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A review of COFRADIC techniques targeting protein N-terminal acetylation.

Van Damme P, Van Damme J, Demol H, Staes A, Vandekerckhove J, Gevaert K - BMC Proc (2009)

Bottom Line: Acetylation of nascent protein Nalpha-termini is a common modification among archae and eukaryotes and can influence the structure and function of target proteins.This modification has been studied on an individual protein or (synthetic) peptide level or on a proteome scale using two-dimensional polyacrylamide gel electrophoresis.We recently developed mass spectrometry driven proteome analytical approaches specifically targeting the amino (N) terminus of proteins based on the concept of diagonal reverse-phase chromatography.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Medical Protein Research, VIB, B-9000 Ghent, Belgium. petra.vandamme@ugent.be

ABSTRACT
Acetylation of nascent protein Nalpha-termini is a common modification among archae and eukaryotes and can influence the structure and function of target proteins. This modification has been studied on an individual protein or (synthetic) peptide level or on a proteome scale using two-dimensional polyacrylamide gel electrophoresis. We recently developed mass spectrometry driven proteome analytical approaches specifically targeting the amino (N) terminus of proteins based on the concept of diagonal reverse-phase chromatography. We here review how this so-called combined fractional diagonal chromatography (COFRADIC) technique can be used in combination with differential mass-tagging strategies as to both qualitatively and quantitatively assess protein Nalpha-acetylation in whole proteomes.

No MeSH data available.


Related in: MedlinePlus

A, B and C: Representative MS spectra of N-terminal peptides from partially alpha-N-acetylated proteins. The MS-spectrum from the N-terminal peptide (doubly charged precursor) of the DnaJ homolog subfamily C member 2 (1MLLLPSAADGR11, 44% acetylated) reveals two distinguishable isotopic envelopes of the two isotopomers (i.e. the acetylated (Ac) and trideutero-acetylated forms (AcD3)) (A). Panels B & C show MS-spectra of the N-terminal peptide (triply charged precursor) of the signal recognition particle 68 kDa protein (2AAEKQVPGGGGGGGSGGGGGSGGGGSGGGR31) when trideutero-acetylated (B) or 13C2D3-acetylated (C) (53% acetylated). Panel B illustrates the need for increased mass spacing by heavier isotopomers. D: Comparison of protein N-trideuteroacetylation and N-propionylation. A protein that is partially in vivo α-N-acetylated can be modified in vitro by N-trideutero-acetylation or N-propionylation. When trideutero-acetylation is used, the RP-HPLC elution profiles of the α-N-acetylated and α-N-trideutero-acetylated variants are indistinguishable and the peptide variants only segregate upon MS analysis by their 3 Da mass difference. In contrast, when N-propionylation is used the α-N-acetylated and α-N-propionylated variants segregate upon RP-HPLC, with the propionylated variant eluting at a later time because of increased hydrophobicity. E.: The use of methionine oxidation to segregate methionine-containing from non-methionine-containing N-terminal peptides. Methionine oxidation, when applied in between the primary and secondary RP-HPLC separations and after TNBS modification, causes methionine-sulfoxide containing N-terminal peptides to shift to earlier elution times on RP-HPLC. As a result the N-terminal peptides of NatB (Met-Asn, Met-Asp and Met-Glu) and NatC (Met-Ile, Met-Leu and Met-Phe) substrates, as well as of substrates of yet unidentified NATs (e.g. Met-Lys) migrate out of the primary collection intervals and are in this way enriched from the non-methionine-containing NatA substrates.
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Figure 2: A, B and C: Representative MS spectra of N-terminal peptides from partially alpha-N-acetylated proteins. The MS-spectrum from the N-terminal peptide (doubly charged precursor) of the DnaJ homolog subfamily C member 2 (1MLLLPSAADGR11, 44% acetylated) reveals two distinguishable isotopic envelopes of the two isotopomers (i.e. the acetylated (Ac) and trideutero-acetylated forms (AcD3)) (A). Panels B & C show MS-spectra of the N-terminal peptide (triply charged precursor) of the signal recognition particle 68 kDa protein (2AAEKQVPGGGGGGGSGGGGGSGGGGSGGGR31) when trideutero-acetylated (B) or 13C2D3-acetylated (C) (53% acetylated). Panel B illustrates the need for increased mass spacing by heavier isotopomers. D: Comparison of protein N-trideuteroacetylation and N-propionylation. A protein that is partially in vivo α-N-acetylated can be modified in vitro by N-trideutero-acetylation or N-propionylation. When trideutero-acetylation is used, the RP-HPLC elution profiles of the α-N-acetylated and α-N-trideutero-acetylated variants are indistinguishable and the peptide variants only segregate upon MS analysis by their 3 Da mass difference. In contrast, when N-propionylation is used the α-N-acetylated and α-N-propionylated variants segregate upon RP-HPLC, with the propionylated variant eluting at a later time because of increased hydrophobicity. E.: The use of methionine oxidation to segregate methionine-containing from non-methionine-containing N-terminal peptides. Methionine oxidation, when applied in between the primary and secondary RP-HPLC separations and after TNBS modification, causes methionine-sulfoxide containing N-terminal peptides to shift to earlier elution times on RP-HPLC. As a result the N-terminal peptides of NatB (Met-Asn, Met-Asp and Met-Glu) and NatC (Met-Ile, Met-Leu and Met-Phe) substrates, as well as of substrates of yet unidentified NATs (e.g. Met-Lys) migrate out of the primary collection intervals and are in this way enriched from the non-methionine-containing NatA substrates.

