A peptide affinity column for the identification of integrin alpha IIb-binding proteins.
To understand the regulation of integrin alpha(IIb)beta(3), a critical platelet adhesion molecule, we have developed a peptide affinity chromatography method using the known integrin regulatory motif, LAMWKVGFFKR. Using standard Fmoc chemistry, this peptide was synthesized onto a Toyopearl AF-Amino-650 M resin on a 6-aminohexanoic acid (Ahx) linker. Peptide density was controlled by acetylation of 83% of the Ahx amino groups. Four recombinant human proteins (CIB1, PP1, ICln and RN181), previously identified as binding to this integrin regulatory motif, were specifically retained by the column containing the integrin peptide but not by a column presenting an irrelevant peptide. Hemoglobin, creatine kinase, bovine serum albumin, fibrinogen and alpha-tubulin failed to bind under the chosen conditions. Immunodetection methods confirmed the binding of endogenous platelet proteins, including CIB1, PP1, ICln RN181, AUP-1 and beta3-integrin, from a detergent-free platelet lysate. Thus, we describe a reproducible method that facilitates the reliable extraction of specific integrin-binding proteins from complex biological matrices. This methodology may enable the sensitive and specific identification of proteins that interact with linear, membrane-proximal peptide motifs such as the integrin regulatory motif LAMWKVGFFKR.
Daxecker
Heide
H
Molecular and Cellular Therapeutics, Royal College of Surgeons in Ireland, Dublin, Republic of Ireland. hdaxecker@rcsi.ie
Raab
Markus
M
Bernard
Elise
E
Devocelle
Marc
M
Treumann
Achim
A
Moran
Niamh
N
D000595:Amino Acid Sequence; D000818:Animals; D001792:Blood Platelets / Q000737:chemistry; D002352:Carrier Proteins / Q000302:isolation & purification*; D002846:Chromatography, Affinity / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D010455:Peptides / Q000138:chemical synthesis; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000032:analysis*; D054645:Protein Phosphatase 1 / Q000737:chemistry*; D040901:Proteomics / Q000379:methods; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry
D002352:Carrier Proteins; D010455:Peptides; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D011994:Recombinant Proteins; D054645:Protein Phosphatase 1