Comparison of Reversed-Phase and Mixed-Mode SPE for Enrichment and Clean-Up of Surrogate Peptides in CysC Quantified by LC-MS/MS.

Accurate quantification of low-abundance protein Cystatin-C (CysC) in serum by liquid chromatography tandem mass spectrometry (LC-MS/MS) method is very difficult. After sample processing and tryptic digestion, the matrix of CysC surrogate peptides is very complexity, and the concentrations of them are very low, so solid-phase extraction (SPE) must be used to make the surrogate peptides purification and enrichment. In this paper, we used C18 reversed-phase SPE (RP-SPE) and mixed-mode SPE as SPE cartridges. We quantitatively assessed and compared the CysC surrogate peptides recoveries and matrix effects by different SPE cartridges. The sequence of CysC surrogate peptide is ALDFAVGEYNK, and sequence-specific subions y6 (VGEYNK+, m/z 709.3) and y9 (DFAVGEYNK+, m/z 1042.4) were selected for quantification of CysC, because the two fragment ions showed the highest sensitivity. In neat solution, the highest recoveries were similarly for y9 and y6 when used RP-SPE and mixed-mode SPE. However, in serum matrix, the recoveries were significantly higher when used mixed-mode SPE than RP-SPE, which was caused by matrix effects. Results showed that both RP-SPE and mixed-mode SPE were resulted in ion enhancement for CysC surrogate peptides quantification by LC-MS/MS, but mixed-mode SPE reduced more matrix effects. So mixed-mode SPE was more suitable SPE type for purification and enrichment of CysC surrogate peptides.