Mentions: The group of Fred Regnier previously reported on the use of trideutero-acetylation of so-called signature peptide N-termini for quantitative proteomics [14]. However, when this reaction is performed at the protein level rather than the peptide level (Figure 1), one can distinguish between in vivo acetylated and their chemically trideutero-acetylated N-terminal counterparts (in vivo free protein N-termini) [11]. Both types of peptides behave extremely similarly during all enrichments steps and only segregate during mass spectrometric analysis. Thus, in MS-spectra very often these two types of N-terminal peptides are easily distinguished (Figure 2A) and the relative amount of each of these can be weighed, which finally provides information on the degree of protein Nα-terminal acetylation [13].


A review of COFRADIC techniques targeting protein N-terminal acetylation.

Van Damme P, Van Damme J, Demol H, Staes A, Vandekerckhove J, Gevaert K - BMC Proc (2009)

A, B and C: Representative MS spectra of N-terminal peptides from partially alpha-N-acetylated proteins. The MS-spectrum from the N-terminal peptide (doubly charged precursor) of the DnaJ homolog subfamily C member 2 (1MLLLPSAADGR11, 44% acetylated) reveals two distinguishable isotopic envelopes of the two isotopomers (i.e. the acetylated (Ac) and trideutero-acetylated forms (AcD3)) (A). Panels B & C show MS-spectra of the N-terminal peptide (triply charged precursor) of the signal recognition particle 68 kDa protein (2AAEKQVPGGGGGGGSGGGGGSGGGGSGGGR31) when trideutero-acetylated (B) or 13C2D3-acetylated (C) (53% acetylated). Panel B illustrates the need for increased mass spacing by heavier isotopomers. D: Comparison of protein N-trideuteroacetylation and N-propionylation. A protein that is partially in vivo α-N-acetylated can be modified in vitro by N-trideutero-acetylation or N-propionylation. When trideutero-acetylation is used, the RP-HPLC elution profiles of the α-N-acetylated and α-N-trideutero-acetylated variants are indistinguishable and the peptide variants only segregate upon MS analysis by their 3 Da mass difference. In contrast, when N-propionylation is used the α-N-acetylated and α-N-propionylated variants segregate upon RP-HPLC, with the propionylated variant eluting at a later time because of increased hydrophobicity. E.: The use of methionine oxidation to segregate methionine-containing from non-methionine-containing N-terminal peptides. Methionine oxidation, when applied in between the primary and secondary RP-HPLC separations and after TNBS modification, causes methionine-sulfoxide containing N-terminal peptides to shift to earlier elution times on RP-HPLC. As a result the N-terminal peptides of NatB (Met-Asn, Met-Asp and Met-Glu) and NatC (Met-Ile, Met-Leu and Met-Phe) substrates, as well as of substrates of yet unidentified NATs (e.g. Met-Lys) migrate out of the primary collection intervals and are in this way enriched from the non-methionine-containing NatA substrates.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 2: A, B and C: Representative MS spectra of N-terminal peptides from partially alpha-N-acetylated proteins. The MS-spectrum from the N-terminal peptide (doubly charged precursor) of the DnaJ homolog subfamily C member 2 (1MLLLPSAADGR11, 44% acetylated) reveals two distinguishable isotopic envelopes of the two isotopomers (i.e. the acetylated (Ac) and trideutero-acetylated forms (AcD3)) (A). Panels B & C show MS-spectra of the N-terminal peptide (triply charged precursor) of the signal recognition particle 68 kDa protein (2AAEKQVPGGGGGGGSGGGGGSGGGGSGGGR31) when trideutero-acetylated (B) or 13C2D3-acetylated (C) (53% acetylated). Panel B illustrates