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suffi cient diversity is given to bind potentially all proteins within
the sample. As the amount of each specifi c hexapeptide is limited
in a given amount of bead volume, high-abundant proteins satu-
rating the beads will be reduced, whereas low-abundance proteins
will be quantitatively trapped. Although the exact binding mechanism
of complex samples to a hexapeptide library is not entirely under-
stood ( 6 ), this technology provides a powerful tool to get access to
the low-abundant protein fraction. Metaphorically speaking, this
approach eases the problem of fi nding a needle in a haystack by
removing most of the hay (high-abundant proteins) and attracting
the needle (low-abundant protein) at the same time. In addition,
this sample preparation method fulfi lls two important prerequisites
for quantitative proteomics: it is highly reproducible ( 7 ), and the
proportionality of the initial sample composition concerning the
low-abundant proteins is preserved during the fractionation
procedure ( 8 ). Therefore, this sample preparation approach can be
combined in an ideal setting with 2D DIGE to allow valid and
sensitive differential profi ling of low-abundance proteins.
2. Materials
2.1. Serum Samples
Human blood samples were provided from healthy volunteers.
The study was conducted in accordance with ethical guidelines of
the Heinrich-Heine-University of Duesseldorf. Sample collection
was exclusively performed in 2.7-mL S-Monovettes ® (Sarstedt,
Nuembrecht, Germany) according to a standard operation proce-
dure (see Note 1).
1.
Prepacked spin-columns fi lled with hexapeptide-coupled beads
for different starting protein amounts as well as bulk beads for
customized applications are available under the commercial
trademark ProteoMiner™ (Bio-Rad Laboratories, Hercules,
CA, USA). In this study, prepacked columns containing 500
2.2. Hexapeptide
Library Sample
Preparation
L
of bead slurry (20% beads, 20% (v/v) aqueous ethanol, 0.05%
(w/v) sodium azide) were used (see Notes 2 and 3).
μ
2.
Deionized water.
3.
Wash buffer: 150 mM NaCl, 10 mM NaH 2 PO 4 , pH 7.4.
4.
2D DIGE lysis buffer: 25 mM Tris-base, 4% (w/v) CHAPS,
7 M urea, and 2 M thiourea supplemented with 1% (v/v) phos-
phatase inhibitor 1 and 2 (Sigma Aldrich, Taufkirchen,
Germany) and 1 tablet (per 50 mL) Complete™ protease
inhibitor cocktail (Roche, Mannheim, Germany). Store at
−20°C (see Note 4).
5.
Advanced Protein Assay (Cytoskeleton, Distributor: TeBu-bio,
Offenbach, Germany).
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