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Protein-phosphotyrosine proteome profiling by superbinder-SH2 domain affinity purification mass spectrometry, sSH2-AP-MS
- Source :
- PROTEOMICS. 17:1600360
- Publication Year :
- 2017
- Publisher :
- Wiley, 2017.
-
Abstract
- Recently, "superbinder" SH2 domain variants with three amino acid substitutions (sSH2) were reported to have 100-fold or greater affinity for protein-phosphotyrosine (pY) than natural SH2 domains. Here we report a protocol in which His-tagged Src sSH2 efficiently captures pY-peptides from protease-digested HeLa cell total protein extracts. Affinity purification of pY-peptides by this method shows little bias for pY-proximal amino acid sequences, comparable to that achieved by using antibodies to pY, but with equal or higher yield. Superbinder-SH2 affinity purification mass spectrometry (sSH2-AP-MS) therefore provides an efficient and economical approach for unbiased pY-directed phospho-proteome profiling without the use of antibodies.
- Subjects :
- Proteomics
0301 basic medicine
Proteome
Biology
Mass spectrometry
SH2 domain
Biochemistry
Chromatography, Affinity
Mass Spectrometry
src Homology Domains
03 medical and health sciences
0302 clinical medicine
Affinity chromatography
Humans
Amino Acid Sequence
Phosphotyrosine
Molecular Biology
chemistry.chemical_classification
Tandem affinity purification
Chromatography
Isotope-coded affinity tag
Amino acid
030104 developmental biology
chemistry
Peptides
030217 neurology & neurosurgery
HeLa Cells
Proto-oncogene tyrosine-protein kinase Src
Subjects
Details
- ISSN :
- 16159853
- Volume :
- 17
- Database :
- OpenAIRE
- Journal :
- PROTEOMICS
- Accession number :
- edsair.doi.dedup.....dc6db88a2a4ab192709f3732762b1d7a