Improving the Phosphoproteome Coverage for Limited Sample Amounts Using TiO2-SIMAC-HILIC (TiSH) Phosphopeptide Enrichment and Fractionation

Kasper Engholm-Keller, Martin R Larsen

Publikation: Bidrag til bog/antologi/rapport/konference-proceedingBidrag til bog/antologiForskningpeer review

Resumé

Obtaining high phosphoproteome coverage requires specific enrichment of phosphorylated peptides from the often extremely complex peptide mixtures generated by proteolytic digestion of biological samples, as well as extensive chromatographic fractionation prior to liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Due to the sample loss resulting from fractionation, this procedure is mainly performed when large quantities of sample are available. To make large-scale phosphoproteomics applicable to smaller amounts of protein we have recently combined highly specific TiO2-based phosphopeptide enrichment with sequential elution from immobilized metal affinity chromatography (SIMAC) for fractionation of mono- and multi-phosphorylated peptides prior to capillary scale hydrophilic interaction liquid chromatography (HILIC) based fractionation of monophosphorylated peptides. In the following protocol we describe the procedure step by step to allow for comprehensive coverage of the phosphoproteome utilizing only a few hundred micrograms of protein.

OriginalsprogEngelsk
TitelPhospho-Proteomics : Methods and Protocols
RedaktørerLouise von Stechow
Vol/bindPart III
ForlagSpringer
Publikationsdato2016
Udgave2.
Sider161-177
Kapitel11
ISBN (Trykt)978-1-4939-3048-7
ISBN (Elektronisk)978-1-4939-3049-4
DOI
StatusUdgivet - 2016
NavnMethods in Molecular Biology
Vol/bind1335
ISSN1064-3745

Fingeraftryk

Phosphopeptides
Affinity Chromatography
Liquid Chromatography
Metals
Peptides
Tandem Mass Spectrometry
Complex Mixtures
Proteins

Citer dette

Engholm-Keller, K., & Larsen, M. R. (2016). Improving the Phosphoproteome Coverage for Limited Sample Amounts Using TiO2-SIMAC-HILIC (TiSH) Phosphopeptide Enrichment and Fractionation. I L. V. Stechow (red.), Phospho-Proteomics : Methods and Protocols (2. udg., Bind Part III, s. 161-177). Springer. Methods in Molecular Biology, Bind. 1335 https://doi.org/10.1007/978-1-4939-3049-4_11
Engholm-Keller, Kasper ; Larsen, Martin R. / Improving the Phosphoproteome Coverage for Limited Sample Amounts Using TiO2-SIMAC-HILIC (TiSH) Phosphopeptide Enrichment and Fractionation. Phospho-Proteomics : Methods and Protocols. red. / Louise von Stechow. Bind Part III 2. udg. Springer, 2016. s. 161-177 (Methods in Molecular Biology, Bind 1335).
@inbook{921cac68c6314bbc8739cc0b19485a3e,
title = "Improving the Phosphoproteome Coverage for Limited Sample Amounts Using TiO2-SIMAC-HILIC (TiSH) Phosphopeptide Enrichment and Fractionation",
abstract = "Obtaining high phosphoproteome coverage requires specific enrichment of phosphorylated peptides from the often extremely complex peptide mixtures generated by proteolytic digestion of biological samples, as well as extensive chromatographic fractionation prior to liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Due to the sample loss resulting from fractionation, this procedure is mainly performed when large quantities of sample are available. To make large-scale phosphoproteomics applicable to smaller amounts of protein we have recently combined highly specific TiO2-based phosphopeptide enrichment with sequential elution from immobilized metal affinity chromatography (SIMAC) for fractionation of mono- and multi-phosphorylated peptides prior to capillary scale hydrophilic interaction liquid chromatography (HILIC) based fractionation of monophosphorylated peptides. In the following protocol we describe the procedure step by step to allow for comprehensive coverage of the phosphoproteome utilizing only a few hundred micrograms of protein.",
author = "Kasper Engholm-Keller and Larsen, {Martin R}",
year = "2016",
doi = "10.1007/978-1-4939-3049-4_11",
language = "English",
isbn = "978-1-4939-3048-7",
volume = "Part III",
series = "Methods in Molecular Biology",
publisher = "Springer",
pages = "161--177",
editor = "Stechow, {Louise von}",
booktitle = "Phospho-Proteomics",
address = "Germany",
edition = "2.",

