Towards comprehensive non-target screening using heart-cut two-dimensional liquid chromatography for the analysis of organic atmospheric tracers in ice cores.

Non-target screening of secondary organic aerosol compounds in ice cores is used to reconstruct atmospheric conditions and sources and is a valuable tool to elucidate the chemical profiles of samples with the aim to obtain as much information as possible from one mass spectrometric measurement. The coupling of mass spectrometry to chromatography limits the results of a non-target screening to signals of compounds within a certain polarity range based on the utilized stationary phases of the columns. Comprehensive two-dimensional liquid chromatography (LCxLC) introduces a second column of different functionality to enable the analysis of a broader range of analytes. Conventional LCxLC requires complex instrumental setups and is difficult to implement for most laboratories. In this work we demonstrate an approach to approximate a comprehensive non-target screening using a simple instrumental setup employing two columns of orthogonal functionalities (HILIC and reversed-phase), an additional pump, and an additional six-port valve. The void volume of the first dimension is transferred to the reversed-phase column to analyze low-polarity compounds during the re-equilibration of the HILIC. Method validation showed adequate repeatability and detection limits for two selected void volume markers and application to snow samples collected at the high-alpine research station Jungfraujoch yielded a total of 270 signals. Comparison to the one-dimensional HILIC approach revealed 175 signals exclusively detected in the two-dimensional method, of which 23 were detected in the second dimension. Detailed analysis of the chemical composition showed consistency with expected compounds in snow samples like lignin or cellulose combustion products from biomass burning or secondary organic aerosol constituents. The results confirmed that one-dimensional chromatography was not sufficient to cover the entire range of compounds and the developed two-dimensional approach will improve the information content from non-target screening while maintaining time of analysis and a simple instrumental setup.

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

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 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 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 (N = 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 SOLAµTM 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 (P > 0.05) regarding protein recovery between both SPE methods. However, only glaucoma-associated marker 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 in the verification experiment using 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 ZIPTIP® C18 method.