| Autor: |
Tran TT; Department of Biosciences, University of Oslo, Norway. bernd.thiede@ibv.uio.no., Bollineni RC; Department of Biosciences, University of Oslo, Norway. bernd.thiede@ibv.uio.no., Koehler CJ; Department of Biosciences, University of Oslo, Norway. bernd.thiede@ibv.uio.no., Thiede B; Department of Biosciences, University of Oslo, Norway. bernd.thiede@ibv.uio.no. |
| Jazyk: |
angličtina |
| Zdroj: |
The Analyst [Analyst] 2018 Sep 21; Vol. 143 (18), pp. 4359-4365. Date of Electronic Publication: 2018 Aug 16. |
| DOI: |
10.1039/c8an01081a |
| Abstrakt: |
Absolute targeted proteomics typically employs known amounts of synthetic stable isotopically labeled peptides which are mixed with the analyte and analysed by LC-MS to determine the concentration of proteins. In order to obtain more data, we evaluated the use of two different stable isotopes of the same peptide as spike-in for absolute quantification. For this purpose, peptide labeling by reductive amination was applied, which is a mild reaction for dimethylation of amine groups with very high yield. Three different forms can be generated with e.g., light and heavy labels for spike-in peptides, and medium label for endogenous peptides. The method was studied with peptides of apolipoprotein A-I, apolipoprotein B-100, and leucine-rich alpha-2-glycoprotein without and with serum. In serum, the endogenous protein concentrations were measured across four orders of magnitude by the two-point quantification method. Less than 20% of coefficient of variation (CV) values and strong correlation with R 2 of 0.99 across three analytical replicates was observed. Most importantly, the two-point quantification method allows an internal quality control of the spike-in peptide as strong deviations in ratios calculated between the first and second reference indicate a methodical error. Because of the significant lower costs than synthetically stable isotopically labeled peptides, this approach might be particularly interesting for the absolute quantification of multiple proteins. |
| Databáze: |
MEDLINE |
| Externí odkaz: |
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