Combinatorial peptide ligand libraries for urine proteome analysis: investigation of different elution systems.
Proteome treatments with peptide libraries in view of reducing high-abundance proteins and increasing the concentration of rare species involve the adsorption on solid-phase material. Subsequent elution of captured proteins may not be fully effective except when sequences of eluting agents are used. The standard way utilized up to the present has been a three- to four-step, sequential elution system consisting of various agents mixed together such as urea, thiourea, CHAPS, sodium chloride, citric or acetic acid and some polar solvents such as ACN and isopropanol. Elution sequences produce distinct fractions adding to the burden of having to analyze all of them. An alternative, highly effective, single elution to reduce the workload is here reported for the first time, namely elution in boiling 10% SDS added with 3% DTE. This single step elutes almost quantitatively the adsorbed proteins, thus ensuring, for all practical purposes, a full recovery. This high efficiency is believed to be due to the fact that the SDS micelles bury the polypeptide chains within their hydrophobic core, thus shielding them from the surroundings and impeding accidental adsorption to surfaces. Suggestions for selecting the best method to eliminate the excess of SDS for further protein analysis are also evaluated. The merits and limits of this novel system are assessed and discussed.
Candiano
Giovanni
G
Laboratory on Pathophysiology of Uremia and Department of Nephrology, G. Gaslini Children's Hospital, Genoa, Italy.
Dimuccio
Veronica
V
Bruschi
Maurizio
M
Santucci
Laura
L
Gusmano
Rosanna
R
Boschetti
Egisto
E
Righetti
Pier Giorgio
PG
Ghiggeri
Gian Marco
GM
D000327:Adsorption; D000328:Adult; D011232:Chemical Precipitation; D002793:Cholic Acids / Q000737:chemistry; D019343:Citric Acid / Q000737:chemistry; D020650:Combinatorial Chemistry Techniques / Q000379:methods*; D015180:Electrophoresis, Gel, Two-Dimensional; D005260:Female; D019791:Guanidine / Q000737:chemistry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008297:Male; D019151:Peptide Library; D011507:Proteinuria / Q000652:urine*; D020543:Proteome / Q000032:analysis*; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry; D013890:Thiourea / Q000737:chemistry; D014508:Urea / Q000737:chemistry
D002793:Cholic Acids; D019151:Peptide Library; D020543:Proteome; D019343:Citric Acid; D012967:Sodium Dodecyl Sulfate; D014508:Urea; D013890:Thiourea; D019791:Guanidine; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate