The binding of avidin to biotin is the strongest known noncovalent biological interaction [dissociation constants (K_d) approximately 10^-15 M] between a protein and ligand (1). The high affinity and specificity of the avidin-biotin interaction is utilized for enriching biotin-containing proteins from mixtures (2,3)- Water-soluble N-hydroxysulfosuccinimide (sulfo-NHS) derivatives of biotin are compatible with biological systems, providing the opportunity to label proteins in biological samples with biotin as a component of purification protocols. Sarbarth et al. (4), for example, used the biotinylation of Helicobacter proteins to isolate and identify the proteins expressed at the cell surface. Immobilized tetrameric avidin was used to capture the biotinylated proteins, and proteins were eluted using 8 M guanidine HC1 at pH 1.5. More recently, immobilized monomeric avidin with a lower binding affinity for biotin (K_d approximately 10^-8 M) has become commercially available. This reagent permits recovery of biotinylated proteins and peptides using milder elution conditions, thus preserving native protein conformation and enzyme activity (5). In experiments to biotinylate proteins using biotin conjugates with different spacer arm lengths, we have observed differential protein recovery when immobilized polymeric or monomeric avidin is used. Our results illustrate the importance of selecting the optimal combination of biotinylation and avidin reagents for the recovery of the proteins of interest in proteomics studies.

To biotinylate the outer membrane proteins of Shewanella oneidensis MR-1, 1 g (wet weight) of cells were harvested and biotinylated as described by Sabarth et al. (4) using sulfo-succinimidyl-6-(biotinamido) hexanoate (EZ-Link® Sulfo-NHS-LC-Biotin; Pierce Chemical, Rockford, IL, USA), sulfo-succinimidyl-6′-(biotinamido)-6-hexanamodo hexanoate (EZ-Link Sulfo-NHS-LC-LC-Biotin; Pierce Chemical) or a 50:50 combination of the two reagents. After biotinylation, cells were sonicated, and the membrane fraction was isolated using differential centrifugation (9000× g for 10 min at 4°C to remove unbroken cells; 40,000× g for 30 min at 4°C to recover the membranes). Membranes were suspended in 500 µL 50 mM Tris-HC1, 150 mM KC1, and 10 mM EDTA, pH 7.4. Biotinylated proteins were released by adding 1 mL 2% Zwittergent® 3-14 (Calbiochem, San Diego, CA, USA) and incubating for 1–2 h at 4°C. Soluble biotinylated proteins were collected in supernatants after ultracentrifugation at 100,000× g for l h at 4°C.

For polyvalent avidin capture of biotinylated proteins, supernatants diluted 1:5 with 100 mM NaPO₄, 150 mM NaCl, pH 7.2 [phosphate-buffered saline (PBS)] were loaded onto Immunopure® AffinityPak™ Immobilized Avidin columns (Pierce Chemical) equilibrated with PBS plus 0.2% Zwittergent. After incubation for 30 min at room temperature, the columns were washed five times with PBS plus 0.2% Zwittergent. Biotinylated proteins were eluted at 37°C with five column volumes of PBS plus 0.2% Zwittergent containing 5 mM D-biotin, followed by five column volumes of 8 M guanidine hydrochloride buffer, pH 1.5, at room temperature. Combined protein eluates were concentrated by acetone precipitation.

Alternatively, biotinylated proteins were captured using Immunopure Immobilized Monomeric Avidin Gel (Pierce Chemical). After blocking the irreversible biotin binding sites, columns were loaded with biotinylated protein samples diluted 1:5 and incubated for 1 h at room temperature. Columns were washed six times with PBS containing 0.2% Zwittergent. Biotinylated proteins were eluted with 10 column volumes of PBS containing 0.2% Zwittergent and 5 mM D-biotin, and combined protein eluates were concentrated by acetone precipitation.

Acetone-precipitated proteins were solubilized in 100 µL 9 M urea, 4% Nonidet™ P40 (NP40), 2% mercaptoethanol, and 2% ampholyte, pH 8.0–10.0 (Bio-Rad Laboratories, Hercules, CA, USA). Five-microliter aliquots were separated by two-dimensional gel electrophoresis (2-DE) (6,7). After 2-DE, proteins were transferred to Hybond®-C membranes (Amersham Biosciences, Piscataway, NJ, USA) by semi-dry electroblotting. The membranes were then incubated for 30 min with 20 mM Tris-HC1, 140 mM NaCl, and 5 mM KC1, pH 7.0, containing 0.05% Tween® 20, followed by incubation with horseradish peroxidase-conjugated neutravidin (1:400 dilution) for 1 h at room temperature. Reactive proteins were detected by chemiluminescence with SuperSignal® West Femto maximum sensitivity substrate (Pierce Chemical). Blots were exposed to X-ray film for 1, 5, and 10 min to generate images that revealed the full range of proteins from high to low abundance. The X-ray film images were digitized using an Eikonix 1412 charge-coupled device scanner interfaced with a VAX 4000–90 workstation (Hewlett-Packard, Loveland, CO, USA).