Comparison of Two Solid-Phase Extraction (SPE) Methods for the Identification and Quantification of Porcine Retinal Protein Markers by LC?MS/MS

Abstract

Proper sample preparation protocols represent a critical step for liquid chromatography mass spectrometry (LC-MS)-based proteomic study designs and influence the speed, performance and automation of high-throughput data acquisition. Main objective of this study was to compare two commercial Solid-Phase Extraction (SPE)-based sample preparation protocols (comprising SOLAμTM HRP SPE spin plates from Thermo Fisher Scientific and ZIPTIP® C18 pipette tips from Merck Millipore) for analytical performance, reproducibility and analysis speed. The house swine (Sus scrofa domestica) represents a promising animal model for studying human eye diseases including glaucoma and provides excellent requirements for the qualitative and quantitative MS based comparison in terms of ocular proteomics. In total 6 technical replicates of two protein fractions (extracted with 0.1% dodecyl-s-maltoside (DDM) or 1% trifluoroacetic acid (TFA)) of porcine retinal tissues were subjected to in-gel trypsin digestion and purified with both SPE-based workflows (N=3) prior LC-MS/MS analysis. On average both protein fractions (DDM and TFA) provided the identification of 550 ± 70 and 305 ± 48 proteins after ZIPTIP® purification protocol and SOLAμTM workflow resulted in the detection of 513 ± 55 and 300 ± 33 proteins (FDR0.05) regarding protein recovery between both SPE methods. However, only glaucoma protein marker methyl-CpG-binding protein 2 (MECP2) showed a significant (P=0.02) higher abundance in ZIPTIP®-purified replicates in comparison to SOLAμTM-treated study samples. Nevertheless, this result was not confirmed by in-gel trypsin digestion of recombinant MECP2 (P=0.24). In conclusion, both SPE-based purification methods worked equally well in terms of analytical performance and reproducibility, whereas the analysis speed and the semi‑automation of the SOLAμTM spin plates workflow is much more convenient in comparison to the manual ZIPTIP® C18 pipette tip protocol.