Phosphopeptide enrichment by immobilized metal affinity chromatography

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Resumé

Immobilized metal affinity chromatography (IMAC) has been the method of choice for phosphopeptide enrichment prior to mass spectrometric analysis for many years and it is still used extensively in many laboratories. Using the affinity of negatively charged phosphate groups towards positively charged metal ions such as Fe3+, Ga3+, Al3+, Zr4+, and Ti4+ has made it possible to enrich phosphorylated peptides from peptide samples. However, the selectivity of most of the metal ions is limited, when working with highly complex samples, e.g., whole-cell extracts, resulting in contamination from nonspecific binding of nonphosphorylated peptides. This problem is mainly caused by highly acidic peptides that also share high binding affinity towards these metal ions. By lowering the pH of the loading buffer nonspecific binding can be reduced significantly, however with the risk of reducing specific binding capacity. After binding, the enriched phosphopeptides are released from the metal ions using alkaline buffers of pH 10–11, EDTA, or phosphate-containing buffers. Here we describe a protocol for IMAC using Fe 3+ for phosphopeptide enrichment. The principles are illustrated on a semi-complex peptide mixture.

OriginalsprogEngelsk
TitelPhospho-Proteomics : Methods and Protocols
RedaktørerLouise von Stechow
ForlagHumana Press
Publikationsdato2016
Udgave2.
Sider123-133
Kapitel8
ISBN (Trykt)978-1-4939-3048-7
ISBN (Elektronisk)978-1-4939-3049-4
DOI
StatusUdgivet - 2016
NavnMethods in Molecular Biology
Vol/bind1355
ISSN1064-3745

Fingeraftryk

Phosphopeptides
Affinity Chromatography
Metals
Peptides
Ions
Cell Extracts
Complex Mixtures
Edetic Acid

Citer dette

Thingholm, T. E., & Larsen, M. R. (2016). Phosphopeptide enrichment by immobilized metal affinity chromatography. I L. von Stechow (red.), Phospho-Proteomics: Methods and Protocols (2. udg., s. 123-133). Humana Press. Methods in Molecular Biology, Bind. 1355 https://doi.org/10.1007/978-1-4939-3049-4_8
Thingholm, Tine E. ; Larsen, Martin R. / Phosphopeptide enrichment by immobilized metal affinity chromatography. Phospho-Proteomics: Methods and Protocols. red. / Louise von Stechow. 2. udg. Humana Press, 2016. s. 123-133 (Methods in Molecular Biology, Bind 1355).
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Thingholm, TE & Larsen, MR 2016, Phosphopeptide enrichment by immobilized metal affinity chromatography. i L von Stechow (red.), Phospho-Proteomics: Methods and Protocols. 2. udg, Humana Press, Methods in Molecular Biology, bind 1355, s. 123-133. https://doi.org/10.1007/978-1-4939-3049-4_8

Phosphopeptide enrichment by immobilized metal affinity chromatography. / Thingholm, Tine E.; Larsen, Martin R.

Phospho-Proteomics: Methods and Protocols. red. / Louise von Stechow. 2. udg. Humana Press, 2016. s. 123-133 (Methods in Molecular Biology, Bind 1355).

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

TY - CHAP

T1 - Phosphopeptide enrichment by immobilized metal affinity chromatography

AU - Thingholm, Tine E.

AU - Larsen, Martin R.

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N2 - Immobilized metal affinity chromatography (IMAC) has been the method of choice for phosphopeptide enrichment prior to mass spectrometric analysis for many years and it is still used extensively in many laboratories. Using the affinity of negatively charged phosphate groups towards positively charged metal ions such as Fe3+, Ga3+, Al3+, Zr4+, and Ti4+ has made it possible to enrich phosphorylated peptides from peptide samples. However, the selectivity of most of the metal ions is limited, when working with highly complex samples, e.g., whole-cell extracts, resulting in contamination from nonspecific binding of nonphosphorylated peptides. This problem is mainly caused by highly acidic peptides that also share high binding affinity towards these metal ions. By lowering the pH of the loading buffer nonspecific binding can be reduced significantly, however with the risk of reducing specific binding capacity. After binding, the enriched phosphopeptides are released from the metal ions using alkaline buffers of pH 10–11, EDTA, or phosphate-containing buffers. Here we describe a protocol for IMAC using Fe 3+ for phosphopeptide enrichment. The principles are illustrated on a semi-complex peptide mixture.

AB - Immobilized metal affinity chromatography (IMAC) has been the method of choice for phosphopeptide enrichment prior to mass spectrometric analysis for many years and it is still used extensively in many laboratories. Using the affinity of negatively charged phosphate groups towards positively charged metal ions such as Fe3+, Ga3+, Al3+, Zr4+, and Ti4+ has made it possible to enrich phosphorylated peptides from peptide samples. However, the selectivity of most of the metal ions is limited, when working with highly complex samples, e.g., whole-cell extracts, resulting in contamination from nonspecific binding of nonphosphorylated peptides. This problem is mainly caused by highly acidic peptides that also share high binding affinity towards these metal ions. By lowering the pH of the loading buffer nonspecific binding can be reduced significantly, however with the risk of reducing specific binding capacity. After binding, the enriched phosphopeptides are released from the metal ions using alkaline buffers of pH 10–11, EDTA, or phosphate-containing buffers. Here we describe a protocol for IMAC using Fe 3+ for phosphopeptide enrichment. The principles are illustrated on a semi-complex peptide mixture.

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Thingholm TE, Larsen MR. Phosphopeptide enrichment by immobilized metal affinity chromatography. I von Stechow L, red., Phospho-Proteomics: Methods and Protocols. 2. udg. Humana Press. 2016. s. 123-133. (Methods in Molecular Biology, Bind 1355). https://doi.org/10.1007/978-1-4939-3049-4_8