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Analytical methods

Published on 21 August 2020
Protein identification
Most current proteomics methods rely on mass spectrometry (MS) to measure the molecular weight of a polypeptide of interest. Mass spectrometry can also provide structural information by fragmenting the molecules analysed and determining the masses of their fragments. This technique is known as tandem mass spectrometry (or MS/MS). In general, an enzymatic digestion step breaking down the proteins of interest precedes MS analysis. In this step, a specific enzyme, such as trypsin, cuts proteins at pre-determined positions along their sequence to produce a complex mixture of peptides (or small fragments of proteins). The complexity of these samples is managed by separating their constituent peptides by liquid chromatography (LC) before injection into the source of the mass spectrometer. Each peptide is analysed by MS and MS/MS to determine its mass and those of its fragments. This information can be used to search databases of protein sequences, and thus identify the protein from which the peptide is derived.

Sample preparation and MS analyses are performed automatically thanks to the instrument platform.


Relative protein quantification
Relative protein quantification consists in measuring the relative differences in abundance for proteins between, most often, two analogous samples in differential conditions, e.g. "disease"/"healthy" (DECanBio, CT-Lymph projects), "wild-type"/"mutant" (Chloroplast project), "normal environment"/"stressed environment" (Plantox-Ura project). This can involve large sample series (over two hundred samples), for example to follow variations in protein abundance during a time-course.
Spectral count is a simple, semi-quantitative method. It consists in estimating a protein's relative abundance in a sample based on the number of MS/MS spectra associated with it (more abundant peptides will be fragmented more frequently by the mass spectrometer). This estimation, which is now automated in our process, is included in our identification reports. All other relative quantification methods use intensity measurements, the choice of method will depend on the biological project.

- Label-free methods: AMT (Accurate Mass Tag) is a label-free method identifying peptides based on their chromatographic retention time and high-precision mass, determined using a Fourier Transform-based mass spectrometer. Quantification does not require peptide fragmentation. This accelerates throughput, which is a definite advantage for projects with large numbers of samples (AT-Chloro, DECanBio projects).

- Labelled methods: these methods consist in using stable isotopes to mark the proteins - or peptides - in one of the samples to be compared (
SILAC, 15N) (Algomics project). Because of the label, samples can then be mixed (labelled with non-labelled) before mass spectrometry analysis. This reduces variability due to differences introduced during sample preparation, but it also increases the complexity of search algorithms, and ad hoc computing developments are necessary to treat the corresponding data.
 
Absolute protein quantification
Absolute protein quantification methods are used for previously identified proteins (biomarkers, toxins, hormones) when the precise amount contained in the sample is to be determined. These methods involve the addition of isotopically labelled standard peptides (AQUA) or proteins (PSAQ™). The PSAQ™ method (Protein Standard for Absolute Quantification) developed in the laboratory uses a whole recombinant protein, biochemically identical to the native protein. PSAQ™-based quantification is highly specific, sensitive and precise, and was found to be an excellent alternative to antibody-based assays. The method was patented and is now offered by the start-up Promise Advanced Proteomics.

These methods are used in projects assessing/quantifying biomarkers (CardioPSAQ™ project).


Targeted analyses
… of proteins.
SRM, or Selected Reaction Monitoring, analysis consists in detecting proteins of interest, even in a complex sample. SRM is a specific mass spectrometry mode, in which the machine is configured to detect only peptides (MS mode) and fragments (MS/MS mode) characteristic of a specific protein. Particular care is taken in choosing two to three representative (or "proteotypic") peptides for the protein, and two to three representative fragments for each of these peptides. SRM analyses are generally part of a quantification strategy.

… of modifications.
Post-translational modifications (PTMs) of proteins play a key role in all cellular processes. Identification of modified sites on proteins, and how these modifications change over time have become a major, essential part of proteomics. Thus, some of our projects centre on targeted analysis of specific PTMs, in particular: phosphorylations, acetylation (Epigam project), methylations (Epigam project), and glycosylations. PTMs can be studied in LC-MS/MS type acquisitions or in SRM mode.

… of peptides.
Identification of a protein's N-terminal peptide reveals whether maturation of the protein has taken place, such as excision of a signal pre-sequence during transfer of the protein to the mitochondria, the endoplasmic reticulum, or the chloroplast (in photosynthetic organisms). Identification of these peptides is not easy, requiring specific methods to enrich samples for these peptides and/or appropriate search and validation criteria during comparison of MS data with sequence databases. N-terminal peptide targeting was used to create a training set for PredAlgo, a tool predicting subcellular localisation in algae.