Method for concentration of low-molecular-weight proteins and peptides in body fluid sample

Abstract

(Problem to be Solved) There is to be provided a method for extracting low-molecular-weight proteins/peptides contained in a body fluid sample, particularly, in serum or plasma. (Means for Solution) The method according to the present invention comprises the steps of (a) to (e): (a) adding reagent 1 containing urea and thiourea and reagent 2 containing a reducing agent to the body fluid sample, mixing them, subsequently dropping the mixture into reagent 3 containing 90% or more of an organic solvent, and mixing them; (b) stirring at a low temperature the mixed solution obtained in step (a); (c) centrifuging at a low temperature the stirred solution obtained in step (b) and removing the supernatant; (d) adding reagent 4 containing an organic solvent and an acid to the precipitate obtained in step (c) and mixing them; (e) stirring at a low temperature the mixed solution obtained in step (d); and (f) centrifuging at a low temperature the stirred solution obtained in step (e) and recovering the supernatant.

1 .- 23 . (canceled) 
     
     
         24 . A kit for extracting low-molecular-weight proteins/peptides contained in a body fluid sample, wherein the kit comprises reagent 1 containing urea and thiourea, reagent 2 containing a reducing agent, reagent 3 containing 90% or more of an organic solvent, and reagent 4 containing an organic solvent and an acid. 
     
     
         25 . The kit according to  claim 24 , wherein reagent 1 has a urea concentration of 1 to 8 M and a thiourea concentration of 0.5 to 3 M; reagent 2 contains the reducing agent in such an amount that the concentration of reducing agent in the mixture of the body fluid sample, reagent 1, and reagent 2 is 1 mM to 20 mM; the organic solvent in reagent 3 is selected from the group consisting of acetone, ethanol, methanol, 2-propanol, acetonitrile, and mixtures thereof; the organic solvent in reagent 4 is selected from the group consisting of acetonitrile, methanol, ethanol, isopropanol, and mixtures thereof and has a concentration of 50 to 99%; and the acid is selected from the group consisting of hydrochloric acid, TFA, formic acid, acetic acid, and TCA. 
     
     
         26 . The kit according to  claim 24 , wherein reagent 1 has a urea concentration of 3 to 8 M and a thiourea concentration of 1 to 3 M; when the reducing agent in reagent 2 is dithiothreitol (DTT), dithioerythritol (DTE), 2-mercaptoethanol (2-ME), or 2-mercaptoethanolamine (2-MEA), reagent 2 contains the reducing agent in a concentration of 5 mM to 20 mM in the mixture of the body fluid sample, reagent 1, and reagent 2 or when the reducing agent is Tris(2-carboxyethyl)phosphine HCl (TCEP HCl) or tri-n-butylphosphine (TBP), reagent 2 contains the reducing agent in a concentration of 1 mM to 10 mM in the mixture of the body fluid sample, reagent 1, and reagent 2; the organic solvent in reagent 3 is acetone and has a concentration of 98% or more; and the organic solvent in reagent 4 is acetonitrile and has a concentration of 60 to 80%. 
     
     
         27 . A kit for preparing an analysis sample of low-molecular-weight proteins/peptides contained in a body fluid sample, wherein the kit comprises reagent 1 containing urea and thiourea, reagent 2 containing a reducing agent, reagent 3 containing 90% or more of an organic solvent, and reagent 4 containing an organic solvent and an acid and further comprises reagent 5 containing a component, reagent 5 being added to a lyophilized product obtained by the method according to claim  2 , and the component being selected from the group consisting of TPA, hydrochloric acid, formic, acid, acetic acid, and TCA.