Dataset Information

Membrane Fraction Complexome of Nitrosocaldus cavascurensis

ABSTRACT: Biomass of N. cavascurensis grown in a bioreactor was lysed and membrane fractions were harvested using an ultracentrifuge. Membrane proteins were extracted using n-dodecyl-beta-D-maltoside (DDM) and loaded on a 3-12% gradient blue native PAGE gel. Selected bands were cut out and analyzed via mass spectrometry.

INSTRUMENT(S):

ORGANISM(S): Candidatus Nitrosocaldus Cavascurensis

SUBMITTER: Logan Hodgskiss

LAB HEAD: Christa Schleper

PROVIDER: PXD034632 | Pride | 2023-02-13

REPOSITORIES: Pride

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			Action	DRS
	20210903_QExHFX2_LC4_Hodgskiss_Schleper_ext_Uni_MPL_Ncav_B10_30p.raw	Raw
	20210903_QExHFX2_LC4_Hodgskiss_Schleper_ext_Uni_MPL_Ncav_B11_30p.raw	Raw
	20210903_QExHFX2_LC4_Hodgskiss_Schleper_ext_Uni_MPL_Ncav_B12_30p_2.raw	Raw
	20210903_QExHFX2_LC4_Hodgskiss_Schleper_ext_Uni_MPL_Ncav_B13_30p_2.raw	Raw
	20210903_QExHFX2_LC4_Hodgskiss_Schleper_ext_Uni_MPL_Ncav_B14_30p_2.raw	Raw

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Project description:Proper sample preparation protocols represent a critical step for 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 6 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® 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.

Dataset Information

Membrane Fraction Complexome of Nitrosocaldus cavascurensis

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