TY - JOUR
T1 - Analysis of in vivo-derived amyloid-β polypeptides by on-line two-dimensional chromatography-mass spectrometry
AU - Clarke, Nigel J.
AU - Crow, Frank W.
AU - Younkin, Steven
AU - Naylor, Stephen
PY - 2001/11/1
Y1 - 2001/11/1
N2 - The presence of senile plaques composed of amyloid-β (Aβ) polypeptides within brain tissue is normally used as a definitive postmortem diagnosis for Alzheimer's Disease (AD). Therefore, these polypeptides have been investigated as potential biomarkers of the disease state. However, at present, there is a lack of a robust assay for the detection of such polypeptides derived from in vivo sources. Such an assay is essential for analysis of biological samples from model AD systems. To overcome this problem we have developed a new single-step assay utilizing two dimensional-chromatography in conjunction with mass spectrometry. The method consists of on-line size-exclusion chromatography (SEC) to provide initial separation of analytes from the sample (based on their molecular weight) coupled with sample preconcentration prior to analysis by microbore high-performance liquid chromatography-mass spectrometry (HPLC-MS). This provides an extremely versatile and powerful assay which can separate specific analytes from cell lysate in a single step without further sample handling. The use of mass spectrometry as the detection system yields much more structural information than can be obtained from traditional ELISA and sandwich ELISA antibody assays. Furthermore, the on-line sample cleanup protocol minimizes sample handling and facilitates assay automation. Utilizing this new assay we have been able to detect Aβ 1-40 and Aβ 1-42 at cellular concentration levels directly from cell lysates. Moreover, we have detected multiple peptide responses within the same analysis, some of which have been tentatively identified as other ragged C-termini Aβ polypeptides derived from Aβ 1-42, based on their molecular weight, as well as oxidized Aβ polypeptides.
AB - The presence of senile plaques composed of amyloid-β (Aβ) polypeptides within brain tissue is normally used as a definitive postmortem diagnosis for Alzheimer's Disease (AD). Therefore, these polypeptides have been investigated as potential biomarkers of the disease state. However, at present, there is a lack of a robust assay for the detection of such polypeptides derived from in vivo sources. Such an assay is essential for analysis of biological samples from model AD systems. To overcome this problem we have developed a new single-step assay utilizing two dimensional-chromatography in conjunction with mass spectrometry. The method consists of on-line size-exclusion chromatography (SEC) to provide initial separation of analytes from the sample (based on their molecular weight) coupled with sample preconcentration prior to analysis by microbore high-performance liquid chromatography-mass spectrometry (HPLC-MS). This provides an extremely versatile and powerful assay which can separate specific analytes from cell lysate in a single step without further sample handling. The use of mass spectrometry as the detection system yields much more structural information than can be obtained from traditional ELISA and sandwich ELISA antibody assays. Furthermore, the on-line sample cleanup protocol minimizes sample handling and facilitates assay automation. Utilizing this new assay we have been able to detect Aβ 1-40 and Aβ 1-42 at cellular concentration levels directly from cell lysates. Moreover, we have detected multiple peptide responses within the same analysis, some of which have been tentatively identified as other ragged C-termini Aβ polypeptides derived from Aβ 1-42, based on their molecular weight, as well as oxidized Aβ polypeptides.
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U2 - 10.1006/abio.2001.5357
DO - 10.1006/abio.2001.5357
M3 - Article
C2 - 11673892
AN - SCOPUS:0035499465
SN - 0003-2697
VL - 298
SP - 32
EP - 39
JO - Analytical Biochemistry
JF - Analytical Biochemistry
IS - 1
ER -