Remigiusz Serwa, PhD
Core Facility Leader
PROTEOMICS CORE FACILITY
The Proteomic Core Facility is a specialized laboratory dedicated to the analysis of complex mixtures of peptides and proteins.
Our laboratory provides the following services:
data analysis and mining.
Please visit our webpage for more details.
We conduct research in chemical biology. Our main interest lies in the development of new chemoproteomic tools and methods and their application for the interrogation of interactions between biomolecules (e.g. metabolites, co-factors, post-translational modifiers) or synthetic bioactive molecules (e.g. approved medicines, drug candidates) with proteins in living cells, whole organisms, or disease model systems. Currently our study focuses on polyamine-protein interactions. Polyamines are ubiquitous small molecules that are essential for many physiological processes, while dysregulation of their cellular metabolism has been implicated in human diseases. Our research aims to develop and apply a unified set of molecular probes suitable for system-wide exploration of the biological networks involved in polyamine-protein interactions by proteomics.
All the measurements performed at the PCF IMol PAS are made using a Dionex UltiMate 3000 nano-LC system (equipped with a fraction collector) coupled to a Q-Exactive HF-X via an EASY-Spray ion source (all Thermo Fisher Scientific).
Q-Exactive HF-X is a high-performance hybrid quadrupole-orbitrap instrument characterised by superb sensitivity and scan speed. Dionex UltiMate 3000 nano-LC system equipped with a fraction collector allows automated 2D separation of peptides prior to their measurement on the Q-Exactive HF-X to provide increased depth and reproducibility of results. EASY-Spray ion source uses integrated, temperature-controlled column-emitter units to maximize nano-LC performance by generating narrow peaks, maximum separation efficiency and outstanding MS sensitivity.
This analytical platform is ideal for shotgun proteomics. Shotgun proteomics utilizes digestion of proteins into peptides which are then sequenced in the mass spectrometer and act as surrogates for the presence of proteins from which they are derived. Besides insights into protein expression levels and the extent of posttranslational modifications, this mode of analysis can be applied to provide information about protein turnover, subcellular localization, as well as protein-protein and protein-small molecule interactions.
Data analysis (1h)
Biotinylated protein/protein enrichment
Phosphopeptide enrichment (incl. tryptic digest of up to 2 mg of protein)
TMT labelling (up to 18 samples per set)
Protein concentration determination (if not provided)
Protease surcharge (for digests with protease other than trypsin)
LC-MS/MS measurement - subsequent 0,5/1h
LC-MS/MS measurement - initial 1h (incl. tryptic digest)
RULES AND REGULATIONS
The lab rules and regulations provide a uniform set of rules in relation to the operations and services provided by the PCF IMol PAS to internal and external users.
The lab rules and regulations provide a guideline for soliciting services, ordering services, and paying for services provided by the PCF IMol PAS.
Direct use of the equipment allocated at the PCF IMol PAS is restricted to the PCF IMol PAS Staff.
The equipment allocated at the PCF IMol PAS may be used to provide services to internal and external users.
Proposals of scientific collaborations between external users and heads of research groups at IMol PAS, requiring the use of the resources allocated at the PCF IMol PAS, need the approval of the committee consisting of the Director of IMol PAS, his or her deputies, and the head of the PCF IMol PAS.
The list of prices for the services provided by the PCF IMol PAS is set by Director’s Order.
Users are obliged to familiarize themselves with rules 1-22 and confirm this fact by clicking the accept icon on the online interest submission form.
Provision of services
The services offered by the PCF IMol PAS will be adapted to the users’ needs. They scope of services includes
basic services (sample measurement, standard sample preparation: tryptic digest, peptide clean up),
additional preparative services (e.g. isobaric TMT labelling of peptides, enrichment of phosphorylated / biotinylated / ubiquitinated / etc. peptides/proteins, peptide fractionation),
consulting services (experimental design, customized sample preparation protocols, and post-experiment recommendations),
data analytical services (results are returned to users as MaxQuant output tables free of charge, any further data analysis/mining will be considered additional service.
Prices for the common services are listed here and users are free to inquire about the cost of less common services that have not been listed. The PCF IMol PAS Staff is responsible for scheduling the services. Generally, user requests are being processed on a first come, first served basis. In justified cases (e.g. optimization of instrument usage or to ensure the reproducibility of serial measurements) an exception can be made and a divergent scheduling order may be applied.
The head of the PCF IMol PAS reserves the right to reject requests for the provision of services in objectively justified cases. In the event of a refusal, a constructive feedback will be provided to the user.
Registration of intent to use services
All users (internal or external) intending to submit samples for the measurement at the PCF IMol PAS are first requested to submit a brief proteomic experiment description via an online submission form.
Proposals submitted via the online form will be evaluated and the information regarding the acceptance or rejection will be sent within 14 days by the PCF IMol PAS staff to the e-mail address provided in the form.
In case of acceptance, the user will be invited to a remote meeting. During this meeting the most optimal analytical strategy is agreed as well as the involvement of PCF IMol PAS Staff and the estimated cost of services, calculated based on the Pricelist. A twenty-minute consultation per dataset is included in the measurement price. This time is sufficient to discuss most experiments. Consultations for more complex experiments, which may need beyond 20 min, will be considered as an additional service, charged based on the current pricelist.
Before the samples are handed over, the user needs to agree to cover the cost of services by signing a purchase order form sent by the PCF IMol PAS Staff following the consultation. The form needs to be signed, scanned and emailed back to the address it was sent from.
Users are required to follow sample preparation protocol approved by the PCF IMol PAS head. Any modification (e.g. resulted from a human error) should be reported before the samples are handed over for the measurement. User who are discovered not to stick to this important rule, will be banned from submitting future samples.
Users can request bespoke sample preparation trainings. The cost is estimated based on specific needs of the user.
Results and Payment
Upon completion of services users will be provided with results (in the form agreed during the remote meeting, see rule 11). We aim to deliver the results within 21 days, counting from the day the samples are received by the PCF IMol PAS Staff.
Within two weeks of the completion of services, users will be emailed (to the address provided in the online interest submission form) invoices. Upon request, original invoice documents will be sent by mail (Poczta Polska).
Data storage & security
The results, MaxQuant output tables (and raw files from the measurements, if requested) are being made available to the users in adequate form (e-mail, web-based file transfer, direct deposition on external hard drives, etc.).
Raw data files from the measurements of external users’ samples will be stored by PCF IMol PAS for at least 3 months. If these raw data files are not requested by the user within this time, the PCF IMol PAS reserves the right to delete them.
Publication of data
Acknowledgment: Any service provided by the PCF IMol PAS must be clearly indicated in the acknowledgments section of resulting publications. This is true even if relatively basic service is provided (standard sample preparation and/or standard measurement and/or standard data processing to produce MaxQuant output tables). Upon request, users will be provided with a generic statement about the instrumentation, data acquisition, and search parameters will be provided by the PCF IMol PAS Staff.
Co-authorship: When a significant intellectual contribution from PCF Imol PAS Staff has been requested and delivered, their work must be acknowledged by naming them as co-authors of resulting publications. This is a default mode of agreement for experiments involving development or optimization of methods (in relation to sample preparation or data acquisition) or extensive data processing and/or mining. A selection of publication-ready figures and an input into writing of the relevant sections of the paper will be provided.