Strategies for the identification of arginine ADP-ribosylation sites.
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Strategies for the identification of arginine ADP-ribosylation sites. / Laing, Sabrina; Koch Nolte, Friedrich; Haag, Friedrich; Buck, Friedrich.
In: J PROTEOMICS, Vol. 75, No. 1, 1, 2011, p. 169-176.Research output: SCORING: Contribution to journal › SCORING: Journal article › Research › peer-review
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TY - JOUR
T1 - Strategies for the identification of arginine ADP-ribosylation sites.
AU - Laing, Sabrina
AU - Koch Nolte, Friedrich
AU - Haag, Friedrich
AU - Buck, Friedrich
PY - 2011
Y1 - 2011
N2 - Mono-ADP-ribosylation of arginine is a protein modification in eukaryotic cells regulating protein activity and thereby influencing signal transduction and metabolism. Due to the complexity of the modification and the fragmentation pattern in MS/MS CID experiments, the identification of ADP-ribosylation sites in complex mixtures is difficult. Here we describe a two-step strategy, in the first step enriching and identifying potentially ADP-ribosylated proteins and in the second step identifying the sites of modification by a combination of LC/MS-, LC/MS(E) (MS at elevated fragmentation energy)- and LC/MS/MS experiments. Using this technique we could identify two ADP-ribosylation sites in TNF? digested with trypsin, protease V8 and both proteases and thereby demonstrate the specific ADP-ribosylation of TNF?. In complex samples the detection of ADP-ribosylated peptides requires further enrichment of the modified peptides. We tested various materials routinely used for the isolation of phosphopeptides. IMAC as well as TiO(2) chromatography were successfully applied for the selective enrichment of ADP-ribosylated model peptides.
AB - Mono-ADP-ribosylation of arginine is a protein modification in eukaryotic cells regulating protein activity and thereby influencing signal transduction and metabolism. Due to the complexity of the modification and the fragmentation pattern in MS/MS CID experiments, the identification of ADP-ribosylation sites in complex mixtures is difficult. Here we describe a two-step strategy, in the first step enriching and identifying potentially ADP-ribosylated proteins and in the second step identifying the sites of modification by a combination of LC/MS-, LC/MS(E) (MS at elevated fragmentation energy)- and LC/MS/MS experiments. Using this technique we could identify two ADP-ribosylation sites in TNF? digested with trypsin, protease V8 and both proteases and thereby demonstrate the specific ADP-ribosylation of TNF?. In complex samples the detection of ADP-ribosylated peptides requires further enrichment of the modified peptides. We tested various materials routinely used for the isolation of phosphopeptides. IMAC as well as TiO(2) chromatography were successfully applied for the selective enrichment of ADP-ribosylated model peptides.
KW - Animals
KW - Humans
KW - Mice
KW - Amino Acid Sequence
KW - Molecular Sequence Data
KW - Tumor Necrosis Factor-alpha/metabolism
KW - Mass Spectrometry/methods
KW - Titanium/chemistry
KW - Chromatography, Liquid/methods
KW - ADP Ribose Transferases/metabolism
KW - Adenosine Diphosphate Ribose/analysis/chemistry/metabolism
KW - Arginine/analysis/chemistry/metabolism
KW - Phosphopeptides/chemistry/isolation & purification/metabolism
KW - Trypsin/metabolism
KW - alpha-Defensins/metabolism
KW - Animals
KW - Humans
KW - Mice
KW - Amino Acid Sequence
KW - Molecular Sequence Data
KW - Tumor Necrosis Factor-alpha/metabolism
KW - Mass Spectrometry/methods
KW - Titanium/chemistry
KW - Chromatography, Liquid/methods
KW - ADP Ribose Transferases/metabolism
KW - Adenosine Diphosphate Ribose/analysis/chemistry/metabolism
KW - Arginine/analysis/chemistry/metabolism
KW - Phosphopeptides/chemistry/isolation & purification/metabolism
KW - Trypsin/metabolism
KW - alpha-Defensins/metabolism
M3 - SCORING: Journal article
VL - 75
SP - 169
EP - 176
JO - J PROTEOMICS
JF - J PROTEOMICS
SN - 1874-3919
IS - 1
M1 - 1
ER -
