are available for everybody from the academic and non-academic areas.
Our Services:
- Preparation of samples for analysis (cells, body fluids, tissues, plants etc.)
- Targeted or untargeted proteomics and metabolomics analysis
- Data analysis and basic statistical evaluation
- Consultation of experimental design and the results
Proteomics:
We are modern well equipped laboratory with the technical background able to perform most of the currently used untargeted and targeted proteomic measurements. We provide full proteomics service, including protein isolation from the supplied sample to evaluation and consultation of the measured data. Since 2015, we handle approximately 300 orders per year (roughly 3000 samples). Results based on measurements from our laboratory were used in dozens of publications. To consult your plan, please contact Karel Harant ([email protected]) or Pavla Talacko ([email protected]).
Metabolomics:
As a service, we provide targeted and untargeted analysis of small molecules. Thanks to the combination of equipment for LC-MS and GCxGC-MS, it is possible to measure a wide range of small molecules. We use several types of C18 and HILIC stationary phase columns on LC. For GC, we have various columns for different types of analytes. Individual measuremnts must always be consulted in advance, an individual approach and method must be chosen for each group of molecules. Please contact Olga Součková ([email protected]) for LC-MS or Petr Žáček ([email protected]) for GCxGC-MS.
All our services are available for everybody from the academic and non-academic areas.
Contact us before you start your first experiment: +420 325 873 925!
23.7.2024 Waiting time from “sample to result” is approximately 6-8 weeks.
BIOCEV: BIOCEV OMICS Mass Spectrometry Core Facility
Services
- Preparation of samples for analysis
- Untargeted and targeted proteomics
- Untargeted and targeted metabolomics
- Data analysis and basic statistical processing
- Consultation of experiment design and measured data
Untargeted proteomics
Up to 8000 identified proteins in one injection.
A basic type of proteomic experiment. Complete lysate is digested with trypsin. The resulting peptides are desalted and separated on a reverse phase column. Due to the low amount of proteins and the high complexity of the sample, liquid chromatography is currently most often used in nanoflow - hundreds of nanoliters per minute. Liquid chromatography is directly connected to the electrospray of the Orbitrap Fusion or Orbitrap Ascend mass spectrometer. The mass spectrometer measures alternately in two modes: MS¹, where peptide masses are measured in the Orbitrap with high resolution and accuracy, and MS², in which peptides selected from the previous MS¹ scan are fragmented. These are measured either again in the Orbitrap or in the ion trap. The ion trap provides lower accuracy and resolution than the Orbitrap, but is faster and more sensitive.
We offer untargeted proteomics measurement and analysis of post-translational modifications in different types of samples (cells, tissues, body fluids, plant samples, etc.).
Our newest service is "Thermal proteome profiling (TPP)" that allows you direct detection of drug-protein interactions in living cells.
Examples of analyses:
- LFQ (label free quantification)
- TMT (tandem mass tags)
- TPP (Thermal proteome profiling)
- phosphoproteome profiling
- pull-down experiments
Tips for sample handling:
- do not use trypsin for cell harvesting
- inform us about presence of detergent
- always keep in mind same protein loading for pull-down experiments (eg. Co-immunoprecipitation)
- do not use phosphatase inhibitors for phosphoproteomic experiments
Targeted proteomics
Accurate quantification of selected proteins.
Method aims to validate the results from an untargeted approach. It is performed on a triple quadrupole in SRM mode or using the MRM method on an Orbitrap. It is an an alternative approach to common techniques such as western blot. The advantage of this approach is the possibility to analyze several proteins (dozens) at once during one injection. This eliminates the problem of finding suitable antibodies.
The targeted proteomics service is always project specific and we offer it after prior meeting with the customer.
Parallel analysis of peptides labeled with heavy amino acids is essential. These serve as an internal standards.
The amount of proteins that can be targeted in one injection using a triple quadrupole, is strongly dependent on the concentration of the target proteins in the sample. The lower the expression of the target proteins, the less it is possible to analyze them in one injection due to the need for higher loading times. Information of fragmentation properties of targeted peptides is also important. This is usually obtained from an untargeted experiment, which should precede the targeted proteomic analysis. If the protein of interest was not detected during an untargeted experiment, then its targeted analysis is very difficult. However, in some cases fragmentation information can be obtained from publicly available databases.
Tips for sample handling:
- do not use trypsin for cell harvesting
- inform us about presence of detergent
- always keep in mind same protein loading for pull-down experiments (eg. Co-immunoprecipitation)
- do not use phosphatase inhibitors for phosphoproteomic experiments
BIOCEV: Introducing BIOCEV OMICS Mass Spectrometry Core Facility - Targeted proteomics.
BIOCEV: Introducing BIOCEV OMICS Mass Spectrometry Core Facility - Targeted proteomics.
Untargeted metabolomics
The goal of untargeted metabolomics is to cover the widest possible spectrum of metabolites present in the sample, their identification, and quantification. The development of this approach was made possible by the recent advances in instrumentation and computing. Our laboratory equipment enables non-targeted analyses by liquid and gas chromatography.
We offer untargeted analyses of metabolites in various biological matrices and basic statistical evaluation of the data (Figure 2, 3, 4).
Our newest service is stable isotope tracing (Figure 1).
