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Hit-Gel: Streamlining in-gel protein digestion for high-throughput proteomics experiments.

ABSTRACT: Traditional in gel-digestion procedures require extensive sample handling, are prone to contamination and not compatible with high-throughput sample preparation. To address these shortcomings, we have modified the conventional in-gel digestion procedure for high-throughput proteomics studies. The modified method, termed “High Throughput in Gel digestion” (HiT-Gel), is based on a 96-well plate format which results in a drastic reduction in labour intensity and sample handling. Direct comparison revealed that HiT-Gel reduces technical variation and significantly decreases sample contamination over the conventional in-gel digestion method. HiT-Gel also produced superior results when a single protein band was excised from a gel and processed by in-gel digestion. Moreover, we applied Hit-Gel for a mass spectrometry analysis of Arabidopsis thaliana protein complexes separated by native PAGE in 24 fractions and four biological replicates. We show that the high throughput capacity of HiT-Gel facilitates large scale studies with high sample replication or detailed fractionation. Our method can easily be implemented as it does not require specialised laboratory equipment.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Arabidopsis Thaliana (mouse-ear Cress)

TISSUE(S): Leaf

SUBMITTER: Michal Gorka

LAB HEAD: Dr. Alexander Graf

PROVIDER: PXD009510 | Pride | 2018-10-16

REPOSITORIES: Pride

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Hit-Gel: Streamlining in-gel protein digestion for high-throughput proteomics experiments.

Swart Corné C Martínez-Jaime Silvia S Gorka Michal M Zander Kerstin K Graf Alexander A

Scientific reports 20180605 1

In-gel digestion has been used as a standard method for the preparation of protein samples for mass spectrometry analysis for over 25 years. Traditional in gel-digestion procedures require extensive sample handling, are prone to contamination and not compatible with high-throughput sample preparation. To address these shortcomings, we have modified the conventional in-gel digestion procedure for high-throughput proteomics studies. The modified method, termed "High Throughput in Gel digestion" (H ...[more]

PMID: 29872109

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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.

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Dataset Information

Hit-Gel: Streamlining in-gel protein digestion for high-throughput proteomics experiments.

Dataset's files

Publications

Hit-Gel: Streamlining in-gel protein digestion for high-throughput proteomics experiments.

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