Multiple heart-cutting two dimensional liquid chromatography and isotope dilution tandem mass spectrometry for the absolute quantification of proteins in human serum.

We evaluate here the combination of two-dimensional liquid chromatography (2D-LC) in the multiple heart cutting mode and isotope dilution tandem mass spectrometry for the direct analysis of tryptic digests of serum samples. As a proof of concept, we attempt the quantification of proteotypic peptides of Apolipoprotein AIV (APOA4), Complement C3 (C3) and Vitronectin (VTN) which have been previously identified as potential candidate biomarkers of glaucoma. Using this 2D-LC strategy, analyte enrichment steps are avoided and the sample preparation involved after enzymatic digestion amounted to a simple centrifugation, evaporation of the supernatant and reconstitution in the 1D mobile phase. A mobile phase not compatible with the ESI source (10 mM KHPO at pH 2.7) was used in the first dimension as it provided a satisfactory chromatographic resolution of the peptides and a high buffering capacity avoiding changes in retention times when analyzing complex matrices like human serum. We also demonstrate that using coeluting labelled analogues of the target peptides, protein concentrations were not affected by slight retention time shifts affecting the amount of target peptides transferred to the second dimension. Satisfactory results were obtained when analyzing fortified serum samples (recoveries from 98 to 113%). Precisions in the range of 1-9% RSD were obtained when replicating the analysis of a pooled serum sample. The comparative analysis of serum samples from n = 94 control subjects and n = 91 patients diagnosed with primary open-angle glaucoma did not show significant differences in the APOA4, VTN and C3 concentrations in contrast with previous studies using immunoassays.