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A micro-flow, high-pH, reversed-phase peptide fractionation and collection system for targeted and in-depth proteomics of low-abundance proteins in limiting samples

We present a method and a simple system for high-pH RP-LC peptide fractionation of small sample amounts (30–60 µg), at micro-flow rates with micro-liter fraction collection using ammonium bicarbonate as an optimized buffer for system stability and robustness. The method is applicable to targeted mas...

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Autores principales: Zurawska, Marta, Basik, Mark, Aguilar-Mahecha, Adriana, Dadlez, Michal, Domanski, Dominik
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Elsevier 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10413349/
https://www.ncbi.nlm.nih.gov/pubmed/37577163
http://dx.doi.org/10.1016/j.mex.2023.102306
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author Zurawska, Marta
Basik, Mark
Aguilar-Mahecha, Adriana
Dadlez, Michal
Domanski, Dominik
author_facet Zurawska, Marta
Basik, Mark
Aguilar-Mahecha, Adriana
Dadlez, Michal
Domanski, Dominik
author_sort Zurawska, Marta
collection PubMed
description We present a method and a simple system for high-pH RP-LC peptide fractionation of small sample amounts (30–60 µg), at micro-flow rates with micro-liter fraction collection using ammonium bicarbonate as an optimized buffer for system stability and robustness. The method is applicable to targeted mass spectrometry approaches and to in-depth proteomic studies where the amount of sample is limited. Using targeted proteomics with peptide standards, we present the method's analytical parameters, and potential in increasing the detection of low-abundance proteins that are difficult to quantify with direct targeted or global LC-MS analyses. This fractionation system increased peptide signals by up to 18-fold, while maintaining high quantitative precision, with high fractionation reproducibility across varied sample sets. In real applications, it increased the detection of targeted endogenous peptides by two-fold in a 25 cell-cycle-control protein panel, and in-depth MS analyses of nuclear extracts, it allowed the detection of up to 8,896 proteins with 138,417 peptides in 24-concatenated fractions compared to 3,344 proteins with 23,093 peptides without fractionation. In a relevant biological problem of CDK4/6-inhibitors and breast cancer, the method reproduced known information and revealed novel insights, highlighting that it can be successfully applied in studies involving low-abundance proteins and limited samples. • Tested nine high-pH buffer/solvent systems to obtain a robust, effective, and reproducible micro-flow fractionation method which was devoid of commonly encountered LC clogging/pressure issues after months of use. • Peptide enrichment method to improve detection and quantitation of low-abundance proteins in targeted and in-depth proteomic studies. • Can be applied to diverse protein samples where the available amount is limited.
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spelling pubmed-104133492023-08-11 A micro-flow, high-pH, reversed-phase peptide fractionation and collection system for targeted and in-depth proteomics of low-abundance proteins in limiting samples Zurawska, Marta Basik, Mark Aguilar-Mahecha, Adriana Dadlez, Michal Domanski, Dominik MethodsX Biochemistry, Genetics and Molecular Biology We present a method and a simple system for high-pH RP-LC peptide fractionation of small sample amounts (30–60 µg), at micro-flow rates with micro-liter fraction collection using ammonium bicarbonate as an optimized buffer for system stability and robustness. The method is applicable to targeted mass spectrometry approaches and to in-depth proteomic studies where the amount of sample is limited. Using targeted proteomics with peptide standards, we present the method's analytical parameters, and potential in increasing the detection of low-abundance proteins that are difficult to quantify with direct targeted or global LC-MS analyses. This fractionation system increased peptide signals by up to 18-fold, while maintaining high quantitative precision, with high fractionation reproducibility across varied sample sets. In real applications, it increased the detection of targeted endogenous peptides by two-fold in a 25 cell-cycle-control protein panel, and in-depth MS analyses of nuclear extracts, it allowed the detection of up to 8,896 proteins with 138,417 peptides in 24-concatenated fractions compared to 3,344 proteins with 23,093 peptides without fractionation. In a relevant biological problem of CDK4/6-inhibitors and breast cancer, the method reproduced known information and revealed novel insights, highlighting that it can be successfully applied in studies involving low-abundance proteins and limited samples. • Tested nine high-pH buffer/solvent systems to obtain a robust, effective, and reproducible micro-flow fractionation method which was devoid of commonly encountered LC clogging/pressure issues after months of use. • Peptide enrichment method to improve detection and quantitation of low-abundance proteins in targeted and in-depth proteomic studies. • Can be applied to diverse protein samples where the available amount is limited. Elsevier 2023-07-31 /pmc/articles/PMC10413349/ /pubmed/37577163 http://dx.doi.org/10.1016/j.mex.2023.102306 Text en © 2023 The Authors. Published by Elsevier B.V. https://creativecommons.org/licenses/by/4.0/This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Biochemistry, Genetics and Molecular Biology
Zurawska, Marta
Basik, Mark
Aguilar-Mahecha, Adriana
Dadlez, Michal
Domanski, Dominik
A micro-flow, high-pH, reversed-phase peptide fractionation and collection system for targeted and in-depth proteomics of low-abundance proteins in limiting samples
title A micro-flow, high-pH, reversed-phase peptide fractionation and collection system for targeted and in-depth proteomics of low-abundance proteins in limiting samples
title_full A micro-flow, high-pH, reversed-phase peptide fractionation and collection system for targeted and in-depth proteomics of low-abundance proteins in limiting samples
title_fullStr A micro-flow, high-pH, reversed-phase peptide fractionation and collection system for targeted and in-depth proteomics of low-abundance proteins in limiting samples
title_full_unstemmed A micro-flow, high-pH, reversed-phase peptide fractionation and collection system for targeted and in-depth proteomics of low-abundance proteins in limiting samples
title_short A micro-flow, high-pH, reversed-phase peptide fractionation and collection system for targeted and in-depth proteomics of low-abundance proteins in limiting samples
title_sort micro-flow, high-ph, reversed-phase peptide fractionation and collection system for targeted and in-depth proteomics of low-abundance proteins in limiting samples
topic Biochemistry, Genetics and Molecular Biology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10413349/
https://www.ncbi.nlm.nih.gov/pubmed/37577163
http://dx.doi.org/10.1016/j.mex.2023.102306
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