Large scale protein identification and quantification
Current proteomics methods and instrumentation represent today a powerful tool to get new insights into complex biological systems, in particular for:
- Characterization of complex samples (proteomes, sub-proteomes, biological fluids, protein complexes…) with maximal analytical depth (number of proteins identified)
- Precise relative quantification of each protein in different samples using repeatable and reproducible analytical methods
- Semi-absolute quantification of different proteins in a complex mixture
The ProFI teams have expertise in discovery proteomics, e.g. all methods dedicated to the systematic, non-hypothesis-driven, and large-scale characterization of complex mixtures of proteins, in a qualitative and quantitative manner. Thus, we apply bottom-up strategies based on the enzymatic digestion of proteins with a specific protease (e.g. trypsin) and high-throughput analysis of resulting peptides by liquid nanochromatography coupled to mass spectrometry (nanoLC-MS), using fast-sequencing instruments. According to the project, we design optimized analytical workflows including the following steps:
- Sample preparation (total cellular lysates, sub-cellular fractionation, enrichment techniques, handling of biological fluids etc.)
- Analytical pre-fractionation. In order to increase the analytical depth for highly complex proteomes, samples can be fractionated at protein or peptide level, prior to nanoLC-MS analysis (gel electrophoresis, IEF, SCX, HPLC)
- Quantitative methods using label-free or isotopic labelling methods (SILAC, iTRAQ, TMT)
- NanoLC-MS analysis with benchmarked and quality controlled systems to ensure a sustained level of performance and with optimum chromatographic gradients and MS modes (DDA, DIA).
- Bioinformatic processing. We use several software tools installed on clusters and/or multicore servers to process large scale data. Our in-house developed tools (Proline, MS-Angel, mz-Scope) offer an optimized pipeline solution for data handling, processing and inspection.
- Statistical analysis. We offer support to interpret large-scale proteomic data using state-of-the-art statistical methods and tools, in order to identify differentially abundant proteins and control false-discovery rate.
Targeted strategies have emerged ten years ago and are best suited when precise, ideally absolute, quantification of finite sets of proteins are required with highest sensitivity. Targeted strategies are widely applied in the biomarker field as potential biomarker candidates identified during discovery experiments need to be verified and validated on large sample cohorts, sometimes even in a clinical environment.
The ProFI teams have expertise in targeted proteomics for the development of robust targeted assays using Selected/Multiple Reaction Monitoring (SRM/MRM) on triple-quadrupole-type instruments or high resolution accurate mass targeted monitoring modes such as Parallel Reaction Monitoring (PRM) or DIA (Data Independent Acquisition) on high resolution instruments. In that context our objectives are to provide precise and absolute quantification of finite sets of proteins of interest, to develop a robust targeted quantification assay and to transfer quantification assays to the clinics for biomarker monitoring. We develop targeted assays on complex protein mixtures (whole cell lysates, blood, plasma, urine, CSF etc.), enzymatically digested (e.g. trypsin) and eventually fractionated at protein and/or peptide levels. The resulting peptide mixtures are analyzed on liquid chromatography systems (nano- or micro-scale) coupled to dedicated mass spectrometers run in Selected/Multiple Reaction Monitoring (SRM/MRM) or high resolution full scan MS/MS (HR-MRM, HD-MRM, PRM or DIA) modes. According to the project, we design optimized analytical workflows including the following steps:
- Choice of proteins and peptides to be targeted. We use all existing resources to refine the choice of tracer peptides, including prior shotgun data screening (in-house acquired or available on ProteomeExchange), public and dedicated repositories (such as SRMAtlas, PASSEL), software for prediction
- Sample preparation. We can implement various protein extraction methods, as for discovery experiments. Of note is that for a robust quantification, sample preparation should be minimal as each step introduces additional variability.
- Design and synthesis of heavy labelled standards (e.g. AQUA peptides, PSAQ standards).
- LC-MS-MS analysis. We use state-of-the art LC-MS/MS couplings, based on powerful chromatographic systems and last generation mass spectrometers that are benchmarked and quality controlled to ensure a sustained level of performance and with optimum chromatographic gradients, chromatographic scales (nano or micro) and MS modes.
Post-translational modifications: detection and quantification
Proteins are very often the subject of various post-translational modifications (PTMs) that can impact their association with other proteins and globally other biomolecules, their subcellular localization and their stability. Both the protein abundance and the levels of PTMs can vary between cellular states, such as cells submitted to an environmental stimulus or left otherwise, between differentiation states or between normal and diseased tissues.
