Performance of combinatorial peptide libraries in capturing the low-abundance proteome of red blood cells. 1. Behavior of mono- to hexapeptides.
For a better understanding of the behavior of solid-phase combinatorial peptide ligands for capturing the red blood cell low-abundance soluble proteome, combinatorial peptides of different lengths from a single amino acid up to a hexapeptide were evaluated. A red blood cell lysate (6 g total protein) was loaded in a cascade fashion to the six columns, which were individually eluted with 8 M urea, 2% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (v/w), and 50 mM citric acid. Each eluate was analyzed via sodium dodecyl sulfate polyacrylamide gel electrophoresis, two-dimensional maps, and nanoLC-MS/MS.


RESULTS
mixed beads with a single amino acid attached showed the capture of a non-negligible portion of the proteome. A progressive increasing of the length of the peptide bait enlarges the pool of captured proteins. Above a length of four amino acids, a plateau is progressively reached, suggesting that not much could be gained with baits longer than six amino acids. Interestingly, whereas the beads laden with a single amino acid seem to be able to capture large-size proteins (>40 kDa), beads with progressively longer peptides capture additional proteins in the smaller size range (10-50 kDa). This suggests that interactions already begin with a single amino acid, but selectivity requires baits of proper length, at least above four amino acids. Plain beads, with a spacer arm carrying a primary amino terminal group for anchoring the baits, are essentially unable to capture proteins, suggesting that the peptide baits do not act by a mechanism of ion exchange but rather via a complex mixed mode, yielding a specific capture.
Simó
Carolina
C
Department of Chemistry, Materials and Chemical Engineering Giulio Natta, Polytechnic of Milano, 20131 Milano, Italy.
Bachi
Angela
A
Cattaneo
Angela
A
Guerrier
Luc
L
Fortis
Frederic
F
Boschetti
Egisto
E
Podtelejnikov
Alexander
A
Righetti
Pier Giorgio
PG
D001798:Blood Proteins / Q000737:chemistry / Q000302:isolation & purification; D002853:Chromatography, Liquid / Q000379:methods; D020650:Combinatorial Chemistry Techniques; D004591:Electrophoresis, Polyacrylamide Gel; D004912:Erythrocytes / Q000737:chemistry*; D009842:Oligopeptides / Q000737:chemistry*; D019151:Peptide Library; D020543:Proteome; D012680:Sensitivity and Specificity; D053719:Tandem Mass Spectrometry
D001798:Blood Proteins; D009842:Oligopeptides; D019151:Peptide Library; D020543:Proteome