En bloc elution of proteomes from combinatorial peptide ligand libraries.
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.
Farinazzo
Alessia
A
Department of Chemistry, Materials and Chemical Engineering Giulio Natta, Politecnico di Milano, Via Mancinelli 7, Milan, Italy.
Fasoli
Elisa
E
Kravchuk
Alexander V
AV
Candiano
Giovanni
G
Aldini
Giancarlo
G
Regazzoni
Luca
L
Righetti
Pier Giorgio
PG
D003544:Cysteic Acid / Q000737:chemistry; D004912:Erythrocytes / Q000378:metabolism; D005561:Formates / Q000737:chemistry; D006801:Humans; D019151:Peptide Library; D010940:Plant Proteins / Q000032:analysis; D020543:Proteome / Q000378:metabolism*; D053719:Tandem Mass Spectrometry / Q000379:methods; D003313:Zea mays / Q000378:metabolism
D005561:Formates; D019151:Peptide Library; D010940:Plant Proteins; D020543:Proteome; C030544:formic acid; D003544:Cysteic Acid