Fractionation of depleted human plasma using strong cation-exchange resin (S Ceramic HyperD 20) shows that most plasma proteins bind at pH 4.8 and subsequently elute at pH 6.5, 7.0, and 7.5 (Figure 2B). The resin was slightly overloaded so the flow-through fraction is similar in composition to the depleted plasma. Overall ~73% of total protein loaded is recovered with the bulk in the pH 6.5 and 7.0 fraction.
Fractionation of undepleted human plasma using HyperD 20 strong anion-exchange resin showed that albumin is eluted at pH 5.0 and masks other proteins that migrate near albumin in 1-D gel electrophoresis (data not shown). Prior depletion of the albumin improves detection of other proteins that elute in fractions near the pI of albumin.
The depletion of human albumin and IgG from human plasma results in better ion-exchange fractionation as compared to whole plasma. Thus, the combination of HSA + IgG depletion and Q or S fractionation results in three or four plasma fractions each showing significant reduction in protein complexity.
The effectiveness of this protocol was demonstrated by spiking a low concentration (20 ng/µL) of ovatransferrin into depleted plasma prior to anion fractionation. After fractionation the presumed ovatransferrin band from 1-D SDS-PAGE was subjected to trypsin digestion and MALDI analysis of resultant peptides.
A peptide map search algorithm identified the band as ovatransferrin (data not shown). Therefore, the current strategy can be used for the fractionation and detection of low concentration protein (~1% of total protein). Thus, a combined approach of simple depletion and fractionation results in detection of lower concentration proteins for proteomic analysis.
To achieve improved protein detection from plasma or serum with minimal sample manipulation, a method combining HSA and IgG depletion followed by batch mode ion-exchange fractionation was developed. These depletion and fractionation steps are both simple and cost-effective. All reagents are single-use, reducing the possibility of sample cross-contamination. The use of a minimum number of steps is intended to decrease protein loss and improve reproducibility. Both methods are sufficiently flexible to accommodate a variety of sample volumes and downstream applications.
In addition, the depletion and fractionation schemes can be used in both single-sample and high-throughput processing. This data demonstrates that depletion of the two most abundant proteins from human plasma results in the visualization of a higher number of proteins in 1-D and 2-D gel electrophoresis (data not shown).
Further fractionation of depleted human plasma by ion-exchange resin results in significant complexity reduction in each fraction and thus, the enrichment of medium to low abundant plasma proteins. Optimization of the ratio of resin volume to protein load for this fractionation protocol reveals that a minimum resin volume of 25 µL of HyperD 20 Q or S anion exchange resin can efficiently fractionate ~1 mg of protein sample. This method can be scaled up as needed.