Amyloid-β (Aβ) in human plasma was detected and quantified by an antibody-free method, selected reaction monitoring mass spectrometry (SRM-MS) in the current study. Due to its low abundance, SRM-based quantification in 10μL plasma was a challenge. Prior to SRM analysis, human plasma proteins as a whole were digested by trypsin and high pH reversed-phase liquid chromatography (RPLC) was used to fractionate the tryptic digests and to collect peptides, Aβ1-5, Aβ6-16, Aβ17-28 and Aβ29-40(42) of either Aβ1-40 or Aβ1-42. Among those peptides, Aβ17-28 was selected as a surrogate to measure the total Aβ level. Human plasma samples obtained from triplicate sample preparations were analyzed, obtaining 4.20ngmL-1 with a CV of 25.3%. Triplicate measurements for each sample preparation showed CV of <5%. Limit of quantification was obtained as 132pM, which corresponded to 570pgmL-1 of Aβ1-40. Until now, most quantitative measurements of Aβ in plasma or cerebrospinal fluid have required antibody-based immunoassays. Since quantification of Aβ by immunoassays is highly dependent on the extent of epitope exposure due to aggregation or plasma protein binding, it is difficult to accurately measure the actual concentration of Aβ in plasma. Our diagnostic method based on SRM using a surrogate peptide of Aβ is promising in that actual amounts of total Aβ can be measured regardless of the conformational status of the biomarker.
Bibliographical noteFunding Information:
This study was supported by a grant ( 2E24582 ) from Open Research Project funded by KIST , and grants ( 2012M3A9B9036679 and 2013M3A9B9044846 ) from Proteogenomics Program funded by NRF , MSIP .
All Science Journal Classification (ASJC) codes
- Analytical Chemistry
- Environmental Chemistry