sample-preparation-for-mass-spectrometry-based-proteomics-analysis

Sample Preparation for Mass-spectrometry-based Proteomics Analysis of Ocular Microvessels

The use of isolated ocular blood vessels in vitro to decipher the pathophysiological state of the eye using advanced technological approaches has greatly ...

University Medical Centre of the Johannes Gutenberg University Mainz

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. The main objective of this study was to compare two commercial solid-phase extraction (SPE)-based sample preparation protocols (comprising SOLAµ 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 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 six technical replicates of two protein fractions [extracted with 0.1% dodecyl-ß-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 ( = 3) prior to LC-MS analysis. On average, 550 ± 70 proteins (1512 ± 199 peptides) and 305 ± 48 proteins (806 ± 144 peptides) were identified from DDM and TFA protein fractions, respectively, after ZIPTIP C18 purification, and SOLAµ workflow resulted in the detection of 513 ± 55 proteins (1347 ± 180 peptides) and 300 ± 33 proteins (722 ± 87 peptides), respectively (FDR < 1%). Venn diagram analysis revealed an average overlap of 65 ± 2% (DDM fraction) and 69 ± 4% (TFA fraction) in protein identifications between both SPE-based methods. Quantitative analysis of 25 glaucoma-related protein markers also showed no significant differences ( > 0.05) regarding protein recovery between both SPE methods. However, only glaucoma-associated marker  showed a significant ( = 0.02) higher abundance in ZIPTIP-purified replicates in comparison to SOLAµ-treated study samples. Nevertheless, this result was not confirmed in the verification experiment using in-gel trypsin digestion of recombinant  ( = 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 SOLAµ spin plates workflow is much more convenient in comparison to the ZIPTIP C18 method.

Carsten Schmelter

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