Chromatographic and electrophoretic separation methods

Aoa-OH·DCHA by acid extraction. To this purpose, 500 mg Boc-N[Me]-Aoa-OH·DCHA were dissolved in a mixture of 35 mL 0.2 M HCl and 35 mL ethyl acetate and vigorously shaken. The organic phase from 3 extractions was evaporated and the remaining residue (Boc-N[Me]-Aoa-OH) was dissolved in DCM and lyophilized. Boc-N[Me]-Aoa-OH was coupled manually in the same conditions as the other amino acids. The resulting carrier peptide Aoa-GFAKKG-NH2 was cleaved from the resin, dissolved in 60 % aqueous ACN and lyophilized. Lactosylation was performed by incubating 2.5 mmol peptide with 25 mmol lactose in 1.5 mL 0.1 M NaOAc (pH 4.6) for 72 hours at 37 ºC. The unreacted Aoa residues were capped by addition of 25 mmol N-ethylmaleimide and incubation for 18 hours at 37 ºC. The glycopeptide was purified by semipreparative RP-HPLC, immediately neutralized with 10 mM NH4HCO3 to prevent acid degradation and lyophilized.

3.6 Chromatographic and electrophoretic separation methods

3.6.1 Reversed phase high performance liquid chromatography

Purification of synthetic peptides by preparative RP-HPLC was carried out on a Knauer HPLC system (Bad Homburg, Germany), using a 250 x 20 mm, 10 μm preparative C18 column (GROM-SIL 120 ODS-4 SE from Grom, Herrenberg-Kayh, Germany) at a flow rate of 10 mL/min. Semipreparative RP-HPLC of synthetic peptides was performed on an UltiMate 3000 system (Dionex, Germering, Germany) at a flow rate of 2 mL/min, using C8 and C18 Grace-Vydac TP1010 columns (250 x 20 mm, 10 μm). Homogeneity of the purified peptides was confirmed by analytical RP-HPLC performed on an UltiMate 3000 system (Dionex, Germering, Germany) at a flow rate of 1 mL/min., using a 250 × 4 mm, 7 μm analytical C18 column (Macherey-Nagel, Düren, Germany). Chromatograms on all HPLC systems were recorded by UV detection at 220 nm. The same binary solvent system was employed for all separations: 0.1 % aqueous TFA (solvent A) and 80 % ACN, 0.1 % TFA in water (solvent B). The following gradient was used: 0 min 0 % B; 5 min. 0 % B; 65 min. 65

% B, 70 min. 100 % B. Sequence and homogeneity of the purified peptides were confirmed by MALDI-MS and ESI-ion trap MS/MS.

3.6.2 Sample concentration and desalting

Prior to MALDI-MS analysis peptide and protein samples were desalted and concentrated using 10 μL pipette tips with ~0.6 μL C4 or C18 resin packed into the narrow end of the tip. The tips were purchased from Millipore (ZipTip^® pipette tips) or Agilent (OMIX pipette tips). The desalting procedure consisted of five steps: wetting and equilibration of the pipette tip resin, binding of the peptides and/or proteins to the tip resin, removal of salts by washing and finally, elution of bound peptides. The lyophilized sample was dissolved in 20-50 μL aqueous TFA (0.1-0.5 %), to a final pH

~2. The ZipTip^®/OMIX tip, attached to a 10 μL pipette, was conditioned through 2-3 aspiration-dispensing cycles, first with 75 % aqueous ACN (wetting), then with 0.1 % aqueous TFA (equilibration). To bind the analyte, the sample was aspirated and dispensed in the sample tube for 10-20 cycles. The salts trapped in the tip were removed by 2-5 cycles of aspirating and discarding 0.1 % aqueous TFA. The peptides were eluted from the tip with 5 μL 50-90 % ACN in 0.1 % TFA in water.

3.6.3 One-dimensional gel electrophoresis

1D-SDS-PAGE was employed for the analysis of protein fractions from affinity chromatography. A Mini-PROTEAN3 system and a Powerpac 1000 power supply (Bio-Rad, München, Germany) were used for casting and running the gels. The gels were hand cast fresh before each run according to the recipes listed in Table 7, in 10 x 10 x 0.1 cm size. The lyophilized samples were dissolved in 50 mM Tris-HCl, 4

% SDS, 25 % glycerol, 0.02 % bromophenol blue (pH 6.8). The running buffer consisted of 25 mM Tris-HCl, 192 mM glycin and 0.1 % SDS. Gel electrophoresis was first carried out at a constant voltage of 60 V for ~30 min., until the tracking dye (bromophenol blue) entered the separating gel and then at 120 V for ~2 hours, until the tracking dye reached the end of the separating gel.

Table 7. Solutions employed for polyacrylamide gel casting [216].

Solution Stacking gel

5 %

Separating gel 10 %

Separating gel 12 %

4× Stacking gel buffer ^(a) 2.5 mL - -

4×Separating gel buffer ^(b) - 6 mL 6 mL

Deionized water 5.8 mL 10 mL 8.4 mL

Acrylamide solution ^(c) 1.7 mL 8 mL 9.6 mL

APS ^(d) 85 μL 125 μL 125 μL

TEMED ^(e) 20 μL 20 μL 20 μL

(a) 0.5 M Tris-HCl, 0.4 % SDS, ~0.001 % bromophenol blue (pH 6.8) (b) 1.5 M Tris-HCl, 0.4 % SDS (pH 8.8)

(c) 30 % acrylamide and N,N'-Methylenebisacrylamide (37.5:1) aqueous solution (d) 10 % (NH4)2S2O8

(e) N, N, N’, N’-tetramethylethylenediamine

After separation the protein bands were visualized by colloidal Coomassie staining and the stained gels were scanned using a GS-710 Calibrated Imaging Densitometer (Bio-Rad, München, Germany). Gels prepared for subsequent N-terminal sequence analysis of protein spots were electroblotted immediately (without staining) on PVDF membranes.

3.6.4 Colloidal Coomassie staining

Staining of polyacrylamide gels was performed according to Neuhoff [217].

First, the gels were fixed for 1 hour in 12 % TCA. The staining solution, enough for two 10 x 10 x 0.1 cm size gels, was prepared by mixing 200 mL of 10 % ammonium sulfate with 2 % phosphoric acid and 2 mL of 5 % aqueous Coomassie Brilliant Blue G250. The solution was shaken vigorously and 50 mL methanol were added. The fixing solution was then discarded and the gels were incubated with the staining solution overnight, under gentle shaking. The gels were finally washed with 25 % methanol. The methanol solution was refreshed periodically, until the protein bands had a good contrast against the gel background.

For staining of protein bands on PVDF membranes, the membranes were washed after transfer with water for 15 min. and then with methanol for ~10 s.

Incubation with 0.1 % Coomassie Brilliant Blue R-250 in 40 % methanol was then

carried out until the protein bands became visible. The membranes were afterwards washed with 50 % methanol, until the protein bands had a good contrast against the membrane background. Next, the membranes were dried under a stream of nitrogen and the protein bands were excised and put in sample tubes. The bands were destained completely in 75 % methanol, dried and stored at 4 °C.

3.7 Chemical modification and enzymatic fragmentation of