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Cuantificación absoluta de péptidos mediante dilución isotópica

Resumen

Tipo:
Oferta Tecnológica
Referencia:
TOES20150904001
Publicado:
07/10/2015
Caducidad:
06/10/2016
Resumen:
Un organismo español de transferencia de tecnología ha desarrollado una metodología de cuantificación absoluta de péptidos mediante dilución isotópica para industrias que necesitan cuantificar péptidos en una muestra, como agricultura, farmacia, biomedicina y seguridad alimentaria. La metodología se basa en la combinación del uso de péptidos mínimamente marcados en 13C o 15N, la monitorización de reacciones múltiples (MRM) a baja resolución en el primer analizador de masas y la aplicación de regresión lineal múltiple. Se buscan empresas de química clínica, farmacia o biomedicina interesadas en establecer acuerdos de licencia.

Details

Tittle:
Absolute quantification of peptides by isotope dilution
Summary:
A Spanish technology transfer organization has developed a methodology of absolute quantification of peptides by isotope dilution which is useful in industries where the quantification of peptides in a sample is required such as agriculture, pharmacy, biomedicine and food safety. Clinical chemistry, pharmacy or biomedicine companies are sought for license agreement.
Description:
The methodology is based on the combination of using minimally labeled peptides 13C or 15N, the multiple reaction monitoring (MRM) at low resolution in the first mass analyzer and application of multiple linear regression.

The objective of the invention is the absolute, accurate, and precise quantification, directly traceable to the International System of units of peptides, defined as chains of amino acids with less than 50 units. The method can also be used for the determination of one or more proteins present in biological samples by quantifying their characteristic peptides after the hydrolysis of the protein.

This method allows directly obtaining the molar ratio between the peptide to be determined and the isotopically enriched peptide by calculating the isotopic composition of the mixture.

In one embodiment of the present invention, the liquid chromatography coupled to tandem mass spectrometry is used as in some of the methods described in the prior art but, unlike these methods, the isotopically labeled peptides employed are enriched in a small number of 13C or 15N atoms (between 1 and 4). This fact, in combination with multiple reaction monitoring (MRM) using FWHM (Full Width at Half Maximum) values between 2 and 20 in the first mass analyzer, enables the precise and accurate measurement of the isotopic abundances of the natural isotopic abundance peptide, the isotopically enriched peptide and in the isotopically diluted peptide.

Furthermore, the present invention may be applicable to the determination of proteins when the tryptic digestion is used to obtain the characteristic peptides of the protein of interest.

The use of isotopically enriched peptides with spectral overlap has two advantages:
1. Isotope effects between peptide natural isotopic abundance and isotopically enriched peptide are minimized.
2. The transfer of the entire cluster of precursor ions of the isotopically diluted peptide is favored through the first mass analyzer when the FWHM is increased to values between 2 and 20.

This concept is totally new and contrary to the methods described in the prior art where one needs to avoid at all costs the spectral overlap between the compound of natural isotope abundance peptide and the one isotopically enriched peptide. To do so, isotopically enriched peptides with a high number of enriched isotopes are employed (for example, six 13C atoms in the AQUA method) and this may lead to isotope effects during the sample preparation or the chromatographic separation. Therefore, the present invention involves a quantitative improvement over current methods.



Moreover, using multiple linear regression allows to directly obtain the molar fractions of the natural isotopic abundance peptide and of the isotopically enriched peptide from the product ion mass spectra. This provides two major advantages over the methods described in the prior art:
i) The molar ratio is obtained from the ratio of the molar fractions instead from the peak area ratio (AQUA method), providing improved and accurate determinations
ii) We avoid the need of constructing a methodological calibration to assess the dependence between intensities and molar ratios, which significantly reduces the analysis time.

It is especially relevant in clinical chemistry due to the number of proteins acting as biomarkers of diseases. This is growing exponentially in recent years.

The method can also be very useful for the validation of simple procedures. Even for application as routine method in clinical laboratories. It can as well be very useful in industries where the quantification of peptides in a sample is required: agriculture, pharmacy, biomedicine and food safety. It is ready for licensing and commercial exploitation. The partnership sought is a license agreement with clinical chemistry, pharmacy or biomedicine companies.

Advantages and Innovations:
- Allows obtaining directly the mole ratio between the peptide and its enriched analogue.

- Allows obtaining, precise and accurately, of isotopic abundance profiles in: the peptide of natural isotopic abundance, the isotopically enriched in peptide and in peptide isotopically diluted.

- Useful to the determination of proteins when used tryptic digestion.

- Using multiple linear regression allows to obtain directly the mole fractions::

- Molar ratio is obtained from the ratio of the molar fractions instead of peak area ratio.

- Performing a calibration methodology is avoided, which significantly reduces the analysis time.
Stage of Development:
Available for demonstration
IPs:
Patent(s) applied for but not yet granted
CommeR Statunts Regarding IPR Status:
PCT Applied

Partner sought

Type and Role of Partner Sought:
- Type of partner sought: Clinical chemistry, pharmacy or biomedicine companies with a solid and well-structured R-Y-D department.
- Specific area of activity of the partner: Diagnostic, therapeutic, other medical/health related
- Task to be performed: The main task is to obtain a kit to precise accurately, isotopic abundance of peptide profiles.

Client

Type and Size of Client:
Other
Already Engaged in Trans-National Cooperation:
Si
Languages Spoken:
English
Spanish

Keywords

Technology Keywords:
06002001 Bioquímica / biofísica
06003002 Expresión genética, investigación proteómica
05001001 Química analítica