The ProFI teams have developed expertise in the analysis of several types of PTMs, in particular phosphorylations, methylations, acetylations, O-glycosylations, and also in assessing the possible cleavage of protein N-termini to yield the mature sequences. The large-scale LC-MS/MS analysis of one type of PTM usually requires the enrichment of the proteolytic peptides bearing the PTM of interest, by implementing a step of affinity purification. Besides, some PTMs produce specific fragments that can be tracked in MS/MS to pinpoint peptide sequences containing the PTM of interest. The ProFI teams have been developing methods at the two levels to increase the specificity, sensitivity and thus coverage of the proteome bearing the above PTMs. PTMs investigations are also based on our strategies and instrumentation for large-scale and targeted quantification.
Interactomics and protein complexes
Proteins mainly act in association with other proteins in a dynamic way to reach a tight control of cellular processes. To address these issues modern mass spectrometry-based proteomics strategies are combined with efficient biochemical approaches, like affinity-purification methods.
We have expertise in interactomics, ie the analysis of protein complexes and protein networks, in a qualitative and quantitative manner.
- Identify protein partners using one protein of interest as a bait
- Determine the dynamics of protein complexes (variations in protein partners abundances in different conditions or in time)
- Determine the stoichiometry of associated proteins using quantitative proteomics
We apply affinity-purification approaches combined with quantitative mass spectrometry analysis (AP-MS) to identify proteins associated to a bait protein of interest. Depending on the sample and the bait protein several purification methods can be applied (immunopurification of endogenous proteins, affinity-purification of tagged proteins using various tags). In any case, a relevant control experiment is conducted to distinguish contaminant protein associations from true interactors. Differential quantitative proteomics analysis between the bait and the control assays then allows the identification of specific protein partners of the bait protein.
The ProFI teams have been developing strategies to maintain labile interactions in vivo using crosslinking agents, to analyze specific interactions across various samples using abundance correlation profiling, to cluster proteins with similar dynamics.
Protein complexes and interactomics investigations rely on our quantitative proteomics strategies and instrumentation used for large-scale and targeted quantification.
Characterization of intact proteins and complexes
The next frontiers in proteome research are the study of protein-ligand interactions, protein-peptide and protein-protein complexes, together with entire protein interaction networks by high-throughput methods. Structural MS is a quite recent research area that consists in deriving structural information from MS based data and usually focuses on highly purified proteins or protein complexes, most of the time from recombinant origin.
- Protein characterization (intact mass analysis)
- Multiprotein complex analysis (stoichiometry, conformation, interacting regions, dynamics)
Mass spectrometry plays a pivotal role in purified protein or protein complexes analysis. Even though MS-based approaches are rather low-resolution techniques, they represent a potential alternative to get structural information that is complementary to classical biophysical methods. Different MS-based approaches for their structural characterization are available within the ProFI consortium:
- Native MS and ion mobility
- Hydrogen Deuterium eXchange-MS
- Covalent chemical labeling (crosslinking)
- Top/middle-down MS (proteoform characterization)
How to submit a request
ProFI is a national infrastructure developing methodological innovations in the field of proteomic analysis, particularly in data processing (Proline link). ProFI offers a service to the scientific community, from academic or private sectors. ProFI provides its expertise (trained staff and advanced instrumentation) for the realization of projects requiring proteomic analyzes.
The ProFI infrastructure proposes its services in collaboration or as fee-for-service.
- Collaboration: concerns analyses that will be carried out in a context where intellectual property is shared.
- Service: concerns analyzes that will be carried out in a context where there is no sharing of intellectual property
For these two service offers, depending on the scope of the request, either a contract (e.g. ANR projects, INCa etc.) or a quote with an invoice will be established. For any request with a quote, this one will be issued by one of the ProFI sites, signed by the customer and accompanied by a purchase order before any realization. The rates applied are specific to each type of offer and will be the subject of an estimate.
ProFI offers the following services:
- Protein identification
- Large-scale relative quantification
- Targeted quantification
- Detection and quantification of post-translational modifications interactomics
- Characterization of intact proteins and complexes
- Other types of analysis (to be specified)
If a given ProFI platform is chosen, the request will be taken into account directly by the manager of the platform concerned. Otherwise, ProFI platform managers will come back to you to offer you a ProFI platform, according to the expertise and schedule required.
The request is validated, or invalidated, by the platform managers and / or the ProFI steering committee.
When a request is validated, a project manager is appointed and takes charge of carrying out the analysis, up to the results report, which can be made available on the ProFI customer area (secure access).
A discussion with a platform manager in order to assess the feasibility, to plan the optimal experimental plan is recommended before any project acceptance.