Using guanidine-hydrochloride for fast and efficient protein digestion and single-step affinity-purification mass spectrometry
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Using guanidine-hydrochloride for fast and efficient protein digestion and single-step affinity-purification mass spectrometry. / Poulsen, Jon Wriedt; Madsen, Christian Toft; Young, Clifford; Poulsen, Flemming Martin; Nielsen, Michael L.
I: Journal of Proteome Research, Bind 12, Nr. 2, 2013, s. 1020-30.Publikation: Bidrag til tidsskrift › Tidsskriftartikel › Forskning › fagfællebedømt
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TY - JOUR
T1 - Using guanidine-hydrochloride for fast and efficient protein digestion and single-step affinity-purification mass spectrometry
AU - Poulsen, Jon Wriedt
AU - Madsen, Christian Toft
AU - Young, Clifford
AU - Poulsen, Flemming Martin
AU - Nielsen, Michael L
PY - 2013
Y1 - 2013
N2 - Protein digestion is an integral part of the "shotgun" proteomics approach and commonly requires overnight incubation prior to mass spectrometry analysis. Quadruplicate "shotgun" proteomic analysis of whole yeast lysate demonstrated that Guanidine-Hydrochloride (Gnd-HCl) protein digestion can be optimally completed within 30 min with endoprotease Lys-C. No chemical artifacts were introduced when samples were incubated in Gnd-HCl at 95 °C, making Gnd-HCl an appropriate digestion buffer for shotgun proteomics. Current methodologies for investigating protein-protein interactions (PPIs) often require several preparation steps, which prolongs any parallel operation and high-throughput interaction analysis. Gnd-HCl allow the efficient elution and subsequent fast digestion of PPIs to provide a convenient high-throughput methodology for affinity-purification mass spectrometry (AP-MS) experiments. To validate the Gnd-HCl approach, label-free PPI analysis of several GFP-tagged yeast deubiquitinating enzymes was performed. The identification of known interaction partners demonstrates the utility of the optimized Gnd-HCl protocol that is also scalable to the 96 well-plate format.
AB - Protein digestion is an integral part of the "shotgun" proteomics approach and commonly requires overnight incubation prior to mass spectrometry analysis. Quadruplicate "shotgun" proteomic analysis of whole yeast lysate demonstrated that Guanidine-Hydrochloride (Gnd-HCl) protein digestion can be optimally completed within 30 min with endoprotease Lys-C. No chemical artifacts were introduced when samples were incubated in Gnd-HCl at 95 °C, making Gnd-HCl an appropriate digestion buffer for shotgun proteomics. Current methodologies for investigating protein-protein interactions (PPIs) often require several preparation steps, which prolongs any parallel operation and high-throughput interaction analysis. Gnd-HCl allow the efficient elution and subsequent fast digestion of PPIs to provide a convenient high-throughput methodology for affinity-purification mass spectrometry (AP-MS) experiments. To validate the Gnd-HCl approach, label-free PPI analysis of several GFP-tagged yeast deubiquitinating enzymes was performed. The identification of known interaction partners demonstrates the utility of the optimized Gnd-HCl protocol that is also scalable to the 96 well-plate format.
U2 - 10.1021/pr300883y
DO - 10.1021/pr300883y
M3 - Journal article
C2 - 23186134
VL - 12
SP - 1020
EP - 1030
JO - Journal of Proteome Research
JF - Journal of Proteome Research
SN - 1535-3893
IS - 2
ER -
ID: 46282673