Trace polypeptide or protein-enriched and its direct analyzing method

A protein and enrichment technology, applied in the field of biochemical analysis, can solve the problems of affecting the sensitivity of mass spectrometry identification, cumbersome operation process, difficult to be eluted, etc., and achieve the effect of overcoming the sample adsorption discrimination effect, simple operation, and increasing the negative charge density

Inactive Publication Date: 2005-01-05
FUDAN UNIV
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Problems solved by technology

Solvent evaporation methods mainly include vacuum drying and freeze-drying. These methods are time-consuming and labor-intensive, and the adsorption on the surface of the container will cause a large amount of peptide loss during the drying process, which will affect the sensitivity of mass spectrometry identification.
The chromatographic concentration method is to use hydrophilic / hydrophobic interactions to adsorb peptides, and then use a small amount of organic solvent to elute. The operation process is cumbersome, and it is difficult to elute samples with strong hydrophobicity. Weak force and poor recovery

Method used

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  • Trace polypeptide or protein-enriched and its direct analyzing method
  • Trace polypeptide or protein-enriched and its direct analyzing method
  • Trace polypeptide or protein-enriched and its direct analyzing method

Examples

Experimental program
Comparison scheme
Effect test

example 1-5

[0029] Example 1-5 Effect of Zeolite Nanoparticles on Matrix-Assisted Laser Desorption Ionization-Time-of-Flight Mass Spectrometry (MALDI-TOF / MS) Determination of Standard Peptide Mixture

[0030] Take 0.25 μL of the 50pg / μL three kinds of standard peptide mixed solutions and spot them on the MALDI target plate respectively, and then add 0.25 μL H 2 Suspensions of O, LTL, β-30, β-60 and silicalite-1 (20 mg / mL), and finally covered with an equal volume of saturated α-CHCA solution (0.1% TFA, 50% acetonitrile aqueous solution), and dried for crystallization Afterwards, mass spectrometry was performed. The mass spectrometer used is MALDI-TOF / TOF (4700 Proteomics Analyzer, Applied Biosystems); the laser is Nd-YAG laser, the wavelength is 355nm, and the laser pulse frequency is 200Hz; the accelerating voltage is 20kV; positive ion mode, reflective TOF detection.

example 6-9

[0031] Enrichment and mass spectrometry of example 6-9 polypeptide samples

[0032] Take 1ml of polypeptide sample aqueous solution and add 5μL of 20mg / mL zeolite nanoparticle LTL, β-30, β-60 and silicalite-1 suspension respectively, shake at 37℃ for 90min; centrifuge at 13000g for 10min, discard the upper clear, add 5 μL HO to the zeolite precipitate 2 O, shake to make it suspend. Mix 0.25 μL of the suspension with an equal volume of a saturated solution of α-CHCA and place it on a MALDI target plate, and perform mass spectrometry according to the conditions of Example 1-5.

example 10-14

[0034] Adjust the addition amount of 20 mg / mL zeolite nanoparticles to 1, 2, 10, 20, 50 μL, and keep other conditions unchanged, repeat the above concentration and mass spectrometry experiments.

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Abstract

The invention is a method for carrying on enrichment-groundmass assistant laser analyzing ionized mass spectrum direct analysis to trace peptide and protein product using zeolite nanometer particles as nanometer micro absorbing agent. The zeolite nanometer particle has excellent absorbing effect to the protein and the polypeptide, and it has good compatibility to the groundmass assistant laser analyzing ionized spectrum, the sample absorbed by the zeolite nanometer particle needs no sample rinsing step to carry on groundmass assistant laser analyzing ionized spectrum analysis directly, the operation is simple, avoids the sample loss in rinsing process. The enrichment efficiency to polypeptide in the invention can reach to 1250 times, the protein enzyme sample with 50X10 to the power -12g / micro litre can be testified by the invention.

Description

technical field [0001] The invention belongs to the field of biochemical analysis, and relates to a method for enriching and identifying trace samples. Use a series of zeolite nanoparticles with different surface properties (different crystal structures and silicon-aluminum ratios) to enrich peptide and protein samples, and directly analyze and identify the enriched samples by matrix-assisted laser desorption ionization mass spectrometry . Background technique [0002] The analysis and identification of low-abundance proteins is one of the key and difficult aspects of proteomics research. Proteins that undertake important life activities in organisms are often low-abundance proteins. However, their extremely low content brings difficulties to subsequent analysis and detection, and limits people's research and understanding of them. Therefore, achieving low-abundance Effective concentration of protein is one of the first conditions for its accurate analysis and identificati...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/08
Inventor 王晓燕张亚红樊惠芝唐颐杨芃原
Owner FUDAN UNIV
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