A proteome-scale study on in vivo protein N(α)-acetylation using an optimized method.

Xumin Zhang, Kasper Engholm-Keller, Peter Højrup

Research output: Contribution to journalJournal articleResearchpeer-review

Abstract

Protein N-terminal acetylation (N(α)-acetylation) is among the most common modifications in eukaryotes. We previously described a simple method to enrich N(α)-modified peptides using CNBr-activated Sepharose resin. A limitation of this method is that an optimal ratio of sample to resin had to be determined prior to the analysis since Lys-containing N(α)-modified peptides may be lost. To address this problem, we hereby present an optimized method by the introduction of double incubation at pH 6.0. We demonstrate with the optimized method that the N(α)-modified peptides can be enriched regardless of whether ε-NH(2) is present or not, and the sample to resin ratio optimization is no longer necessary. Another improvement was accomplished by the inclusion of the singly charged precursor for MS/MS fragmentation to alleviate the shortcoming of the reduced charge state of N(α)-modified peptides. We employed a duplicate experiment using 80 μg samples each and identified 922 IPI annotated and 103 IPI unannotated acetylated N-termini from 989 proteins, so far the largest acetylated N-termini data set acquired from a tryptic digest. Furthermore, the reproducibility of the N(α)-acetyl proteome approach was evaluated and its complementarity to the regular proteome approach was analyzed. The unexpected coupling of CNBr-activated Sepharose to His-containing peptides via the imidazole group was discovered.

Original languageDanish
JournalProteomics
Volume11
Pages (from-to)81-93
Number of pages14
ISSN1615-9853
Publication statusPublished - 2011

Cite this

@article{444a4a6efcb340358fab302ad70a6424,
title = "A proteome-scale study on in vivo protein N(α)-acetylation using an optimized method.",
abstract = "Protein N-terminal acetylation (N(α)-acetylation) is among the most common modifications in eukaryotes. We previously described a simple method to enrich N(α)-modified peptides using CNBr-activated Sepharose resin. A limitation of this method is that an optimal ratio of sample to resin had to be determined prior to the analysis since Lys-containing N(α)-modified peptides may be lost. To address this problem, we hereby present an optimized method by the introduction of double incubation at pH 6.0. We demonstrate with the optimized method that the N(α)-modified peptides can be enriched regardless of whether ε-NH(2) is present or not, and the sample to resin ratio optimization is no longer necessary. Another improvement was accomplished by the inclusion of the singly charged precursor for MS/MS fragmentation to alleviate the shortcoming of the reduced charge state of N(α)-modified peptides. We employed a duplicate experiment using 80 μg samples each and identified 922 IPI annotated and 103 IPI unannotated acetylated N-termini from 989 proteins, so far the largest acetylated N-termini data set acquired from a tryptic digest. Furthermore, the reproducibility of the N(α)-acetyl proteome approach was evaluated and its complementarity to the regular proteome approach was analyzed. The unexpected coupling of CNBr-activated Sepharose to His-containing peptides via the imidazole group was discovered.",
author = "Xumin Zhang and Kasper Engholm-Keller and Peter H{\o}jrup",
year = "2011",
language = "Dansk",
volume = "11",
pages = "81--93",
journal = "Proteomics",
issn = "1615-9853",
publisher = "Wiley - V C H Verlag GmbH & Co. KGaA",

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A proteome-scale study on in vivo protein N(α)-acetylation using an optimized method. / Zhang, Xumin; Engholm-Keller, Kasper; Højrup, Peter.

In: Proteomics, Vol. 11, 2011, p. 81-93.

Research output: Contribution to journalJournal articleResearchpeer-review

TY - JOUR

T1 - A proteome-scale study on in vivo protein N(α)-acetylation using an optimized method.

AU - Zhang, Xumin

AU - Engholm-Keller, Kasper

AU - Højrup, Peter

PY - 2011

Y1 - 2011

N2 - Protein N-terminal acetylation (N(α)-acetylation) is among the most common modifications in eukaryotes. We previously described a simple method to enrich N(α)-modified peptides using CNBr-activated Sepharose resin. A limitation of this method is that an optimal ratio of sample to resin had to be determined prior to the analysis since Lys-containing N(α)-modified peptides may be lost. To address this problem, we hereby present an optimized method by the introduction of double incubation at pH 6.0. We demonstrate with the optimized method that the N(α)-modified peptides can be enriched regardless of whether ε-NH(2) is present or not, and the sample to resin ratio optimization is no longer necessary. Another improvement was accomplished by the inclusion of the singly charged precursor for MS/MS fragmentation to alleviate the shortcoming of the reduced charge state of N(α)-modified peptides. We employed a duplicate experiment using 80 μg samples each and identified 922 IPI annotated and 103 IPI unannotated acetylated N-termini from 989 proteins, so far the largest acetylated N-termini data set acquired from a tryptic digest. Furthermore, the reproducibility of the N(α)-acetyl proteome approach was evaluated and its complementarity to the regular proteome approach was analyzed. The unexpected coupling of CNBr-activated Sepharose to His-containing peptides via the imidazole group was discovered.

AB - Protein N-terminal acetylation (N(α)-acetylation) is among the most common modifications in eukaryotes. We previously described a simple method to enrich N(α)-modified peptides using CNBr-activated Sepharose resin. A limitation of this method is that an optimal ratio of sample to resin had to be determined prior to the analysis since Lys-containing N(α)-modified peptides may be lost. To address this problem, we hereby present an optimized method by the introduction of double incubation at pH 6.0. We demonstrate with the optimized method that the N(α)-modified peptides can be enriched regardless of whether ε-NH(2) is present or not, and the sample to resin ratio optimization is no longer necessary. Another improvement was accomplished by the inclusion of the singly charged precursor for MS/MS fragmentation to alleviate the shortcoming of the reduced charge state of N(α)-modified peptides. We employed a duplicate experiment using 80 μg samples each and identified 922 IPI annotated and 103 IPI unannotated acetylated N-termini from 989 proteins, so far the largest acetylated N-termini data set acquired from a tryptic digest. Furthermore, the reproducibility of the N(α)-acetyl proteome approach was evaluated and its complementarity to the regular proteome approach was analyzed. The unexpected coupling of CNBr-activated Sepharose to His-containing peptides via the imidazole group was discovered.

M3 - Tidsskriftartikel

VL - 11

SP - 81

EP - 93

JO - Proteomics

JF - Proteomics

SN - 1615-9853

ER -