HPLC-MS/MS (Figure 2): Analyzes are performed on Thermo Scientific equipment on an Ultimate 3000 liquid chromatograph coupled to an Orbitrap Fusion mass detector. The measured data are evaluated by the Compound Discoverer software.
GCxGC-MS (Figure 3): Analyzes are performed on a two-dimensional comprehensive chromatograph connected to a Pegasus 4D mass detector. We evaluate the data in ChromaTOF v4.5 software.
The liquid chromatography method covers non-volatile substances, generally polar, soluble in water, and polar organic solvents (methanol, acetonitrile...). The gas chromatography method is more suitable for analyzing stable volatile non-polar substances up to a weight of approx. 1000 Da. The range of measured substances can be increased for both methods eg., by chemical modifications (derivatization) of the analyzed molecules, which adjust their polarity and volatility (for GC) and/or stability (for LC). We choose a suitable technique or a combination of several techniques based on prior consultation with the customer.
The untargeted analysis results in a list of substances found in the samples, a proposal of probable structures, and an indication of the quantity of individual compounds. We also provide a basic statistical evaluation focusing on looking for differences between the tested sample groups (Figure 4).
Tips for sample handling:
- do not use phosphate buffers in the sample
- inform us about sample type, growth medium, chemicals used, etc.
BIOCEV: Introducing BIOCEV OMICS Mass Spectrometry Core Facility - Untargeted metabolomics Fig. 1.
BIOCEV: Introducing BIOCEV OMICS Mass Spectrometry Core Facility - Untargeted metabolomics Fig. 2.
BIOCEV: Introducing BIOCEV OMICS Mass Spectrometry Core Facility - Untargeted metabolomics Fig. 3.
BIOCEV: Introducing BIOCEV OMICS Mass Spectrometry Core Facility - Untargeted metabolomics Fig. 4.
Targeted metabolomics
Targeted metabolomics includes analyses of selected metabolites, which are then detected selectively and with greater sensitivity than applying an untargeted approach. It is also possible to quantify substances both relatively and absolutely. In the case of absolute quantification, it is necessary to supply a suitable labeled analog of the determined substance.
We offer analysis of primary cellular metabolites - see List of substances.
We analyze metabolites in tissues, cell pellets, incubation media, and body fluids (plasma, urine...).
We also offer the possibility of developing a method for the analysis of substances that are not in the attached list or for other matrices.
The attached list contains substances with the analysis of which we have experience. We adapt each method to the type of experiment and matrix. We can develop a method for any substance or group of substances capable of being detected by liquid and gas chromatography techniques (both connected to a mass detector or a UV detector in liquid chromatography). In the case of the development of a new method, it is necessary to supply the standards of these substances.
Tips for sample handling:
- do not use phosphate buffers in the sample
- inform us about sample type, growth medium, chemicals used, etc.
BIOCEV: Introducing BIOCEV OMICS Mass Spectrometry Core Facility - Targeted metabolomics: List of substances.
Equipment
Mass spectrometers
Orbitrap Ascend
Thermo Scientific Orbitrap Ascend Tribrid mass spectrometer, offering new capabilities for multiplexed proteomics and native protein characterization. The mass spectrometer enables improved sample throughput, versatility and ease-of-use — producing high quality data with simplicity. More samples can be analyzed at lower concentrations, enabling faster, more sensitive analysis, and macromolecule experiments, that were not possible before, can now be achieved. The new Auto-Ready ion source for calibration maximizes convenience, allowing the user to spend more time on the results, and less time on instrument set-up.
Orbitrap Fusion
Mass spectrometer combining Quadrupole, Ion trap and Orbitrap. The quadrupole is used as a mass filter for the selection of precursors, after which the instrument branches and the ions can travel either to the Orbitrap - a very accurate and high-resolution mass analyzer that uses the Fourier transform to determine their mass, or to the collision cell, where they can be fragmented. From there, the path leads either back to the Orbitrap or further to the linear ion trap. This serves as the second mass analyzer in the device. It is less accurate and low-resolution, but provides greater sensitivity and the possibility of ion fragmentation. Fragmentation products can be isolated in the trap and fragmented again. This enables analysis in MS³ and even higher levels of fragmentation.
TSQ Quantiva Triple Quadrupole Mass Spectrometer
Leco - Pegasus® 4D GCxGC-TOFMS
Liquid chromatography
- nano LC Thermo Dionex Ultimate 3000
- LC Thermo Dionex Ultimate 3000
Sample preparation
- KingFisher Flex
- vacuum concentrator
- Low flow fraction collector - Spider
- Bioruptor pico - sonication bath
Software
- Spectronaut 19
- FreeStyle
- ChromaTOF 4.5
- Proteome Discoverer 2.2
- Scaffold Q+S 4.6.1
- Compound Discoverer 3.3
- TraceFinder
Software - Freeware
- LipidHunter
- MaxQuant
- Perseus
- Skyline
Team
Mgr. Karel Harant
- Head of Proteomic Core Facilty
Mgr. Pavel Talacko
- Non-targeted proteomics, targeted proteomics
RNDr. Petr Žáček, Ph.D.
- GC / MS specialist, small molecule analysis
Mgr. Veronika Ševců
- researcher, preparation of proteomic samples
RNDr. Petr Žáček, Ph.D.
Mgr. Veronika Ševců
- researcher, preparation of proteomic samples
Ing. Olga Součková, Ph.D.
- LC/MS specialist, small molecule analysis
