Down syndrome (DS) is an abnormality of the 21st chromosome that commonly occurs in children born to older women. Thus, amniotic fluid (AF) is usually collected from such women for prenatal diagnosis. This study analyzed human AF supernatants (AFS) using a mass spectrometric (MS) approach to search ...
Down syndrome (DS) is an abnormality of the 21st chromosome that commonly occurs in children born to older women. Thus, amniotic fluid (AF) is usually collected from such women for prenatal diagnosis. This study analyzed human AF supernatants (AFS) using a mass spectrometric (MS) approach to search for candidate biomarkers of a DS pregnancy. The AFS were collected from older pregnant women at weeks 16-18 of their gestation by amniocentesis for cytogenetic analysis. The AFS from the pregnancies carrying DS (n=4) or chromosomally normal (n=6) fetuses, as revealed by the cytogenetic analysis, were then subjected to global protein profiling based on liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS). Affinity chromatography was also applied prior to the LC-ESI-MS/MS to minimize the masking effect of highly abundant albumin and immunoglobulin and thereby increase the diversity of the identified proteins. As a result, at least 30 new AFS proteins were identified and 44 AFS proteins were found to be differentially expressed between the DS and normal cases, where 6 of the proteins were unique to the DS cases and 11 were unique to the chromosomally normal cases. In addition, in the DS cases, 19 AFS proteins were downregulated and 8 were upregulated to varying degrees. A Western blot analysis confirmed the LC-ESI-MS/MS data, indicating that the combined detection of apolipoprotein A-II (apoA-II) and alpha-fetoprotein (AFP) could be a potential tool for diagnosing DS cases.
Down syndrome (DS) is an abnormality of the 21st chromosome that commonly occurs in children born to older women. Thus, amniotic fluid (AF) is usually collected from such women for prenatal diagnosis. This study analyzed human AF supernatants (AFS) using a mass spectrometric (MS) approach to search for candidate biomarkers of a DS pregnancy. The AFS were collected from older pregnant women at weeks 16-18 of their gestation by amniocentesis for cytogenetic analysis. The AFS from the pregnancies carrying DS (n=4) or chromosomally normal (n=6) fetuses, as revealed by the cytogenetic analysis, were then subjected to global protein profiling based on liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS). Affinity chromatography was also applied prior to the LC-ESI-MS/MS to minimize the masking effect of highly abundant albumin and immunoglobulin and thereby increase the diversity of the identified proteins. As a result, at least 30 new AFS proteins were identified and 44 AFS proteins were found to be differentially expressed between the DS and normal cases, where 6 of the proteins were unique to the DS cases and 11 were unique to the chromosomally normal cases. In addition, in the DS cases, 19 AFS proteins were downregulated and 8 were upregulated to varying degrees. A Western blot analysis confirmed the LC-ESI-MS/MS data, indicating that the combined detection of apolipoprotein A-II (apoA-II) and alpha-fetoprotein (AFP) could be a potential tool for diagnosing DS cases.
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제안 방법
A semiquantitative analysis of the protein profile data was performed by comparing the total peptide intensity with the peptides of an identified protein. The total peptide intensity was obtained by summing up the peptide intensities of the peptide hits for the protein.
Accordingly, this study applied a 1D-LC-ESI-MS/MS approach to extend the existing information on the protein composition of human AFS. To increase the chances of identifying low abundant proteins, affinity chromatography was applied prior to LC-ESI-MS/MS to remove highly abundant proteins, such as albumin and immunoglobulin.
The initial results were autovalidated using the following parameters for the “protein details” mode: SPI (scored percent intensity) >70% for matches with a score >7 for +1, >9 for +2, >9 for +3, >8 for +4, and SPI>90% for a score >6 for +1.
As AFS proteins are produced and secreted by either the fetus or the placenta as the pregnancy progresses, their expression pattern may be a reflection of pathologies, such as Down syndrome, intra-amniotic infection, preterm delivery, pre-eclampsia, neonatal sepsis, and premature rupture of the membrane [6, 7, 13, 35, 42, 47]. The methodology used in this study allowed the assessment of 44 proteins that were differentially expressed between the pregnancies with DS fetuses and chromosomally normal fetuses (Table 1). In the DS cases, 19 of these proteins were downregulated and 8 were upregulated to varying degrees (Table 1B).
대상 데이터
This study was approved by the Institutional Review Board of Kangnam CHA Hospital, Pochon CHA University. The human AF samples (10 ml) were obtained based on written informed consent from 10 women undergoing a routine amniocentesis for genetic karyotyping at gestational weeks 16-18. The samples were centrifuged at 1,800 rpm for 10 min to collect the amniocytes for cytogenetic analysis, and the AFS stored at -70℃ until further analysis.
데이터처리
The Student’s t-test was used to analyze the statistical differences.
이론/모형
Four samples came from pregnancies shown by a conventional cytogenetic analysis to have a fetus with DS, and six came from pregnancies with a chromosomally normal fetus. The total protein concentration of the AFS was determined using a Bradford protein assay. To minimize the masking effects caused by highly abundant proteins, affinity chromatography was applied using an albumin and IgG depletion kit to remove the albumin and IgG as per the manufacturer’s instructions.
However, the identification of more AF proteins is desirable to explore their functions within the AF [2]. Thus, to extend the existing information on the protein composition of human AFS, this study combined the LC-ESI-MS/MS approach with 1D SDS-PAGE [20]. Affinity chromatography was also used prior to the LC-ESI-MS/MS to remove highly abundant proteins, such as albumin and immunoglobulin [51].
성능/효과
In conclusion, when using a modified proteomic approach based on a LC-ESI-MS/MS platform, new AFS proteins were identified that could contribute to a better understanding of the molecular mechanism of AF proteins. The present results also reconfirmed that the global protein profile is altered in the AFS from DS cases.
후속연구
In particular, the present results suggest that the combined detection of apo A-II and AFP could be a possible tool for diagnosing DS cases. However, further studies are needed to investigate the clinical utility of these identified protein biomarkers through systematic analysis of the maternal serum.
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