Proteomic analysis of biological fluids

Abstract

The invention concerns the identification of proteomes of biological fluids and their use in determining the state of maternal/fetal conditions, including maternal conditions of fetal origin, chromosomal aneuploidies, and fetal diseases associated with fetal growth and maturation. In particular, the invention concerns the identification of the proteome of amniotic fluid (multiple proteins representing the composition of amniotic fluid) and the correlation of characteristic changes in the normal proteome with various pathologic maternal/fetal conditions, such as intra-amniotic infection, or chromosomal defects.

1 . A method for determining the state of a maternal or fetal condition, comprising comparing the proteomic profile of a test sample of a biological fluid obtained from a mammalian subject with the proteomic profile of a normal sample, or a reference proteomic profile comprising at least one unique expression signature characteristic of said condition. 
     
     
         2 . The method of  claim 1  wherein said mammalian subject is a pregnant female. 
     
     
         3 . The method of  claim 2  wherein said pregnant female is human. 
     
     
         4 . The method of  claim 3  wherein said maternal condition is selected from the group consisting of intrauterine infection, preeclampsia, and preterm labor. 
     
     
         5 . The method of  claim 3  wherein said fetal condition is selected from the group consisting of chromosomal aneuploidies, congenital malformation, gestational age and fetal maturity. 
     
     
         6 . The method of  claim 5  wherein said chromosomal aneuploidy is selected from the group consisting of Down syndrome, trisomy-13, trisomy-18, Turner syndrome, and Klinefelter syndrome. 
     
     
         7 . The method of  claim 1  wherein said biological fluid is selected from the group consisting of amniotic fluid, serum, plasma, urine, cerebrospinal fluid, breast milk, mucus, and saliva. 
     
     
         8 . The method of  claim 3  wherein said biological fluid is amniotic fluid or maternal serum. 
     
     
         9 . The method of  claim 1  wherein the proteomic profile of the test sample comprises information of at least 2 proteins. 
     
     
         10 . The method of  claim 1  wherein the proteomic profile of the test sample comprises information of at least 5 proteins. 
     
     
         11 . The method of  claim 1  wherein the proteomic profile of the test sample comprises information of at least 10 proteins. 
     
     
         12 . The method of  claim 1  wherein the proteomic profile of the test sample comprises information of at least 20 proteins. 
     
     
         13 . The method of  claim 1  wherein the proteomic profile of the test sample comprises information of at least 50 proteins. 
     
     
         14 . The method of  claim 1  wherein the proteomic profile is a mass spectrum. 
     
     
         15 . The method of  claim 14  wherein the proteomic profile comprises at least one unique expression signature in the 3 to 5 kDa range of the mass spectrum. 
     
     
         16 . The method of  claim 14  wherein the proteomic profile comprises at least one unique expression signature in the 10 to 12 kDa range of the mass spectrum. 
     
     
         17 . The method of  claim 14  wherein the maternal condition is intra-amniotic infection, and the unique expression signature is an extra peak in the 10 to 11 kDa molecular weight range in the test sample, which is indicative of intra-amniotic infection. 
     
     
         18 . The method of  claim 17  wherein the biological fluid is amniotic fluid. 
     
     
         19 . The method of  claim 17  wherein the biological fluid is maternal serum. 
     
     
         20 . The method of  claim 1  wherein the proteomic profile is produced by Western blot analysis. 
     
     
         21 . The method of  claim 1  wherein the biological fluid is that of a human, and proteomic profile includes information of the expression of one or more of the proteins selected from the group consisting of: macrophage capping protein, neutrophil gelatinase-associated lipocalin, myeloperoxidase; L-plastin; azurocidin; antibacterial protein FALL-39; Gp340 variant protein; Ebner salivary gland protein homolog (GenBank Accession No. 355392); leukocyte elastase inhibitor; calgranulin A; calgranulin B; cofilin; moesin; profilin I, cronin-like protein p57; annexin II, fibronectin; glia-derived nexin; antithrombin-III; squamous cell carcinoma antigen 1, squamous cell carcinoma antigen 2; serpin 12; cystatin A; cystatin B; cystatin C; IGFBP-1; Vitamin D-binding protein; apolipoprotein A-I; 14-3-3 protein sigma; 14-3-3 protein zeta/delta; gelsolin; lactotransferrin; phosphoglycerate kinase 1; phosphoglycerate mutase 1; and transketolase; or a fragment, precursor, or naturally occurring variant thereof. 
     
     
         22 . The method of  claim 21  wherein the proteomic profile includes information of the expression of two or more of said proteins. 
     
     
         23 . The method of  claim 21  wherein the proteomic profile includes information of the expression of all of said proteins. 
     
     
         24 . The method of  claim 21  wherein the biological fluid is amniotic fluid. 
     
     
         25 . The method of  claim 24  wherein the proteomic profile is that of the normal sample. 
     
     
         26 . The method of  claim 24  wherein the proteomic profile is that of the test sample or reference sample. 
     
