Dataset Information

Chemical acetylation of ligands and two-step digestion protocol for reducing co-digestion in affinity purification-mass spectrometry

ABSTRACT: We present an effective, fast and user-friendly method to reduce co-digestion of bead-bound ligands, such as antibodies or streptavidin, in affinity purification-mass spectrometry experiments. A short pre-incubation of beads with Sulfo-NHS-Acetate leads to chemical acetylation of lysine residues, making ligands insusceptible to Lys-C-mediated proteolysis. In contrast to similar approaches, our procedure offers the advantage of exclusively using non-toxic chemicals and employing mild chemical reaction conditions. After binding of bait proteins to Sulfo-NHS-Acetate treated beads, we employ a two-step digestion protocol with the sequential use of Lys-C protease for on-bead digestion followed by in-solution digestion of the released proteins with trypsin. The implementation of this protocol results in a strong reduction of contaminating ligand peptides, which allows significantly higher amounts of sample to be subjected to LC-MS analysis, improving sensitivity and quantitative accuracy.

INSTRUMENT(S): Q Exactive HF-X

ORGANISM(S): Homo Sapiens (human) Saccharomyces Cerevisiae (baker's Yeast)

SUBMITTER: Wolfgang Reiter

LAB HEAD: Markus Hartl

PROVIDER: PXD043709 | Pride | 2023-10-23

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	20170707_QexHF2_RSLC2_Hollenstein_Hartl_UW_MFPL_GFP_01mM_1.raw	Raw
	20170707_QexHF2_RSLC2_Hollenstein_Hartl_UW_MFPL_GFP_01mM_2.raw	Raw
	20170707_QexHF2_RSLC2_Hollenstein_Hartl_UW_MFPL_GFP_05mM_1.raw	Raw
	20170707_QexHF2_RSLC2_Hollenstein_Hartl_UW_MFPL_GFP_05mM_2.raw	Raw
	20170707_QexHF2_RSLC2_Hollenstein_Hartl_UW_MFPL_GFP_0mM_1.raw	Raw

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Publications

[Evaluation of the scientific activity through bibliometric indices].

Bordons M M Zulueta M A MA

Revista espanola de cardiologia 19991001 10

The scope of bibliometric studies is the treatment and quantitative analysis of scientific publications. They belong to the so-called "social studies of science", and science policy constitutes one of its main applied fields. These studies efficiently complement the opinions and judgements of experts, thus providing objective and useful tools for evaluating the results of scientific activity. Nevertheless, given the impact that these evaluations have on the assignment of funding for research and ...[more]

PMID: 10563155

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