Proteomics is the study of expressed proteins in a cell, a tissue, an organ or an organism at definite time and conditions. The proteome is the entire set of these proteins.

Proteomics allows identifying, characterizing and quantifying proteins. The proteomics analysis is a dynamic study: a same genome can produce several proteomes depending on studied cells or tissues, physiological or environmental conditions, or physiopathological status. Furthermore, regulation, transcription or translation events as well as post-translational modifications (like phosphorylation, deamination or glycosylation) lead to different functions.

Except their structural role, proteins have functions and are involved in many processes like metabolism, cell division, cell signalisation, cell exchanges.

We propose to study your samples proteomes applying our preparation techniques (extraction, concentration, protein digestion), our separation techniques (electrophoresis, chromatography) and analytical techniques (high resolution mass spectrometry, ELISA)

Interest of proteomic analysis

Proteomics by LC-MS / MS can describe proteomes, qualitatively (identification of proteins and their post-translational modifications) and quantitatively.

Genomic analysis makes it possible to identify the genes of an organism, but does not rule on their expression (transcription) in a given cell and under given conditions. Similarly, transcriptomic analysis (messenger RNA analysis) makes it possible to identify the genes that are expressed or transcribed in a given cell and under given conditions, but does not rule on the production of functional proteins and the content of these proteins.

Transcriptional or translational regulatory events, such as alternative splicing, as well as post-translational modifications, such as phosphorylation, deamination or glycosylation, lead to different functions for the same gene. This assertion is supported by the results of sequencing projects showing that genomes have fewer genes than described biological functions.

From a quantitative point of view, the quantification of messenger RNAs does not reflect the protein content. Indeed, the speed of production of proteins as well as their life span are variable. For example, a protein whose synthesis is slow but the long life can be present in higher content than a protein whose synthesis is fast but short life span.

Our analytical approach

Our approach is made of two parts :

  • « wet laboratory » part : It includes any experimental technique : extraction and protein separation, MS data acquisition.

Our lab has a know-how in protein extraction from complex environment in defining protocols taking into account extraction yields, purification levels and protein preservation.

Several chromatographic methods can be used for protein separation from your samples (micro and nano LC, different column types). Electrophoretic methods may also be used to increase the technique resolution.

We are equipped with an high resolution mass spectrometer. This equipment allows answering to any proteomics question: proteins identification, characterization and quantification.

  • « dry laboratory » part : It includes any bioinformatics methods useful at the beginning when defining experimental conditions and later when analysing the results.

Our lab has know-how in bioinformatics to analyse your results like, for instance, databases queries using different algorithms in order to identify your proteins, or the use of the new DIA method.

This approach can be applied to different samples like biological fluids, tissues or cell cultures from animal or vegetal origin, microorganisms or gel electrophoresis bands.

We are available to discuss our analytical procedures. Feel free to contact us for any information that may support you to know how mass spectrometry can be of use for your projects.

Services and technologies

Protein identification

« Shotgun proteomics : Mass fingerprinting by tandem mass spectrometry (MS/MS)

An LC-MS/MS instrument analyses continuously the peptide fractions obtained from HPLC after enzymatic digestion of a protein extract. The mass spectrometer does every second an MS spectrum followed by 30 MS/MS fractionation on the most intense components. This information generated on the different proteins segments is compared to mass maps and spectra available in databases and it allows the identification of proteins present in samples.

The “shotgun proteomics” method effectiveness and reliability is based on the large number of involved peptides and on the accuracy of each peptide mass measurement. The high resolution mass spectrometer meets these needs.

To increase the number of identified proteins, it is possible to realize two successive chromatographic separations (bidimensional chromatography) or to separate the proteins by electrophoresis and analyze each band by LC-MS/MS.

Protein characterization

Whole proteins mass characterization

This technique is particularly useful to control recombinant proteins. Culture conditions in a bioreactor may modify proteins post-translational modifications such as glycosylation, phosphorylation or cystein bonds and so slightly modify the mass of the protein. The great mass accuracy of high resolution mass spectrometer (1mDa) allows identifying these small variations.

This technique is applicable to purified whole proteins or in low complex mixes. The chromatographic separation is done on low hydrophobic columns. The protein mass is obtained from its MS spectra.

Post-translational modifications characterization

The post-translational modifications (PTM) are chemical modifications of proteins like phosphorylation, deamination or glycosylation which modify, activate or inhibit their biological functions. PTM are involved in several physiological phenomenons like signal cell transmission or oncogenesis.

An enrichment, by specific chromatographic methods applied during extraction step, may be necessary. The fragmentation spectra analysis allows identifying the protein modifications and/or the modification rate.

Proteins quantification

Relative quantification by TMT® labelling

This method is a relative quantification method which allows the protein concentration comparison between several samples. For instance, it may be used to compare a “treated patient” samples to “untreated patient” samples, or several culture conditions…

This method is based on peptides labelling by the TMT® reagent after sample trypsin digestion. During MS/MS fragmentation, the reagent generates a known mass fragment ion. Different isobaric (same mass) TMT® reagents are available that generate fragment ion of different masses. So they can be used to label peptides coming from different digestions that can be mixed in a 1:1 ratio and analysed in a same LC-MS/MS run. The comparison of ion fragment quantities produced by the different labelling allows the relative protein quantification.

Relative « label-free » quantification

This method permits the qualitative and quantitative comparison of different proteomes. Typically this technique is used to compare two conditions, for instance healthy vs unhealthy, and so to identify biomarkers which presence or quantity shows a physiological condition.

The samples LC-MS profiles are compared and the different peptides pikes areas are calculated. These areas are compared to those acquired for the same peptides in a reference sample and so a protein relative quantity is determined.

In the same test, it is possible to identify the proteins by comparison with databases.

DIA Quantification (Data Independent Analysis)

This method allows generating a large number of MS/MS spectra for a unique sample and within a very large mass range. A high resolution mass spectrometer with high speed data acquisition is necessary.

Due to the increase of the assay reproducibility and specificity, this technique, compared to classical “label-free” quantification, allows absolute protein quantification in a sample.

Absolute quantification by targeted method PRM (Parallel Reaction Monitoring)

The PRM type targeted methods are considered as the most selective and sensitive mass spectrometry methods since using the MRM scan mode, only peptides of interest are analyzed and thus improves the sensitivity and signal to noise ratio. The use of a high resolution instrument (Orbitrap) coupled a quadrupole performing accurate ionic filtration of parent ions allows obtaining screening and quantification methods for which the sensitivity/specificity compromise is better than on a triple quadrupole instrument in classical PRM mode. The advantage of this targeted method type is to be able to follow the proteins of interest in a robust and fast way. Furthermore, this method is easily transposable on triple quadrupole instrument type.

Custom Services

Our teams are fully available to define best strategies for your project and answer to your questions.