     
         27 . The method of  claim 21  wherein one or more of said proteins are differentially expressed in said test sample relative to said normal sample. 
     
     
         28 . The method of  claim 27  wherein the proteomic profile includes information of the expression of one or more of the proteins selected from the group consisting of macrophage capping protein; neutrophil gelatinase-associated lipocalin; myeloperoxidase; L-plastin; azurocidin; antibacterial protein FALL-39; leukocyte elastase inhibitor; calgranulin A; calgranulin B; profilin 1, glia-derived nexin; serpin 12; cystatin A; and IGFBP-1; or a fragment, precursor, or naturally occurring variant thereof. 
     
     
         29 . The method of  claim 28  wherein the proteomic profile includes information of the expression of two or more of said proteins. 
     
     
         30 . The method of  claim 28  wherein the proteomic profile includes information of the expression of all of said proteins. 
     
     
         31 . The method of  claim 28  wherein the biological fluid is amniotic fluid. 
     
     
         32 . The method of  claim 31  wherein one or more of said proteins are differentially expressed in said test sample relative to said normal sample. 
     
     
         33 . The method of  claim 32  wherein said subject is diagnosed with intra-amniotic infection. 
     
     
         34 . The method of  claim 32  wherein said patient is diagnosed with a developmental defect. 
     
     
         35 . The method of  claim 34  wherein said developmental defect is a defect in the development of an organ system. 
     
     
         36 . The method of  claim 35  wherein said organ system is the central nervous system or the cardiovascular system. 
     
     
         37 . The method of  claim 34  wherein said developmental defect is a musculoskeletal deformity. 
     
     
         38 . The method of  claim 34  wherein said developmental defect is results from a chromosomal aneuploidy. 
     
     
         39 . The method of  claim 38  wherein said chromosomal aneuploidy is selected from the group consisting of Downsyndrome, trisomy-13, trisomy-18, Turner syndrome, and Klinefelter syndrome. 
     
     
         40 . The method of  claim 31  wherein the proteomic profile of the test sample is essentially the same as the proteomic profile of the normal sample, and the subject is determined to be free of said maternal or fetal condition. 
     
     
         41 . The method of  claim 31  wherein the proteomic profile contains the same unique expression signature as the reference sample. 
     
     
         42 . The method of  claim 41  wherein the unique expression signature is characteristic of intra-amniotic infection. 
     
     
         43 . The method of  claim 42  wherein said subject is diagnosed with intra-amniotic infection. 
     
     
         44 . A method for the diagnosis of intra-amniotic infection, comprising
 (a) comparing the proteomic profile of a test sample of a biological fluid obtained from a pregnant female mammal with the proteomic profile of a normal sample, or a reference proteomic profile, wherein said proteomic profiles provide information of the mass of the proteins present in said samples, or the proteolytic fragments thereof; and   (b) diagnosing said mammal with intra-amniotic infection if the proteomic profile of the test sample shows a unique expression signature in the 3-5 and/or 10-12 KDa molecular weight range.   
     
     
         45 . The method of  claim 44  wherein the proteomic profiles are represented in the form of mass spectra. 
     
     
         46 . The method of  claim 45  wherein said mammal is a primate. 
     
     
         47 . The method of  claim 45  wherein said mammal is human. 
     
     
         48 . The method of  claim 47  further comprising the step of monitoring the course of said intra-amniotic infection. 
     
     
         49 . The method of  claim 44  wherein the biological fluid is amniotic fluid or maternal serum. 
     
     
         50 . A method for the diagnosis of intra-amniotic infection, comprising:
 (a) comparing the proteomic profile of a test sample of a biological fluid obtained from a pregnant female mammal with the proteomic profile of a normal sample; and   (b) diagnosing said mammal with intra-amniotic infection if at least one protein selected from the group consisting of IGFB-1, profilin, ceruloplasmin, L-plastin, and calgranulin, or a fragment, precursor or naturally occurring variant thereof, is differentially expressed in said test sample relative to said normal sample.   
     
     
         51 . The method of  claim 50  wherein said mammal is a primate. 
     
     
         52 . The method of  claim 51  wherein said primate is human. 
     
     
         53 . The method of  claim 45  wherein said biological fluid is amniotic fluid or maternal serum. 
     
     
         54 . The method of  claim 53  wherein at least one of IGFBP-1, profilin, ceruloplasmin, and calgranulin, or a fragment, precursor, or naturally-occurring variant thereof, is overexpressed in said test sample relative to said normal sample. 
     
     
         55 . The method of  claim 53  wherein L-plastin is underexpressed in said test sample relative to said normal sample. 
     
     
         56 . The method of  claim 50  wherein the presence of IGFBP-1 is detected by identifying the proteolytic fragment shown in  FIG. 12 , or a fragment thereof. 
     
     
         57 . The method of  claim 50  further comprising the step of monitoring the state of said intra-amniotic infection. 
     
