JPWO2021228969A5

JPWO2021228969A5 -

Info

Publication number: JPWO2021228969A5
Application number: JP2022568963A
Authority: JP
Publication date: 2024-05-22

Claims

1. A method of preparing a sample for an analytical procedure, the sample comprising at least one protein, polypeptide or peptide molecule, the method comprising: (a) fragmenting the molecule using at least one moving magnetic body, the at least one moving magnetic body performing an alternating or oscillating motion, the motion being induced by a changing or oscillating magnetic field.

The method of claim 1, wherein the magnetic field is generated by an electric current and/or an electromagnet.

3. The method of claim 1 or 2, wherein (i) the fragmenting is a non-enzymatic and non-chemical process, or (ii) a chemical selected from CNBr, formic acid, hydroxylamine, and 2-nitro-5-thiocyanobenzoic acid and/or a protease is added.

4. The method of claim 1, wherein at least one non-magnetic particle is present and the movement of the magnetic body induces collisions between the at least one non-magnetic particle and the molecule.

The sample is of biological origin, preferably
(i) the sample is or comprises a solution or suspension of the molecule, e.g. the sample comprises a mixture of the molecule, protein, polypeptide and/or peptide in purified form or is or comprises a body fluid such as blood, serum, plasma, cerebrospinal fluid, sputum or urine,
(ii) the sample is or comprises cells, such as prokaryotic or eukaryotic cells, e.g., the sample is or comprises a suspension of cells;
(iii) the sample is or comprises a virus, e.g. the sample is or comprises a suspension of a virus, and/or (iv) the sample is or comprises tissue, e.g. muscle tissue or brain tissue,
5. The method according to any one of claims 1 to 4 .

6. The method of any one of claims 1 to 5, wherein the method further comprises the steps of (b) exposing the sample to heat, such as boiling, denaturing the sample, adding a detergent to the sample, and/or adding a chaotropic agent to the sample, preferably wherein step (b) is carried out prior to or simultaneously with step (a).

7. The method of claim 1 , further comprising the step of: (c) chemically modifying said molecule and/or fragments obtained from said molecule.

8. The method of claim 1, wherein the analytical procedure is mass spectrometry (MS).

The chemically modifying step comprises:
(ca) reducing disulfides;
(cb) alkylating thiol groups such as cysteine residues;
(cc) a crosslinking step, and/or (cd) any combination of (ca), (cb) and (cc), with (ca) and (cb) being preferred.
The method according to claim 7, wherein steps (a), (ca) and (cb) are carried out simultaneously.

10. The method according to any one of claims 1 to 9 , wherein an inert viscous liquid; a gel, such as a polyacrylamide gel or an agarose gel; an aerogel and/or a zeolites are added to the sample.

11. The method according to any one of claims 1 to 10, wherein the method further comprises the step (d) of purifying and/or concentrating the obtained fragments, preferably by filtration, non-covalent and/or covalent binding, wherein non-covalent binding is preferably performed on a reversed phase material, a normal phase material, an ion exchange material, an affinity binding material, a material with chelating properties or a paramagnetic particle, and wherein covalent binding is preferably performed with a reagent capable of forming a conjugate with any amine group of the fragments.

the method further comprises the step of (e) labelling said molecule and/or fragments obtained from said molecule, preferably wherein labelling of said fragments is accomplished after said purification and/or enrichment of said fragments ;
12. The method according to any one of claims 1 to 11, wherein the labeling step is carried out by reacting a functional group of the molecule with a reagent capable of forming a conjugate with said functional group, said reagent capable of forming a conjugate with said functional group being a tag preferably detectable by mass spectrometry.

13. The method of claim 1, wherein the magnetic body collides with the molecule and energy is transferred to the molecule, inducing or contributing to the fragmentation of the molecule.

The method according to any one of claims 1 to 13, wherein the size of the magnetic body is between 0.1 mm and 10 cm.

The current is set to 0.02 to 10 ^９9 The method of claim 2, wherein the magnetic field strength is 0.01 to 0.1 A/m.

16. An analytical method comprising the method according to any one of claims 1 to 15 and carrying out mass spectrometry of the obtained fragments.

1. A method for identifying a site on a first protein which is or may be capable of binding to a second protein or binding partner, said method comprising fragmenting said first protein using at least one moving magnetic body, adding said second protein or said binding partner, separating fragments which bind to said second protein or said binding partner from non-binding fragments, and identifying said fragments which bind said second protein or binding partner, thereby identifying said site, wherein said at least one moving magnetic body performs an alternating or oscillating motion , said motion being induced by a changing or oscillating magnetic field, preferably wherein said first protein is an antigen and said second protein is an antibody.

1. Use of a magnetic body and a magnetic means for generating a fluctuating or oscillating magnetic field for fragmenting a protein, polypeptide or peptide molecule , wherein the fluctuating or oscillating motion of said magnetic body is generated by said magnetic field .