Identification of disease biomarkers has significantly increased interest in studying the plasma proteome. The plasma proteome has a large, dynamic range of individual protein concentrations, which span approximately 10 orders of magnitude, making it difficult to identify disease biomarkers that are present in low concentrations.
Therefore, it is important to employ effective sample-preparation procedures to unmask and identify low copy-number proteins of interest. Thus, the processing of human plasma and serum frequently includes the depletion of high abundant proteins such as albumin and IgG in combination with other fractionation techniques.
Conventional depletion and fractionation methods often require large amounts of sample and tend to be expensive. Additionally, they are labor intensive so processing the large number of samples required to gain confidence in the detected differences is difficult.
Despite these challenges, the important role of sample complexity reduction in proteomics is evidenced by the utilization of a wide array of products and methods. To address the challenges associated with traditional depletion strategies, we developed a method to reduce sample complexity while minimizing sample load and resin volume yet increasing flexibility. This method can be used for processing samples in high-throughput or single-sample modes.
Fractionation of complex samples on ion-exchange resins in a 96-well plate can be a useful approach for small amounts of sample and higher sample numbers. A complete depletion and fractionation experiment can be designed in either a spin device or 96-well plate with a low protein-binding membrane affixed at the bottom. Advantages of using multiwell plates for fractionation include time savings, conservation of sample, and the option of exploring several binding and elution conditions for optimal results in downstream applications.
The present strategy involves ligand-specific depletion of human albumin and IgG, followed by ion-exchange (IEX) fractionation using stepwise pH elution (Figure 1). Pall’s Enchant™ Multi-Protein Affinity Separation Kit is used for depletion, and Pall’s ion-exchange chromatography resins are used for the fractionation. The resins are then loaded into either a Nanosep® centrifugal device or AcroPrep™ filter plate with low protein-binding membrane.
Experiments show that strong anion-exchange resin such as Q HyperD® 20 resin is more efficient in the fractionation of depleted human plasma than other IEX chemistries. Depletion alone dramatically improves spot detection on 2-D gel electrophoresis and when combined with Q resin fractionation, the result is three to four protein fractions having distinct protein populations. Thus, not only are more proteins visualized, but the analysis of these fractions will maximize the analytical information gained from small volumes of human plasma.
Improved protein detection and better overall gel quality was observed after the removal of albumin and IgG. Abundant protein depletion also improves the spot detection in 2-D gel electrophoresis.
Batch-mode fractionation of depleted human plasma using strong anion-exchange resin shows that most proteins in depleted plasma are retained at pH 8.5 (Figure 2A). Most of the retained proteins are eluted in three step-wise fractions at pH 5.0, 4.5, and 4.0.
These fractions have a significant number of proteins that are unique to each fraction. The protein recovery data shows ~77% of the loaded protein is recovered, with ~70% in four eluate fractions (pH 5.0, 4.5, 4.0, and 3.5) and ~7% in the flow-through fraction. The pH 5.0, 4.5, and 4.0 eluates have the highest protein concentration and show similar protein complexity.
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.