Detection of Fumarate Ester-Derived Modification in a Test Sample

Abstract

Examination of a test sample to determine the presence or quantity of succination of proteins is described. Examination can be via protein hydrolysis in total succination determination or via enzymatic digestion of isolated proteins and determination of the presence or quantity of modified peptides. The methods can be utilized for determination of excessive succination of lymph system proteins, which can be utilized in prevention or early detection of lymphopenia. Methods can be utilized for test samples of subjects under treatment with dimethyl fumarate suffering from multiple sclerosis. Methods can be utilized as a determination that treatment of the subject with DMF should be slowed or stopped.

1 . A method for determining a succination level in a test sample, comprising:
 hydrolyzing proteins of a test sample;   determining the presence or quantity of modified cysteines in the hydrolyzed proteins, wherein the modified cysteines have been modified by one or more of dimethyl fumarate, monomethyl fumarate, or fumarate.   
     
     
         2 . The method of  claim 1 , the method comprising determining the quantity of modified cysteines in the hydrolyzed sample, the method further comprising comparing the determined quantity to a control quantity. 
     
     
         3 . The method of  claim 1 , the method further comprising isolating one or more components from the test sample prior to hydrolyzing the proteins of the test sample. 
     
     
         4 . The method of  claim 3 , the one or more components comprising peripheral blood mononuclear cells. 
     
     
         5 . The method of  claim 4 , the peripheral blood mononuclear cells comprising lymphocytes, macrophages, monocytes, or a combination thereof. 
     
     
         6 . The method of  claim 1 , wherein the modified cysteines have been modified by dimethyl fumarate and/or monomethyl fumarate. 
     
     
         7 . The method of  claim 1 , wherein the test sample is obtained from a subject that has been diagnosed with an autoimmune disorder and treated with dimethyl fumarate or a monomethyl fumarate prodrug. 
     
     
         8 . The method of  claim 7 , wherein the autoimmune disorder is multiple sclerosis. 
     
     
         9 . The method of  claim 1 , wherein the step of determining the presence or quantity of the modified cysteines comprises isotope dilution mass spectrometry. 
     
     
         10 . The method of  claim 1 , the sample comprising blood, cerebrospinal fluid, urine or a tissue biopsy. 
     
     
         11 . A method for determining the succination level in a test sample, the method comprising:
 isolating peripheral blood mononuclear cells of the sample;   lysing the peripheral blood mononuclear cells;   enzymatically digesting proteins of the lysed peripheral blood mononuclear cells; and   determining the presence or quantity of one or more modified peptides in digested proteins, the one or more modified peptides including at least one cysteine modified with dimethyl fumarate, monomethyl fumarate, or fumarate.   
     
     
         12 . The method of  claim 11 , the one or more modified peptides comprising one or more of SEQ ID Nos: 1-36. 
     
     
         13 . The method of  claim 11 , the one or more modified peptides comprising SEQ ID NO: 1. 
     
     
         14 . The method of  claim 11 , the method comprising determining the quantity of the one or more modified peptides, the method further comprising comparing the quantity to a control quantity. 
     
     
         15 . The method of  claim 11 , the peripheral blood mononuclear cells comprising lymphocytes, macrophages, monocytes, or combinations thereof. 
     
     
         16 . The method of  claim 11 , wherein the modified cysteines have been modified by dimethyl fumarate and/or monomethyl fumarate. 
     
     
         17 . The method of  claim 11 , wherein the test sample is obtained from a subject that has been diagnosed with an autoimmune disorder and treated with dimethyl fumarate. 
     
     
         18 . The method of  claim 17 , wherein the autoimmune disorder is multiple sclerosis. 
     
     
         19 . The method of  claim 11 , wherein the step of determining the presence or quantity of the modified cysteines comprises mass spectrometry. 
     
     
         20 . The method of  claim 11 , the sample comprising blood, cerebrospinal fluid, or urine.