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  • Bergström Lind, SaraUppsala universitet,Institutionen för genetik och patologi,Pettersson (author)

Immunoaffinity Enrichments Followed by Mass Spectrometric Detection for Studying Global Protein Tyrosine Phosphorylation

  • Article/chapterEnglish2008

Publisher, publication year, extent ...

  • 2008-06-11
  • American Chemical Society (ACS),2008
  • printrdacarrier

Numbers

  • LIBRIS-ID:oai:DiVA.org:uu-87323
  • https://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-87323URI
  • https://doi.org/10.1021/pr8000546DOI

Supplementary language notes

  • Language:English
  • Summary in:English

Part of subdatabase

Classification

  • Subject category:ref swepub-contenttype
  • Subject category:art swepub-publicationtype

Notes

  • Phosphorylation of protein tyrosine residues regulates important cell functions and is, when dysregulated, often crucially involved in oncogenesis. It is therefore important to develop and evaluate methods for identifying and studying tyrosine phosphorylated (P-Tyr) proteins. P-Tyr proteins are present at very low concentrations within cells, requiring highly selective enrichment methods to be detected. In this study, we applied immunoaffinity as enrichment step for P-Tyr proteins. Five selected anti-phosphotyrosine antibodies (monoclonal antibodies 4G10, PY100, PYKD1, 13F9 and one polyclonal antiserum) were evaluated with respect to their capability to enrich P-Tyr proteins from cell extracts of the K562 leukemia cell line. The enrichment resulted in the detection of a group of proteins that potentially were tyrosine-phosphorylated (putative P-Tyr proteins). High accuracy identification of actual P-Tyr sites were performed using a highly selective and sensitive liquid chromatography Fourier transform mass spectrometer (LC-FTMS) setup with complementary collision activated dissociation (CAD) and electron capture dissociation (ECD) fragmentations. 4G10 and PY100 antibodies recognized the greatest number of putative P-Tyr proteins in initial screening experiments and were therefore further evaluated and compared in immunoaffinity enrichment of both P-Tyr proteins and peptides. Using the 4G10 antibody for enrichment of proteins, we identified 459 putative P-Tyr proteins by MS. Out of these proteins, 12 were directly verified as P-Tyr proteins by MS analysis of the actual site. Using the PY100 antibody for enrichment of peptides, we detected 67 P-Tyr peptides (sites) and 89 putative P-Tyr proteins. Generally, enrichment at the peptide level made it difficult to reliably determine the identity of the proteins. In contrast, protein identification following immunoaffinity enrichment at the protein level gave greater sequence coverage and thus a higher confidence in the protein identification. By combining all available information, 40 proteins were identified as true P-Tyr proteins from the K562 cell line. In conclusion, this study showed that a combination of immunoaffinity enrichment using multiple antibodies of both intact and digested proteins in parallel experiments is required for best possible coverage of all possible P-Tyr proteins in a sample.

Subject headings and genre

  • Tyrosine phosphorylation
  • anti-phosphotyrosine antibodies
  • immunoaffinity enrichment
  • mass spectrometric characterization
  • MEDICINE
  • MEDICIN

Added entries (persons, corporate bodies, meetings, titles ...)

  • Molin, Magnus (author)
  • Savitski, Mikhail M.Uppsala universitet,Institutionen för cell- och molekylärbiologi (author)
  • Emilsson, LinaUppsala universitet,Institutionen för genetik och patologi(Swepub:uu)linaemil (author)
  • Åström, JonasUppsala BIO (author)
  • Hedberg, LudwigUppsala universitet,Institutionen för genetik och patologi (author)
  • Adams, ChrisUppsala universitet,Institutionen för cell- och molekylärbiologi (author)
  • Nielsen, Michael (author)
  • Engström, ÅkeUppsala universitet,Institutionen för medicinsk biokemi och mikrobiologi(Swepub:uu)akeengs (author)
  • Elfineh, LioudmilaUppsala universitet,Institutionen för genetik och patologi (author)
  • Andersson, EvaUppsala universitet,Institutionen för medicinsk biokemi och mikrobiologi(Swepub:uu)ean20137 (author)
  • Zubarev, RomanUppsala universitet,Institutionen för cell- och molekylärbiologi(Swepub:uu)rozub839 (author)
  • Pettersson, UlfUppsala universitet,Institutionen för genetik och patologi(Swepub:uu)ulfpette (author)
  • Uppsala universitetInstitutionen för genetik och patologi (creator_code:org_t)

Related titles

  • In:Journal of Proteome Research: American Chemical Society (ACS)7:7, s. 2897-29101535-38931535-3907

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