As an initial analysis, aliquots of the eluted fractions were separated by SDS-PAGE and the gel was stained with Deep Purple Total Protein Stain and scanned directly in the Ettan™ DIGE Imager using excitation and emission wavelengths specific for Cy5 and Deep Purple, respectively.
The analysis of three elution fractions from six replicates originating from two different multiwell plates is shown in Figure 2. The majority of the enriched hTf was eluted in the first fraction with target protein recoveries typically above 50%, with an enrichment factor of more than 100 relative to the starting material. This procedure proved to be highly reproducible with relative standard deviations well below 10% for both target protein recovery and specific purity.
For comparison, standard reversed-phase LC-MS analyses were performed using both start material and hTf-enriched material. Start material was diluted with the elution buffer to a suitable protein concentration and thereafter treated the same way as the enriched sample.
Tricarboxyethyl-phosphine was added to reduce the disulfide bonds of the proteins and cysteines were alkylated with iodoacetamide. After alkylation, the proteins were cleaved into smaller fragments by addition of stabilized porcine trypsin and incubated overnight at room temperature. 10 µL of each sample was injected on a C18 enrichment column and desalted on-line using the Ettan MDLC chromatography system.
Bound peptides were then separated on an analytical C18 reversed-phase column (0.075 x 150 mm) with a gradient from 0 to 67% (v/v) acetonitrile in 0.1% formic acid and water during 60 minutes at a flow rate of 200 nL/minute. The effluent was sprayed into the nanoflow electrospray source of an ion trap mass spectrometer. An automatic data-dependent scan method was used to acquire MS and MS/MS spectra.
An automated protein database search completed the identification of the peptide fragments as well as the overall identification of proteins. Human and E.coli databases were used in the search, allowing for the two modifications—oxidized methionines and carboxyamido methylated cysteines.