Evaluation of Sample Preparation Strategies for Human Milk and Plasma Proteomics

Sample preparation is the most critical step in proteomics as it directly affects the subset of proteins and peptides that can be reliably identified and quantified. Although a variety of efficient and reproducible sample preparation strategies have been developed, their applicability and efficacy d...

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Autores principales:	Sanja Milkovska-Stamenova, Michele Wölk, Ralf Hoffmann
Formato:	article
Lenguaje:	EN
Publicado:	MDPI AG 2021
Materias:	bottom-up proteomics filter-aided sample preparation (FASP) Folch extraction in-solution digestion (ISD) milk plasma Organic chemistry QD241-441
Acceso en línea:	https://doaj.org/article/91bd13b5336b47eb9f603943a88e26aa
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spelling	oai:doaj.org-article:91bd13b5336b47eb9f603943a88e26aa2021-11-25T18:27:28ZEvaluation of Sample Preparation Strategies for Human Milk and Plasma Proteomics10.3390/molecules262268161420-3049https://doaj.org/article/91bd13b5336b47eb9f603943a88e26aa2021-11-01T00:00:00Zhttps://www.mdpi.com/1420-3049/26/22/6816https://doaj.org/toc/1420-3049Sample preparation is the most critical step in proteomics as it directly affects the subset of proteins and peptides that can be reliably identified and quantified. Although a variety of efficient and reproducible sample preparation strategies have been developed, their applicability and efficacy depends much on the biological sample. Here, three approaches were evaluated for the human milk and plasma proteomes. Protein extracts were digested either in an ultrafiltration unit (filter-aided sample preparation, FASP) or in-solution (ISD). ISD samples were desalted by solid-phase extraction prior to nRPC-ESI-MS/MS. Additionally, milk and plasma samples were directly digested by FASP without prior protein precipitation. Each strategy provided inherent advantages and disadvantages for milk and plasma. FASP appeared to be the most time efficient procedure with a low miscleavage rate when used for a biological sample aliquot, but quantitation was less reproducible. A prior protein precipitation step improved the quantitation by FASP due to significantly higher peak areas for plasma and a much better reproducibility for milk. Moreover, the miscleavage rate for milk, the identification rate for plasma, and the carbamidomethylation efficiency were improved. In contrast, ISD of both milk and plasma resulted in higher miscleavage rates and is therefore less suitable for targeted proteomics.Sanja Milkovska-StamenovaMichele WölkRalf HoffmannMDPI AGarticlebottom-up proteomicsfilter-aided sample preparation (FASP)Folch extractionin-solution digestion (ISD)milkplasmaOrganic chemistryQD241-441ENMolecules, Vol 26, Iss 6816, p 6816 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	bottom-up proteomics filter-aided sample preparation (FASP) Folch extraction in-solution digestion (ISD) milk plasma Organic chemistry QD241-441
spellingShingle	bottom-up proteomics filter-aided sample preparation (FASP) Folch extraction in-solution digestion (ISD) milk plasma Organic chemistry QD241-441 Sanja Milkovska-Stamenova Michele Wölk Ralf Hoffmann Evaluation of Sample Preparation Strategies for Human Milk and Plasma Proteomics
description	Sample preparation is the most critical step in proteomics as it directly affects the subset of proteins and peptides that can be reliably identified and quantified. Although a variety of efficient and reproducible sample preparation strategies have been developed, their applicability and efficacy depends much on the biological sample. Here, three approaches were evaluated for the human milk and plasma proteomes. Protein extracts were digested either in an ultrafiltration unit (filter-aided sample preparation, FASP) or in-solution (ISD). ISD samples were desalted by solid-phase extraction prior to nRPC-ESI-MS/MS. Additionally, milk and plasma samples were directly digested by FASP without prior protein precipitation. Each strategy provided inherent advantages and disadvantages for milk and plasma. FASP appeared to be the most time efficient procedure with a low miscleavage rate when used for a biological sample aliquot, but quantitation was less reproducible. A prior protein precipitation step improved the quantitation by FASP due to significantly higher peak areas for plasma and a much better reproducibility for milk. Moreover, the miscleavage rate for milk, the identification rate for plasma, and the carbamidomethylation efficiency were improved. In contrast, ISD of both milk and plasma resulted in higher miscleavage rates and is therefore less suitable for targeted proteomics.
format	article
author	Sanja Milkovska-Stamenova Michele Wölk Ralf Hoffmann
author_facet	Sanja Milkovska-Stamenova Michele Wölk Ralf Hoffmann
author_sort	Sanja Milkovska-Stamenova
title	Evaluation of Sample Preparation Strategies for Human Milk and Plasma Proteomics
title_short	Evaluation of Sample Preparation Strategies for Human Milk and Plasma Proteomics
title_full	Evaluation of Sample Preparation Strategies for Human Milk and Plasma Proteomics
title_fullStr	Evaluation of Sample Preparation Strategies for Human Milk and Plasma Proteomics
title_full_unstemmed	Evaluation of Sample Preparation Strategies for Human Milk and Plasma Proteomics
title_sort	evaluation of sample preparation strategies for human milk and plasma proteomics
publisher	MDPI AG
publishDate	2021
url	https://doaj.org/article/91bd13b5336b47eb9f603943a88e26aa
work_keys_str_mv	AT sanjamilkovskastamenova evaluationofsamplepreparationstrategiesforhumanmilkandplasmaproteomics AT michelewolk evaluationofsamplepreparationstrategiesforhumanmilkandplasmaproteomics AT ralfhoffmann evaluationofsamplepreparationstrategiesforhumanmilkandplasmaproteomics
_version_	1718411145775153152

Evaluation of Sample Preparation Strategies for Human Milk and Plasma Proteomics

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