On-line capillary column immobilised metal affinity chromatography/electrospray ionisation mass spectrometry for the selective analysis of histidine-containing peptides |
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Authors: | Kaur-Atwal Gushinder Weston Daniel J Green Philip S Crosland Susan Bonner Philip L R Creaser Colin S |
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Institution: | School of Biomedical and Natural Sciences, Interdisciplinary Biomedical Research Centre, Nottingham Trent University, Nottingham, UK. |
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Abstract: | Capillary column immobilised metal affinity chromatography (IMAC) has been combined on-line with electrospray ionisation/quadrupole time-of-flight mass spectrometry for the fractionation of histidine-containing peptides. IMAC beads (Poros 20MC, 20 microm) containing imidodiacetate chelating groups on a cross-linked poly(styrene-divinylbenzene) support were packed into a fused silica column (250 microm i.d.), which was interfaced to the electrospray ion source of the spectrometer. A Cu(II) activated column was used to isolate histidine-containing peptides from tryptic and other peptide mixtures with an average breakthrough of 9.1%, to reduce the complexity of the mass spectral analysis. The analysis cycle time was reduced to less than 15 min, at an optimum flow rate of 7.5 microL/min, without sacrificing peptide selectivity. Direct coupling of capillary IMAC with MS allows on-line separation, using MS compatible loading and elution buffers, and detection in a high-throughput fashion when compared to off-line strategies. |
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