Ion Exchange for Proteomics

Ion exchange is particularly well suited to the separation of biological molecules; however, limitations of HPLC packing materials have restricted its use. Silica-based ion-exchange materials are pH limited and suffer from nonspecific interactions, although they can have high efficiency. Porous, polymer resin-based materials may be used over a wide range of pH and have a high capacity (dynamic binding capacity ~45 mg/mL); however, they exhibit lower efficiency and have pressure limitations. Nonporous resins with fast mass transfer characteristics are efficient but have extremely low capacity (<5 mg/mL). The desire to analyze large numbers of charged components in proteomic samples has driven the search for improved ion-exchange columns.

Experimental

A nonporous, polymer resin particle was modified to address the needs of ion-exchange separation. First, a 200-Å-thick hydrophilic layer was chemically bonded to the polystyrene/divinylbenzene (PS/DVB) surface to virtually eliminate nonspecific binding with biological molecules. Then, a densely packed, uniform ion-exchange layer was chemically synthesized to the hydrophilic layer. Multiple ion-exchange functional groups were attached to one anchoring point, resulting in a breakthrough in capacity. The four varieties (SAX, WAX, SCX, and WCX) have dynamic binding capacities ranging from ~20 mg/mL to over 50 mg/mL—for example, 20, 38, 53, and 65 mg/mL for Proteomix SCX-NP resins (Sepax Technologies, Newark, DE) with particle sizes of 10, 5, 3, and 1.7 μm, respectively.

Separation efficiency can be described in terms of plate height, H_T in Eq. (1).¹ Efficiency is defined as the standard deviation (σ²) divided by the bed or column length (L), and it is the sum of H_Eddy, H_MT, H_SMT, H_LD, H_SNS, and H_SSP, which are plate heights contributed by eddy diffusion, mobile phase mass transfer, stagnant mobile phase mass transfer, lateral diffusion, stationary phase nonspecific interaction, and stationary phase specific interaction, respectively.

Eliminating the pores minimizes the contribution from stagnant mobile phase mass transfer. The hydrophilic layer makes the nonspecific interaction factor negligible.

Figure 1 - Separation of horse serum (20 μL, 2× diluted) on 300-Å porous SAX column compared to nonporous Proteomix SAX.

Figure 1 compares the separation of horse serum on a traditional 300-Å porous ion-exchange column to that achieved on a nonporous Proteomix SAX. Both are 5-μm, 4.6 × 150 mm columns. Capacities are equivalent, but efficiency and resolution are significantly improved on the nonporous resin.

Figure 2 - Separation of a protein mixture using four Proteomix WCX-NP columns with particle size ranging from 1.7 μm to 10 μm. Separation conditions—Mobile phase A: 20 mM phosphate-buffered saline (PBS), pH 6.5. Mobile phase B: A + 1.0 M NaCl. Gradient: 0–100% B in 20 min. Flow rate: 1.0 mL/min. Pressure: 10 μm, 540 psi; 5 μm, 890 psi; 3 μm, 1600 psi; 1.7 μm, 3100 psi. Detection: UV at 280 nm. Proteins (1.0 mg/mL each): 1) ribonuclease A, 2) Cytochrome C, 3) lysozyme.

Particle size also plays an important role in efficiency and resolution. Decreasing particle size increases efficiency but dramatically increases backpressure. Commercialization of ultrahigh-pressure LC instrumentation enables the use of very small particles.² Traditional resin-based ion-exchange materials have been unable to withstand the additional backpressure. The upper pressure limit for Proteomix nonporous packings is 10,000 psi, which enables their use in UPLC^™ (Ultra Performance LC^™) systems (Waters, Milford, MA) and other high-pressure systems. Figure 2 shows a separation on four different particle sizes of Proteomix WCX-NP from 10 μm to 1.7 μm. Note the resolution of the small peak adjacent to lysozyme.

Figure 3 - Loading capacity test elution of Cytochrome C (20 mg/mL). Separation conditions—Mobile phase A: 20 mM PBS. Mobile phase B: A + 1.0 M NaCl. Gradient: 0–70% B (21 min). Flow rate: 0.35 mL/min, 2800 psi. Detection: UV at 280 nm.

A loading experiment using Cytochrome C, shown in Figure 3, demonstrates a high capacity while maintaining performance for the 1.7-μm nonporous material.

Conclusion

The use of nonporous resins improves mass transfer and allows smaller particle sizes to dramatically improve efficiency in any pH range. Chemical modification to eliminate nonspecific binding and optimize ion-exchange capacity overcomes the limitations of classical nonporous resins. Ion-exchange chromatography using nonporous, specially modified resin-based packings should become a widely used tool for the analysis of large numbers of proteins, low-abundance protein detection, and monitoring of protein post-translational modification, as well as the analysis of other biological molecules. They should also find use in pharmaceutical applications in which small ionic solutes can be difficult to retain by reversed phase.

References

1. Giddings, J.C. United Separation Science; John Wiley & Sons, Inc.: New York, 1991; Chap 12; pp 269–89.
2. Mazzeo, J.R.; Neue, U.D.; Kile, M.; Plumb, R.S. Advancing LC performance with smaller particles and higher pressure. Anal. Chem. A-pages 2005, 77(23), 460A–7A.

Dr. Huang is CTO, and Ms. McKay is Director of Marketing, Sepax Technologies, Delaware Technology Park, 5-100 Innovation Way, Newark, DE 19711, U.S.A.; tel.: 302-366-1101; fax: 302-366-1151; e-mail: [email protected].