Development of affinity columns for the removal of high-abundance proteins in cerebrospinal fluid
2009 (English)In: Biotechnology and applied biochemistry, ISSN 0885-4513, E-ISSN 1470-8744, Vol. 52, no 2, 159-166 p.Article in journal (Refereed) Published
Various approaches for removal of high-abundance components in body fluids are currently available. While most methods are constructed for plasma depletion, there is a need for body-fluid-specific strategies. The aim of the present study was to design an affinity matrix suitable for the depletion of high-abundance proteins in CSIF (cerebrospinal fluid). Hence, molecules with specific affinity towards proteins present at high concentration in CSIF were desired. Affibody(R) molecules are specific binders of small size that have shown high stability under various conditions and are therefore good candidates for such a matrix. The protein composition in CSF resembles that in plasma. However, 20 % of the proteins are brain-derived and are therefore present in higher proportions in CSF than in plasma, whereas larger plasma-derived proteins are less abundant in CSF. Therefore five high-abundance CSIF proteins were chosen for the design of a CSF-specific depletion setup. Affibody(R) molecules with specificity towards HSA (human serum albumin), IgG, transferrin and transthyretin were combined in an affinity column. In addition, polyclonal antibodies against cystatin C were coupled to chromatographic beads and packed in a separate column. Highly reproducible and efficient removal of the five target proteins was observed. The proportion of depleted proteins were estimated to be 99, 95, 74, 92 and 83 % for HSA, IgG, transferrin, transthyretin and cystatin C respectively. SDS/PAGE analysis was used for monitoring and identifying proteins in native CSF, depleted CSIF samples and the captured fractions. Moreover, shotgun proteomics was used for protein identification in native as well as depleted: CSIF and the achieved data were compared. Enhanced identification of lower abundance components was observed in the depleted fraction, in terms of more detected peptides per protein.
Place, publisher, year, edition, pages
2009. Vol. 52, no 2, 159-166 p.
Affibody (R) molecule; affinity resin; biomarker; cerebrospinal fluid (CSF); MS; proteomics
IdentifiersURN: urn:nbn:se:kth:diva-8126DOI: 10.1042/BA20080028ISI: 000263202500008ScopusID: 2-s2.0-60549084220OAI: oai:DiVA.org:kth-8126DiVA: diva2:13364
QC 20100728. Uppdaterad från manuskript till artikel (20100728). Tidigare titel: Development of affinity columns for the removal of high-abundant proteins in cerebrospinal fluid2008-03-192008-03-192010-07-28Bibliographically approved