Phosphopeptide Enrichment by Immobilized Metal Affinity Chromatography.

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Phosphopeptide Enrichment by Immobilized Metal Affinity Chromatography. / Thingholm, Tine; Larsen, Martin R.

I: Methods in Molecular Biology, Vol. 1355, 2016, s. 123-133.

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Thingholm, Tine ; Larsen, Martin R. / Phosphopeptide Enrichment by Immobilized Metal Affinity Chromatography. I: Methods in Molecular Biology. 2016 ; Vol. 1355. s. 123-133.

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TY - JOUR

T1 - Phosphopeptide Enrichment by Immobilized Metal Affinity Chromatography.

AU - Thingholm, Tine

AU - Larsen, Martin R

PY - 2016

Y1 - 2016

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 Fe(3+), Ga(3+), Al(3+), Zr(4+), and Ti(4+) 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 non-phosphorylated 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 Fe(3+), Ga(3+), Al(3+), Zr(4+), and Ti(4+) 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 non-phosphorylated 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.

U2 - 10.1007/978-1-4939-3049-4_8

DO - 10.1007/978-1-4939-3049-4_8

M3 - Article

VL - 1355

SP - 123

EP - 133

JO - Methods in Molecular Biology

T2 - Methods in Molecular Biology

JF - Methods in Molecular Biology

SN - 1940-6029

ER -