This Month's Feature

Separating Proteins by pI-Values - Can 2D LC Replace 2D GE? (continued)

2D LC of Proteins In the second dimension, if compared to 2D GE, the proteins  are intended to be separated by size. Since size exclusion chromatography (SEC) is a method with a very narrow separation window, with little ability to separate multiple components, reversed-phase chromatography is chosen variably to represent a separation according to size. Zhu and colleagues (15) used nonporous 1.5 µm silica-based C18 beads in the second dimension of their off-line 2D method for proteins, after chromatofocusing. The absence of oxidation of methionine groups to sulphoxides was reported as an important advantage of the LC method, compared to 2D GE (15).  In the present article, large-pore polystyrene-divinylbenzene (PS-DVB) materials were selected in order to avoid silanol interactions on silica-based columns and to mimic some of the properties of monolithic columns, because even large membrane proteins have been demonstrated to elute without problems on PS-DVB monoliths, with TFA in the mobile phase (16). Reversed-phase columns do not separate according to hydrophobicity/size exclusively, but within a group of proteins already selected for their acidic/basic properties, a size-related separation is a reasonable expectation. Figure 6 demonstrates the results of connecting a pH gradient in the first dimension with a reversed-phase separation in the second dimension, on a PS/DVB column. The PS/DVB particles had a pore size of 4000 Å to reduce band broadening caused by slow kinetics, to reduce adsorption in narrow pores, and to avoid exclusion of large proteins from narrow pores. As a result of the low back pressure on the wide-pore particles, high flow-rates allowed short elution times. The timescale in the figure is the total time of analysis. The basic fraction ends at 14 min and the acidic fraction starts at 20 min. The separations of the basic proteins in the second dimension were according to MW. The acidic proteins were also separated according to size except that a-lactalbumin (MW 14200) eluted after the lactoglobulins (MW 18400) and BSA eluted prior to ovalbumin. This demonstrates that expectations of MW-related order of elution on the PS/DVB column must be treated with care. Conclusions Separation and focusing of proteins can be obtained by pH-gradient ion-exchange chromatography when the pH of the sample solution > pI. Hence, pH-gradient IEC has the potential of becoming an important separation technique in proteomic studies, not only for off-line pI fractionation of weakly expressed proteins, but also as a highly efficient reversed-phase compatible dimension in 2D LC systems. Although much more investigation is required to establish the limitations of the technique as demonstrated by the preliminary results, 2D narrow-bore LC can be considered an attractive alternative to traditional 2D GE in the future. It is not likely that the present methods for 2D LC will be able to obtain the same high resolution of  proteins as 2D GE, but for samples where some preseparation has been performed, 2D LC represents a much faster and more easily automated method. Acknowledgements The authors would like to thank the EU research training network HPRN-CT-2001-00180 (COM-CHROM) for financial support. References (1) N.L. Anderson and N.G. Anderson, Electrophoresis 19, 1853–1861 (1998). (2) M. Kinter and N.E. Sherman, Protein Sequencing and Identification Using Tandem Mass Spectrometry (Wiley Interscience, Hoboken, NJ, 2000), 29–63. (3) T. Rabiiioud et al., Electrophoresis 18, 307–316 (1997). (4) D.C. Liebler, Introduction to Proteomics (Humana Press, New Jersey, 2002), 38 (5) M.R. Wilkins et al., Electrophoresis 19, 1501–1505 (1998). (6) J. N. Adkins et al., Mol. Cell Proteomics 1, 947–955 (2002). (7) P.G. Righetti et al., Proteomics 3, 1397–1407 (2003). (8) L.A.A.E. Sluyterman and O. Elgersma, J. Chromatogr. 150, 17 (1978). (9) L.A.A.E. Sluyterman and J. Wijdenes, J. Chromatogr. 206, 429 (1981). (10) J.H. Scott, K.L. Kelner and H.B. Pollard. Anal. Biochem. 149, 163 (1985). (11) Y. Liu and D.J. Anderson, J. Chromatogr., A 762, 47 (1997). (12) L. Shan and D.J. Anderson. Anal. Chem. 74, 5641 (2002). (13) T. Andersen et al., J. Chromatogr., A 1025, 217–226 (2004). (14) J.M.P. Deshusses et al., Proteomics 3, 1418–1424 (2003). (15) K. Zhu et al., J. Chromatogr., A 1053, 133–142 (2004). (16) A. Premstaller et al., Anal. Chem. 73, 2390–02396 (2001). Tyge Greibrokk is professor of analytical chemistry and is currently Head of the Chemistry Department, University of Oslo, Norway. Elsa Lundanes is professor of analytical chemistry, Chemistry Department, University of Oslo. Milaim Pepaj is a Ph.D. student of analytical chemistry at the University of Oslo. Katerina Novotna is a Ph.D. student of analytical chemistry at the University of Pardubice, participating in an EU-research training network coordinated by the Oslo group. Thomas Andersen is a former Ph.D. student of the Greibrokk/Lundanes group and is now a researcher at the G&T Septech company, Oslo, Norway. ♦