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Optimization of immunoaffinity enrichment and detection: toward a comprehensive characterization of the phosphotyrosine proteome of K562 cells by liquid chromatography-mass spectrometry
Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för fysikalisk och analytisk kemi, Analytisk kemi.
Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för immunologi, genetik och patologi.
Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för immunologi, genetik och patologi, Genomik. (Forskargrupp Pettersson)
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2011 (Engelska)Ingår i: The Analyst, ISSN 0003-2654, E-ISSN 1364-5528, Vol. 136, nr 9, s. 1971-1978Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

Phosphorylation of protein tyrosine residues regulates many cell functions and has also been proved to be involved in oncogenesis. Thus, the identification of the phosphotyrosine (pTyr) proteome of cells is a very important task. Since tyrosine phosphorylation represents only around 1% of the total human phosphoproteome, the study of pTyr proteins is rather challenging. Here we report the optimization study of the phosphotyrosine proteome using K562 cells as a model system. A substantial segment of the phosphotyrosine proteome of K562 cells was characterized by immunoaffinity enrichment with 4G10 and PYKD1 antibodies followed by LC-MS/MS analysis. 480 non-redundant pTyr peptides corresponding to 342 pTyr proteins were found. 141 pTyr peptides were not described elsewhere. The mass spectrometry approach involving high-resolving FTMS analysis of precursor ions and subsequent detection of CID fragments in a linear ion trap was considered as optimal. For detection of low abundant pTyr peptides pooling of individual immunoaffinity enrichments for one LC-MS/MS analysis was crucial. The enrichment properties of the monoclonal PYKD1 antibody were presented for the first time, also in comparison to the 4G10 antibody. PYKD1 was found to be more effective for protein enrichment (1.2 and 5% efficiency at peptide and protein level correspondingly), while 4G10 showed better results when peptide enrichment was performed (15% efficiency versus 3.6% at protein level). Substantially different subsets of the phosphoproteome were enriched by these antibodies. This finding together with previous studies demonstrates that comprehensive pTyr proteome characterization by immunoprecipitation requires multiple antibodies to be used for the affinity enrichment.

Ort, förlag, år, upplaga, sidor
2011. Vol. 136, nr 9, s. 1971-1978
Nationell ämneskategori
Analytisk kemi Medicinsk genetik
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URN: urn:nbn:se:uu:diva-151712DOI: 10.1039/c0an00649aISI: 000289365100028PubMedID: 21403953OAI: oai:DiVA.org:uu-151712DiVA, id: diva2:410996
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VetenskapsrådetTillgänglig från: 2011-04-15 Skapad: 2011-04-15 Senast uppdaterad: 2018-01-12Bibliografiskt granskad

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Lind, Sara BergströmPettersson, Ulf

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