Abstract
Two dimensional (2D) liquid chromatography (LC) separations of proteins can be obtained faster and more automated than traditional 2D gel electrophoresis. Previously we have described a 2D LC method for separation of native proteins with separation according to pI by pH-gradient strong anion exchange (SAX) chromatography in the first dimension, and according to hydrophobicity by reversed phase chromatography in the second dimension. Since there are few literature reports on the combination of reduced/alkylated proteins and modern LC, a basic study of the chromatographic properties of a few reduced /alkylated proteins was undertaken with a pH-gradient SAX chromatographic system. Proteins where the disulfide groups were reduced, but not alkylated, were also included. The conditions that separated native proteins according to pI could not be used for neither reduced nor reduced/alkylated proteins. High concentrations of urea (4–8 M) were needed in the mobile phase in order to obtain good peak shapes. Addition of urea had an undesired impact on both the retention of the proteins and the pH gradient profile, with the effect that little correlation between reported pI values and elution pH was found. The conclusion was that proteins should be separated in the native state if good pI–pH correlations are important, and in the alkylated state with urea if other considerations are more important.
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References
Bunai K, Yamane K (2005) J Chromatogr B 815:227–236
Cooper JW, Wang Y, Lee CS (2004) Electrophoresis 25:3913–3926
Righetti PG (2006) J Chromatogr B 841:14–22
Herbert B, Galvani M, Hamdan M, Olivieri E, MacCarthy J, Perdersen S, Righetti PG (2001) Electrophoresis 22:2046–2057
Magnus EM, Khan K (1992) Cereal Chem 69(6):607–612
Hearn MTW, Nguyen T, Fridman M (1988) J Chromatogr 435(2):271–284
Burnouf T, Bietz JA (1984) J Chromatogr 299(1):185–199
Hedström M, Andersson M, Galaev IY, Mattiasson B (2005) J Chromatogr A 1080:117–123
Duan J, Sun L, Liang Z, Zhang J, Wang H, Zhang L, Zhang W, Zang Y (2006) J Chromatogr A 1106:165–174
Andersen T, Pepaj M, Trones R, Lundanes E, Greibrokk T (2004) J Chromatogr A 1025:217–226
Yan JX, Kett WC, Herbert BR, Gooley AA, Packer NH, Williams KL (1998) J Chromatogr A 813:187–200
Nadler T, Blackburn C, Mark J, Gordon N, Regnier FE, Vella G (1996) J Chromatogr A 743:91–98
Romijn EP, Krijgsveld J, Heck AJR (2001) Anal Chem 73:3576–3582
Bosma JC, Wesselingh JA (1998) AIChE J 44:2399–2409
Wait R, Gegum S, Brambilla D, Carabelli AM, Conserva F, Guerini AR, Eberini I, Ballerio R, Gemeiner M, Miller I, Gianazza E (2005) Amino Acids 28:239–272
Chaudhuri AR, Khan IA, Ludueña RF (2001) Biochememistry 40:8834–8841
Eyles SJ, Radford SE, Robinson CV, Dobson CM (1994) Biochemistry 33:13038–13048
Nandel FS, Verma R, Singh B, Jain DVS (1998) Pure Appl Chem 70:659–664
Kadoya T, Okuyama T (1983) Bunseki Kagaku 32:664–669
Acknowledgments
BQT is supported by the Government of Vietnam and the research is partly supported by the EU research training network HPRN-CT-2001-00180 (COM-CHROM). The Q-TOF2 technical support by John Ø Vedde is greatly appreciated.
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Q. Tran, B., Pepaj, M., Lundanes, E. et al. The Behaviour of Reduced, Alkylated and Native Proteins in a pH-Gradient LC System. Chroma 66, 709–715 (2007). https://doi.org/10.1365/s10337-007-0390-8
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DOI: https://doi.org/10.1365/s10337-007-0390-8