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Selective Enrichment of Histidine Phosphorylated Peptides Using Molecularly Imprinted Polymers
Malmö universitet, Fakulteten för hälsa och samhälle (HS), Institutionen för biomedicinsk vetenskap (BMV).ORCID-id: 0000-0003-1723-9803
University of Southern Denmark, Odense.
Malmö universitet, Fakulteten för hälsa och samhälle (HS), Institutionen för biomedicinsk vetenskap (BMV).
Malmö universitet, Fakulteten för hälsa och samhälle (HS), Institutionen för biomedicinsk vetenskap (BMV).
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2021 (Engelska)Ingår i: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 93, nr 8, s. 3857-3866Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

Protein histidine phosphorylation (pHis) is involved in molecular signaling networks in bacteria, fungi, plants, and higher eukaryotes including mammals and is implicated in human diseases such as cancer. Detailed investigations of the pHis modification are hampered due to its acid-labile nature and consequent lack of tools to study this post-translational modification (PTM). We here demonstrate three molecularly imprinted polymer (MIP)-based reagents, MIP1-MIP3, for enrichment of pHis peptides and subsequent characterization by chromatography and mass spectrometry (LC-MS). The combination of MIP1 and β-elimination provided some selectivity for improved detection of pHis peptides. MIP2 was amenable to larger pHis peptides, although with poor selectivity. Microsphere-based MIP3 exhibited improved selectivity and was amenable to enrichment and detection by LC-MS of pHis peptides in tryptic digests of protein mixtures. These MIP protocols do not involve any acidic solvents during sample preparation and enrichment, thus preserving the pHis modification. The presented proof-of-concept results will lead to new protocols for highly selective enrichment of labile protein phosphorylations using molecularly imprinted materials.

Ort, förlag, år, upplaga, sidor
American Chemical Society (ACS), 2021. Vol. 93, nr 8, s. 3857-3866
Nationell ämneskategori
Analytisk kemi
Identifikatorer
URN: urn:nbn:se:mau:diva-41178DOI: 10.1021/acs.analchem.0c04474ISI: 000626269400026PubMedID: 33591162Scopus ID: 2-s2.0-85101877466OAI: oai:DiVA.org:mau-41178DiVA, id: diva2:1536361
Tillgänglig från: 2021-03-10 Skapad: 2021-03-10 Senast uppdaterad: 2024-02-05Bibliografiskt granskad
Ingår i avhandling
1. Amino acid sequence and side chain specific synthetic receptors targeting protein phosphorylation
Öppna denna publikation i ny flik eller fönster >>Amino acid sequence and side chain specific synthetic receptors targeting protein phosphorylation
2021 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
Abstract [en]

Antibodies have become a critical component of many diagnostic assays and are used for therapeutic purposes. Nevertheless they often fail to meet the strict performance demands raised in industry and in the clinic (e.g. stability, reproducibility, selectivity, affinity). These issues are especially notable for assays targeting post translational modifications (PTM) of proteins (phosphorylation, glycosylation, sulfation etc.). Antibody-based technologies suffer from problems of a more general nature associated with the analytical use of biological receptors i.e.: i) limited stability requiring cold chain logistics, ii) high production costs, iii) batch to batch variability. The above emphasizes the need for alternative robust, reproducible and low cost “binders” and assays. The aim in this thesis is to design, develop and test molecularly imprinted polymers (MIPs) which were synthesized epitope and stoichiometric imprinting approaches targeting phosphorylation as a PTM. Protein phosphorylationis one of the most common PTM, which is based on covalent attachment of phosphate group to particular amino acids. Misregulation of phosphorylation process is found related with diseases such as cancer, diabetes, and neurodegeneration. MIPs are synthesized through copolymerization of functional monomers and crosslinkers in the presence of N- and C- terminal protected templates. The key recognition element employed in developed synthetic receptors was 1,3-diaryl urea functionalmonomer 1. This monomer is a potent hydrogen bond donor forming strong cyclichydrogen bonds with oxyanions. Amino acid sequence specific and side chain imprinted binders were prepared targeting phosphorylation on tyrosine (pTyr) and on histidine (pHis). pHis MIP-based approach is proposed as a solution to enrich pHis peptides in the presence of other phosphoesters such as phosphoserine (pSer) in complex mixture without pre-treatment like β-elimination. In pTyr, ZAP-70 (zeta associated 70 kDa protein), which is prognosticator for chronic lymphocytic leukemia (CLL), and pTyr-sequence specific motif Src-SH2 domain were chosen as targets to evaluate regio- or stoichiometric selectivity performance of imprinted polymers. The synthesized polymers are used as effective enrichment tools for target phosphorylated peptides from complex mixture prior to mass spectrometry. Overall, the results demonstrate unique proteomics enrichment tools that link with personalized medicine relying on diagnostic coupled cancer treatment strategies based on kinase inhibitors.

Ort, förlag, år, upplaga, sidor
Malmö: Malmö universitet, 2021. s. 85
Serie
Malmö University Health and Society Dissertations, ISSN 1653-5383 ; 2021:5
Nyckelord
imprinted polymers, synthetic receptors, PTM, phosphorylation, enrichment, diagnosis
Nationell ämneskategori
Biomedicinsk laboratorievetenskap/teknologi
Identifikatorer
urn:nbn:se:mau:diva-46001 (URN)10.24834/isbn.9789178772087 (DOI)9789178772070 (ISBN)9789178772087 (ISBN)
Disputation
2021-10-07, Aulan, Fakulteten Hälsa & samhälle samt livestreamat, Jan Waldenströms g. 25, Malmö, 10:00 (Engelska)
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Anmärkning

Paper III and V in dissertation as manuscript

Tillgänglig från: 2021-09-23 Skapad: 2021-09-23 Senast uppdaterad: 2024-03-01Bibliografiskt granskad

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