Separation of Protein Isoforms from Different Species Using Discovery BIO C5

By: Denise Wallworth, Reporter EU Volume 35

Denise Wallworth

Cytochrome c is a small (ca 12 kDa) mitochondrial protein found in plants, animals and many unicellular organisms, where it is an essential component of the electron transport chain. This protein is present in many closely related variants that can be used as a test for protein selectivity since they are so problematical to separate. Discovery BIO C5 provides an efficient separation that demonstrates the power of this phase for protein and peptide chromatography and the advantages of C5 chemistry over traditional C4 HPLC columns. Figure 1 shows the separation of a series of six types of Cytochrome c on Discovery BIO C5.

Figure 1 Higher Resolution on Discovery BIO C5 Separation of Cytochrome c from Different Sources

Short chain reversed phase wide pore (300 A) columns often provide better separations for many proteins and hydrophobic peptides. The C5 chemistry in Discovery BIO appears to be more stable and have a longer lifetime than C4 phases yet exhibits similar selectivity to traditional C4 phases. Proprietary end capping techniques provide less non-specific interaction between analytes and the silica surface, resulting in better peak shape for these large molecules and in high recoveries. These manufacturing techniques also ensure high reproducibility and good pH stability (2.0 to 8.0): Figure 2 shows the retention time stability for a series of small molecules at pH 8.0 over 14,000 column volumes. A recent study [1] demonstrated a high mean mass recovery of 77%for Discovery BIO C5 for a wide series of different protein types; Discovery BIO C5 also showed the best peak symmetry out of a series of wide pore materials providing higher peak resolution.

Figure 2

Discovery BIO RP HPLC columns are selective, MS compatible and scaleable – all essential for protein and peptide HPLC. In protein and peptide separations, trifluoroacetic acid (TFA) is commonly added to HPLC mobile phases to improve peak shape, but it reduces the LC/ MS signal and so decreases sensitivity. Discovery BIO columns can also provide high efficiency without TFA or other additives for lowest ion suppression. They are also fully compatible with 100 % water.

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  1. E Reh et al, J Chromatogr B 1] , 844 (2006) 204-212

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