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

Abstract 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 p...

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Autores principales:	Corné Swart, Silvia Martínez-Jaime, Michal Gorka, Kerstin Zander, Alexander Graf
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2018
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/5052033028184804a4d3ef43a4698f53
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spelling	oai:doaj.org-article:5052033028184804a4d3ef43a4698f532021-12-02T11:40:37ZHit-Gel: Streamlining in-gel protein digestion for high-throughput proteomics experiments10.1038/s41598-018-26639-32045-2322https://doaj.org/article/5052033028184804a4d3ef43a4698f532018-06-01T00:00:00Zhttps://doi.org/10.1038/s41598-018-26639-3https://doaj.org/toc/2045-2322Abstract 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” (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.Corné SwartSilvia Martínez-JaimeMichal GorkaKerstin ZanderAlexander GrafNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 8, Iss 1, Pp 1-8 (2018)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Corné Swart Silvia Martínez-Jaime Michal Gorka Kerstin Zander Alexander Graf Hit-Gel: Streamlining in-gel protein digestion for high-throughput proteomics experiments
description	Abstract 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” (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.
format	article
author	Corné Swart Silvia Martínez-Jaime Michal Gorka Kerstin Zander Alexander Graf
author_facet	Corné Swart Silvia Martínez-Jaime Michal Gorka Kerstin Zander Alexander Graf
author_sort	Corné Swart
title	Hit-Gel: Streamlining in-gel protein digestion for high-throughput proteomics experiments
title_short	Hit-Gel: Streamlining in-gel protein digestion for high-throughput proteomics experiments
title_full	Hit-Gel: Streamlining in-gel protein digestion for high-throughput proteomics experiments
title_fullStr	Hit-Gel: Streamlining in-gel protein digestion for high-throughput proteomics experiments
title_full_unstemmed	Hit-Gel: Streamlining in-gel protein digestion for high-throughput proteomics experiments
title_sort	hit-gel: streamlining in-gel protein digestion for high-throughput proteomics experiments
publisher	Nature Portfolio
publishDate	2018
url	https://doaj.org/article/5052033028184804a4d3ef43a4698f53
work_keys_str_mv	AT corneswart hitgelstreamliningingelproteindigestionforhighthroughputproteomicsexperiments AT silviamartinezjaime hitgelstreamliningingelproteindigestionforhighthroughputproteomicsexperiments AT michalgorka hitgelstreamliningingelproteindigestionforhighthroughputproteomicsexperiments AT kerstinzander hitgelstreamliningingelproteindigestionforhighthroughputproteomicsexperiments AT alexandergraf hitgelstreamliningingelproteindigestionforhighthroughputproteomicsexperiments
_version_	1718395580805283840

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

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