A proteome-scale study on in vivo protein Nα-acetylation using an optimized method

Xumin Zhang, Juanying Ye, 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 languageEnglish
JournalProteomics
Volume11
Issue number1
Pages (from-to)81-93
Number of pages13
ISSN1615-9853
DOIs
Publication statusPublished - 1. Jan 2011

Fingerprint

Acetylation
Proteome
Peptides
Resins
Proteins
Sepharose
Experiments

Keywords

  • Acetylation
  • Proteins
  • Proteome
  • Proteomics
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
  • Tandem Mass Spectrometry

Cite this

Zhang, Xumin ; Ye, Juanying ; Engholm-Keller, Kasper ; Højrup, Peter. / A proteome-scale study on in vivo protein Nα-acetylation using an optimized method. In: Proteomics. 2011 ; Vol. 11, No. 1. pp. 81-93.
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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.",
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A proteome-scale study on in vivo protein Nα-acetylation using an optimized method. / Zhang, Xumin; Ye, Juanying; Engholm-Keller, Kasper; Højrup, Peter.

In: Proteomics, Vol. 11, No. 1, 01.01.2011, p. 81-93.

Research output: Contribution to journalJournal articleResearchpeer-review

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T1 - A proteome-scale study on in vivo protein Nα-acetylation using an optimized method

AU - Zhang, Xumin

AU - Ye, Juanying

AU - Engholm-Keller, Kasper

AU - Højrup, Peter

N1 - Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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KW - Proteome

KW - Proteomics

KW - Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

KW - Tandem Mass Spectrometry

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