Blanc SVSE 5 - Blanc - SVSE 5 - Physique, chimie du vivant et innovations biotechnologiques

photoSRM: optical fragmentation filtering for quantification by mass spectrometry of protein clinical biomarker in biological matrices – photo-SRM

Laser photo-dissociation for biomarker quantification by mass spectrometry

Mass spectrometry is a powerful alternative for protein biomarkers in blood and urine. Photo-SRM lies in the specific laser photo-dissociation of peptides tagged with a chromophore. The aim is the improvement of specificity and detection level.

Protein biomarker assay by coupling laser photodissociation to mass spectrometry

A technological jump is today essential to clear the gap between the up-stream proteomic discovery phases, which usually ends up on the identification of tens of putative biomarkers, and the large clinical validation step based on validated immuno-enzymatic tests. Absolute quantification of proteins by mass spectrometry has been recently identified as this possible core technology for intermediate clinical evaluation phases. However, substantial improvement of the detection level is required to discriminate between blood biomarkers in the low nanogram/mL range and the major plasmatic proteins. In order to significantly reduce the matrix effect caused by the bulk of highly concentrated peptides, photo-SRM technology will replace conventional collisional activation by photo-excitation. After tagging of cystein-containing peptides with a proper chromophore group, a limited population will hence be detetected. Commercial or home-synthesized chromophores will be evaluated regarding their photodissociation properties in the visible range. The final purpose is the development of validated methods for multiplexed quantification of proteins in blood and urine <br /> <br /> <br />

Photo-SRM proof of concept is carried out by monitoring human plasma peptides tagged with a commercial chromophore. Signal to noise ratio between conventional SRM and Photo-SRM are compared. A commercial mass spectrometer has been modified to introduce a visible laser beam.

A commercial mass spectrometer has been purchased and in house modified to allow for on axis laser irradiation of the ion cloud. This modification did not induced any decrease of the initial instrumental performances. Chemical synthesis of propriatary chromophores has been initiated while commercial chromophores were evaluated regarding their photo-dissociation behavior. After coupling to cystein-containing peptides, the DABCYL scaffold showed high dissociation yield in the visible range. Thus Photo-SRM monitoring derivatized cystein-containing peptides demonstrated similar or improved signal to noise ratio compared to conventional SRM method tracking the best responding peptides.
A partnership with AB Sciex (Toronto, Canada) is currently under discussion

It is anticipated that mass spectrometry might by a powerful tool for routine testing of biomarkers. In this context, the Photo-SRM is a complementary technique to conventional SRM by providing equivalent or better signal to noise ratio when tracking a sub population of peptides containing a cystein residue. The increased specificity of detection allows in addition to simplify the up-stream sample fractionation. Thus, Photo-SRM might easily applied to multiplexed clinical assay of already validated protein biomakers or to clinical evaluation of biomarker candidates

One article has been submitted in august 2012 to Analytical Chemistry
Title :IMPROVED DETECTION SPECIFICITY OF PLASMA PROTEINS BY TARGETING CYSTEINE-CONTAINING PEPTIDES WITH PHOTO-SRM

PROPOSAL ABSTRACT
A technological jump is today essential to clear the gap between the up-stream proteomic discovery phases, which usually ends up on the identification of tens of putative biomarkers, and the large clinical validation step based on validated immuno-enzymatic tests. Absolute quantification of proteins by mass spectrometry has been recently identified as this possible core technology for intermediate clinical evaluation phases. It lies in the assay of surrogate peptides that are issued from trypsin enzymatic hydrolysis. Quantification is obtained by ion chromatograms reconstructed on chosen fragmentation pathways called transitions (one molecular ion generate one fragment ion catalyzed by gas collision). A transition is then recorded in the so-called Selected Reaction Monitoring mode (SRM). However, substantial improvement of the detection level is required to discriminate between blood biomarkers in the low nanogram/mL range and the major plasmatic proteins. In order to significantly reduce the matrix effect caused by the bulk of highly concentrated peptides, photo-SRM technology will replace conventional collisional activation by photo-excitation. Only cysteine-containing peptide derivatized with a photo-cleavable probe will be excited and dissociated by 532nm UV-laser irradiation. The implementation of laser photo-dissociation in a commercial triple quadrupole mass spectrometer as well as the design and synthesis of the photo-cleavable are two of the three main tasks of photo-SRM proposal. The third task concerns the development and validation of a photo-SRM assay targeting Prostate Specific Antigen (PSA) biomarker across a clinical cohort (n=100).

Project coordinator

Monsieur Jérôme LEMOINE (UNIVERSITE CLAUDE BERNARD - LYON I) – jerome.lemoine@univ-lyon1.fr

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.

Partner

LSA UNIVERSITE CLAUDE BERNARD - LYON I
LASIM UNIVERSITE CLAUDE BERNARD - LYON I
ENS ECOLE NORMALE SUPERIEURE DE LYON

Help of the ANR 542,713 euros
Beginning and duration of the scientific project: - 36 Months

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