Abstract: A method is provided for more easily detecting and quantifying a protein by regioselectively digesting a variable region of an Fab domain of a monoclonal antibody while suppressing proteolysis of an Fc domain. In the method, a porous body in which a monoclonal antibody is immobilized in pores and nanoparticles on which a protease is immobilized are brought into contact with each other in a liquid to perform selective proteolysis of the monoclonal antibody, and a resulting peptide fragment is detected using liquid chromatography-mass spectrometry (LC-MS), and a peptide having an amino acid sequence that includes an amino acid derived from a CDR2 region of a heavy chain or a light chain of the monoclonal antibody is detected.

Abstract: There are numerous proteins having different in vivo effects depending on variants. In order to gain a more accurate understanding of an in vivo effect of a protein, when the protein in a sample is quantified, it is necessary to measure an amount of each variant that can be present. Provided is a method for performing parallel quantification of variants of a protein having 2 or more variants using mass spectrometry. The method includes: protease-digesting a protein in a sample; detecting, using mass spectrometry, among peptides obtained by the protease digestion, peptides having amino acid sequences specific to the variants without separating the peptides; and determining amounts of the variants in the sample based on results of the detection.

Abstract: The present invention provides a method for quantifying a monoclonal antibody, the method including: a step of performing selective protease digestion of a monoclonal antibody as a measurement target by bringing a porous body in which the monoclonal antibody is immobilized in pores into contact with fine particles onto which a specific protease is immobilized; and a step of detecting a peptide fragment that is obtained from the digestion and contains an amino acid derived from a CDR2 region of a heavy chain or a light chain of the monoclonal antibody.

Abstract: A method is provided for more easily detecting and quantifying a protein by regioselectively digesting a variable region of an Fab domain of a monoclonal antibody while suppressing proteolysis of an Fc domain. In the method, a porous body in which a monoclonal antibody is immobilized in pores and nanoparticles on which a protease is immobilized are brought into contact with each other in a liquid to perform selective proteolysis of the monoclonal antibody, and a resulting peptide fragment is detected using liquid chromatography-mass spectrometry (LC-MS), and a peptide having an amino acid sequence that includes an amino acid derived from a CDR2 region of a heavy chain or a light chain of the monoclonal antibody is detected.

Abstract: The present invention provides a highly active protease that can be used in sample preparation for mass spectrometry of a protein and has excellent stability against a change in an external environment. The present invention provides an immobilized protease characterized in that a crudely purified protease or a protease that has not been subjected to a self-digestion resistance treatment is immobilized on surfaces of nanoparticles, and provides a method for producing the immobilized protease. The immobilized protease of the present invention can maintain high activity without being subjected to a change in an external environment, and thus is effective, for example, in preparing a sample to be supplied for mass spectrometry of a protein in a clinical specimen.

Abstract: The present invention provides: a method for increasing a yield, from a target monoclonal antibody, of a peptide fragment that comprises an Fab region or a variable region of the antibody or a portion of the region, the method being characterized by including a process in which digestion of the target monoclonal body is performed by bringing the target monoclonal antibody, which is immobilized on inner surfaces of pores of a porous body, and a protease, which is immobilized on surfaces of nanoparticles having an average particle size larger than an average pore diameter of the porous body, into contact with each other in a liquid by stirring or tapping rotation stirring at a low speed of less than 100 rpm so as to maintain a uniform dispersion, the average pore diameter of the porous body being in a range of 10 nm-200 nm and the average particle size of the nanoparticles being in a range of 50 nm-500 nm; and a kit for use in the method.

Abstract: A sample preparation kit related to the present invention provides a significantly versatile analytical technique that is not affected by the diversity of antibodies, difference in species, matrix and the like. For preparing a sample to be used for detection of a monoclonal antibody through high-performance liquid chromatography-mass spectrometry (LC-MS), the kit includes a porous body for immobilizing a monoclonal antibody to be detected; nanoparticles with an immobilized protease; a reaction vessel for selectively digesting the monoclonal antibody by bringing the porous body and nanoparticles into contact; a buffer to be introduced into the reaction vessel along with the nanoparticles and porous body so that a protease reaction is carried out; and a filtration membrane to remove the porous body and nanoparticles after the proteolysis so as to extract the reaction product and the buffer.

Abstract: A method for detecting epithelial-mesenchymal transition of a cell includes measuring a level of serine in a metabolic substance mixture prepared from culture of the cell, and comparing the level of serine with a reference for serine such that epithelial-mesenchymal transition of the cell is detected based on at least the level of serine compared with the reference.