}

Engholm-Keller, K & Larsen, MR 2016, Improving the Phosphoproteome Coverage for Limited Sample Amounts Using TiO2-SIMAC-HILIC (TiSH) Phosphopeptide Enrichment and Fractionation. i LV Stechow (red.), Phospho-Proteomics : Methods and Protocols. 2. udg, bind Part III, Springer, Methods in Molecular Biology, bind 1335, s. 161-177. https://doi.org/10.1007/978-1-4939-3049-4_11

Improving the Phosphoproteome Coverage for Limited Sample Amounts Using TiO2-SIMAC-HILIC (TiSH) Phosphopeptide Enrichment and Fractionation. / Engholm-Keller, Kasper; Larsen, Martin R.

Phospho-Proteomics : Methods and Protocols. red. / Louise von Stechow. Bind Part III 2. udg. Springer, 2016. s. 161-177 (Methods in Molecular Biology, Bind 1335).

Publikation: Bidrag til bog/antologi/rapport/konference-proceedingBidrag til bog/antologiForskningpeer review

TY - CHAP

T1 - Improving the Phosphoproteome Coverage for Limited Sample Amounts Using TiO2-SIMAC-HILIC (TiSH) Phosphopeptide Enrichment and Fractionation

AU - Engholm-Keller, Kasper

AU - Larsen, Martin R

PY - 2016

Y1 - 2016

N2 - Obtaining high phosphoproteome coverage requires specific enrichment of phosphorylated peptides from the often extremely complex peptide mixtures generated by proteolytic digestion of biological samples, as well as extensive chromatographic fractionation prior to liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Due to the sample loss resulting from fractionation, this procedure is mainly performed when large quantities of sample are available. To make large-scale phosphoproteomics applicable to smaller amounts of protein we have recently combined highly specific TiO2-based phosphopeptide enrichment with sequential elution from immobilized metal affinity chromatography (SIMAC) for fractionation of mono- and multi-phosphorylated peptides prior to capillary scale hydrophilic interaction liquid chromatography (HILIC) based fractionation of monophosphorylated peptides. In the following protocol we describe the procedure step by step to allow for comprehensive coverage of the phosphoproteome utilizing only a few hundred micrograms of protein.

AB - Obtaining high phosphoproteome coverage requires specific enrichment of phosphorylated peptides from the often extremely complex peptide mixtures generated by proteolytic digestion of biological samples, as well as extensive chromatographic fractionation prior to liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Due to the sample loss resulting from fractionation, this procedure is mainly performed when large quantities of sample are available. To make large-scale phosphoproteomics applicable to smaller amounts of protein we have recently combined highly specific TiO2-based phosphopeptide enrichment with sequential elution from immobilized metal affinity chromatography (SIMAC) for fractionation of mono- and multi-phosphorylated peptides prior to capillary scale hydrophilic interaction liquid chromatography (HILIC) based fractionation of monophosphorylated peptides. In the following protocol we describe the procedure step by step to allow for comprehensive coverage of the phosphoproteome utilizing only a few hundred micrograms of protein.

U2 - 10.1007/978-1-4939-3049-4_11

DO - 10.1007/978-1-4939-3049-4_11

M3 - Book chapter

C2 - 26584925

SN - 978-1-4939-3048-7

VL - Part III

T3 - Methods in Molecular Biology

SP - 161

EP - 177

BT - Phospho-Proteomics

A2 - Stechow, Louise von

PB - Springer

ER -

Engholm-Keller K, Larsen MR. Improving the Phosphoproteome Coverage for Limited Sample Amounts Using TiO2-SIMAC-HILIC (TiSH) Phosphopeptide Enrichment and Fractionation. I Stechow LV, red., Phospho-Proteomics : Methods and Protocols. 2. udg. Bind Part III. Springer. 2016. s. 161-177. (Methods in Molecular Biology, Bind 1335). https://doi.org/10.1007/978-1-4939-3049-4_11