the need for increased mass spacing by heavier isotopomers. D: Comparison of protein N-trideuteroacetylation and N-propionylation. A protein that is partially in vivo α-N-acetylated can be modified in vitro by N-trideutero-acetylation or N-propionylation. When trideutero-acetylation is used, the RP-HPLC elution profiles of the α-N-acetylated and α-N-trideutero-acetylated variants are indistinguishable and the peptide variants only segregate upon MS analysis by their 3 Da mass difference. In contrast, when N-propionylation is used the α-N-acetylated and α-N-propionylated variants segregate upon RP-HPLC, with the propionylated variant eluting at a later time because of increased hydrophobicity. E.: The use of methionine oxidation to segregate methionine-containing from non-methionine-containing N-terminal peptides. Methionine oxidation, when applied in between the primary and secondary RP-HPLC separations and after TNBS modification, causes methionine-sulfoxide containing N-terminal peptides to shift to earlier elution times on RP-HPLC. As a result the N-terminal peptides of NatB (Met-Asn, Met-Asp and Met-Glu) and NatC (Met-Ile, Met-Leu and Met-Phe) substrates, as well as of substrates of yet unidentified NATs (e.g. Met-Lys) migrate out of the primary collection intervals and are in this way enriched from the non-methionine-containing NatA substrates.
Mentions: The group of Fred Regnier previously reported on the use of trideutero-acetylation of so-called signature peptide N-termini for quantitative proteomics [14]. However, when this reaction is performed at the protein level rather than the peptide level (Figure 1), one can distinguish between in vivo acetylated and their chemically trideutero-acetylated N-terminal counterparts (in vivo free protein N-termini) [11]. Both types of peptides behave extremely similarly during all enrichments steps and only segregate during mass spectrometric analysis. Thus, in MS-spectra very often these two types of N-terminal peptides are easily distinguished (Figure 2A) and the relative amount of each of these can be weighed, which finally provides information on the degree of protein Nα-terminal acetylation [13].

Bottom Line: Acetylation of nascent protein Nalpha-termini is a common modification among archae and eukaryotes and can influence the structure and function of target proteins.This modification has been studied on an individual protein or (synthetic) peptide level or on a proteome scale using two-dimensional polyacrylamide gel electrophoresis.We recently developed mass spectrometry driven proteome analytical approaches specifically targeting the amino (N) terminus of proteins based on the concept of diagonal reverse-phase chromatography.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Medical Protein Research, VIB, B-9000 Ghent, Belgium. petra.vandamme@ugent.be

ABSTRACT
Acetylation of nascent protein Nalpha-termini is a common modification among archae and eukaryotes and can influence the structure and function of target proteins. This modification has been studied on an individual protein or (synthetic) peptide level or on a proteome scale using two-dimensional polyacrylamide gel electrophoresis. We recently developed mass spectrometry driven proteome analytical approaches specifically targeting the amino (N) terminus of proteins based on the concept of diagonal reverse-phase chromatography. We here review how this so-called combined fractional diagonal chromatography (COFRADIC) technique can be used in combination with differential mass-tagging strategies as to both qualitatively and quantitatively assess protein Nalpha-acetylation in whole proteomes.

No MeSH data available.


Related in: MedlinePlus