Absolute quantitation of proteins by two-point quantitation using dimethylated proteotypic peptides

ABSTRACT: Absolute targeted proteomics typically employs known amounts of synthetic stable isotopically labeled peptides which are mixed with the analyte and analysed by LC-MS. In order to obtain accurate 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 and very little side reactions. Furthermore, three different forms can be generated with e.g., light and heavy labels for spike-in peptides, and medium label for endogenous peptides. The addition of the dimethylated proteotypic peptide in two different versions allows to determine the accuracy of the quantification of endogenous peptides using the relative ratio of the intensities of the two internal standards. 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 accurately measured across four orders of magnitude by the two-point quantitation method. Less than 20% of coefficient of variation (CV) values and strong correlation with R2 of 0.99 across three analytical replicates was observed. The results showed that the accuracy to determine the amount of endogenous peptides can be validated using two references against each other, which cannot be obtained by one-point quantitation. Because of the significant lower costs than synthetic stable isotopically labeled peptides, this approach might be particularly interesting for the absolute quantitation of multiple proteins.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Blood Cell, Blood Serum

SUBMITTER: Trung Tran  

LAB HEAD: Bernd Thiede

PROVIDER: PXD007894 | Pride | 2018-11-27

REPOSITORIES: Pride