     
         58 . A method for the diagnosis of a chromosomal aneuploidy, comprising:
 (a) comparing the proteomic profile of a test sample of a biological fluid obtained from a pregnant female mammal with the proteomic profile of a normal sample, or a reference proteomic profile, wherein said proteomic profiles provide information of the mass of the proteins present in said samples, or the proteolytic fragments thereof, and   (b) diagnosing said mammal with said chromosomal aneuploidy if the proteomic profile of the test sample shows a unique expression signature in the 4 to 15 KDa molecular weight range.   
     
     
         59 . The method of  claim 58  wherein the chromosomal aneuploidy is Down syndrome. 
     
     
         60 . A method for the diagnosis of a developmental defect of a fetus, comprising:
 (a) comparing the proteomic profile of a test sample of a biological fluid obtained from a pregnant female mammal with the proteomic profile of a normal sample, or a reference proteomic profile; and   (b) confirming the presence of said developmental defect if at least one actin-modulating protein, or a fragment, precursor, or naturally occurring variant thereof, is differentially expressed in said test sample relative to said normal sample.   
     
     
         61 . The method of  claim 60  wherein said actin-modulating protein is selected from the group consisting of moesin, p57, gelsolin, and a 14-3-3 protein. 
     
     
         62 . A method for the diagnosis of a maternal or fetal infection or immune-response related disorder, comprising
 (a) comparing the proteomic profile of a test sample of a biological fluid obtained from a pregnant female mammal with the proteomic profile of a normal sample, or a reference proteomic profile; and   (b) confirming the presence of said maternal or fetal infection or immune-response related disorder, if at least one protein selected from the group consisting of macrophage capping protein (MCP), leukocyte elastase, neutrophil gelatinase-associated lipcalcin (NGAL), myeloperoxidase, L-plastin, calgranulin, FALL-39, azyrocidin (CAP37), proteases and protease inhibitors, is differentially expressed in said test sample relative to said normal sample.   
     
     
         63 . A method for the diagnosis of neonatal sepsis, comprising detecting in the proteomic profile of a biological fluid obtained from a pregnant females mammal the presence of Gp-340. 
     
     
         64 . A proteomic profile of a biological fluid comprising information of one or more proteins selected from the group consisting of macrophage capping protein, neutrophil gelatinase-associated lipocalin, myeloperoxidase; L-plastin; azurocidin; antibacterial protein FALL-39; Gp340 variant protein; Ebner salivary gland protein homolog (GenBank Accession No. 355392); leukocyte elastase inhibitor; calgranulin A; calgranulin B; cofilin; moesin; profilin I, cronin-like protein p57; annexin II, fibronectin; glia-derived nexin; antithrombin-III; squamous cell carcinoma antigen 1, squamous cell carcinoma antigen 2; serpin 12; cystatin A; cystatin B; cystatin C; IGFBP-1; Vitamin D-binding protein; apolipoprotein A-I; 14-3-3 protein sigma; 14-3-3 protein zeta/delta; gelsolin; lactotransferrin; phosphoglycerate kinase 1; phosphoglycerate mutase 1; and transketolase; or a fragment, precursor, or naturally occurring variant thereof. 
     
     
         65 . A proteomic profile of a biological fluid comprising information of one or more proteins selected from the group consisting of macrophage capping protein; neutrophil gelatinase-associated lipocalin; myeloperoxidase; L-plastin; azurocidin; antibacterial protein FALL-39; leukocyte elastase inhibitor; calgranulin A; calgranulin B; profilin 1, glia-derived nexin; serpin 12; cystatin A; and IGFBP-1; or a fragment, precursor, or naturally occurring variant thereof. 
     
     
         66 . The proteomic profile of  claim 65  confirming the presence of one or more of said proteins, or a fragment, precursor, or naturally occurring variant thereof. 
     
     
         67 . A proteomic profile of a biological fluid characteristic of intra-amniotic infection, comprising information confirming the presence of a protein selected from the group consisting of IGFB-1, profilin, ceruloplasmin, L-plastin, and calgranulin. 
     
     
         68 . A proteomic profile of a biological fluid characteristic of intra-amniotic infection represented in a form providing information of the molecular weight of proteins present in said biological fluid, or the proteolytic fragments thereof, comprising a unique expression signature in the 3-5 KDa and/or 10-12 KDa molecular weight range. 
     
     
         69 . The proteomic profile of  claim 68  represented as a mass spectrum. 
     
     
         70 . The proteomic profile of  claim 69  wherein the biological fluid is amniotic fluid or maternal serum. 
     
     
         71 . A proteomic profile essentially as shown in any one of  FIGS. 1A-1C . 
     
     
         72 . A proteomic profile essentially as shown in any one of  FIGS. 2A-C . 
     
     
         73 . A proteomic profile essentially as shown in any one of  FIGS. 3A-C . 
     
     
         74 . A proteomic profile essentially as shown in  FIG. 4A  or  4 B. 
     
     
         75 . A proteomic profile essentially as shown in any one of  FIGS. 6-10 . 
     
     
         76 . A proteomic profile of any one of  claims 64 - 75 , analyzed in a microarray format.