TY - JOUR
T1 - En bloc elution of proteomes from combinatorial peptide ligand libraries
AU - Farinazzo, Alessia
AU - Fasoli, Elisa
AU - Kravchuk, Alexander V.
AU - Candiano, Giovanni
AU - Aldini, Giancarlo
AU - Regazzoni, Luca
AU - Righetti, Pier Giorgio
PY - 2009/5/2
Y1 - 2009/5/2
N2 - In contrast to the three to four sequential elution steps routinely adopted for recovering proteomes adsorbed onto combinatorial peptide ligand libraries, we report here two en bloc elution systems, which are able to achieve recoveries in the order of 95% in a single step. One consists of TUC (7 M urea, 2 M thiourea, 3% CHAPS) added with 40 mM formic acid, the other of TUC added with 25 mM cysteic acid (Cys-A). Although both systems are almost equally performing, the formic acid eluant has as a drawback, namely the potential to modify proteins by formylation of Ser and Thr residues. On the contrary, the Cys-A system is unreactive towards proteins. Additionally, Cys-A, due to its very low pI value (1.80) does not interfere with subsequent 2D map analyses since, during the first isoelectric focusing step (in general performed in immobilized pH gradients), it migrates to the anodic compartment and thus vacates the gel. Conversely, formic acid would mostly collect around pH 3 and acetic or citric acid, formerly used in the UCA (9 M urea, 50 mM citric acid) eluant, would condense around pH 4 in the focusing step, interfering thus with 2D map analyses. Elution by boiling SDS of the small amount of protein left over after three sequential elution steps in TUC and 25 mM Cys-A and analysis by nanoLC-MS/MS has demonstrated that these residual proteins are indeed a residue left over from proteins already eluted in TUC-Cys-A and not new species absent in the latter eluate.
AB - In contrast to the three to four sequential elution steps routinely adopted for recovering proteomes adsorbed onto combinatorial peptide ligand libraries, we report here two en bloc elution systems, which are able to achieve recoveries in the order of 95% in a single step. One consists of TUC (7 M urea, 2 M thiourea, 3% CHAPS) added with 40 mM formic acid, the other of TUC added with 25 mM cysteic acid (Cys-A). Although both systems are almost equally performing, the formic acid eluant has as a drawback, namely the potential to modify proteins by formylation of Ser and Thr residues. On the contrary, the Cys-A system is unreactive towards proteins. Additionally, Cys-A, due to its very low pI value (1.80) does not interfere with subsequent 2D map analyses since, during the first isoelectric focusing step (in general performed in immobilized pH gradients), it migrates to the anodic compartment and thus vacates the gel. Conversely, formic acid would mostly collect around pH 3 and acetic or citric acid, formerly used in the UCA (9 M urea, 50 mM citric acid) eluant, would condense around pH 4 in the focusing step, interfering thus with 2D map analyses. Elution by boiling SDS of the small amount of protein left over after three sequential elution steps in TUC and 25 mM Cys-A and analysis by nanoLC-MS/MS has demonstrated that these residual proteins are indeed a residue left over from proteins already eluted in TUC-Cys-A and not new species absent in the latter eluate.
KW - Cysteic acid elution
KW - Formic acid elution
KW - Hexapeptide ligands
KW - Low-abundance proteome
KW - Peptide libraries
UR - http://www.scopus.com/inward/record.url?scp=63649143880&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=63649143880&partnerID=8YFLogxK
U2 - 10.1016/j.jprot.2009.02.009
DO - 10.1016/j.jprot.2009.02.009
M3 - Article
C2 - 19269355
AN - SCOPUS:63649143880
VL - 72
SP - 725
EP - 730
JO - Journal of Proteomics
JF - Journal of Proteomics
SN - 1874-3919
IS - 4
ER -