Biology Reference
In-Depth Information
a
b
Sample
Sample
pH 4
pH 7
pH 9
pH 4
Protein desorption
Protein desorption
Eluate blending
Eluate blending
Proteome analysis
Proteome analysis
Fig.
2
Schematic representation of protein capture at different pHs. Parallel (
a
) or
sequential (
b
) protein capture followed by individual desorption and eluate
blending prior to proteomics analysis. The second approach is adapted for small
amount of proteins, but requires an adjustment of pH after each CPLL contact.
For easier understanding the CPLL contact is here represented as chromato-
graphic columns, but the capture phase can be operated in suspension
increase the amount of captured proteins. The pH is also criti-
cal to modulate the protein capture since it acts on the affi nity
constant of proteins for their corresponding peptide baits. To
enlarge the captured protein coverage it is advised to operate at
three different pHs (e.g., 4, 7, and 9) [
39
]. This treatment can
be performed under different confi gurations as illustrated in
Fig.
2
.
4. The excess of proteins is removed from the hexapeptide ligand
library by centrifugation at about 2,000 ×
g
followed by at least
three washings with 500
L of the same buffer used for protein
capture. In between centrifugations, the bead slurry is shaken
gently for 10 min. All along the washing process CPLLs should
not be left completely dry.
μ
3.3 Elution Protocols
While a standard protein elution protocol is generally applied, cus-
tomized elution protocols are possible as described (global elution,
fractionated elution, or direct on-bead protein digestion).
1. Global elution (several options) [
35
].
(a) Beads are added with 200
L of urea-CHAPS-acetic acid
(8 M-4-0.5 %) and gently shaken for about 1 h at room
temperature. Acetic acid could be replaced by citric acid at
similar concentration. In both cases the pH should be
around 3-3.5.
(b) Alternatively CPLLs are added with 6 M guanidine-HCl,
pH 6.0. This strong dissociating solution at relatively high
ionic strength also allows desorption of most of the proteins.
μ
