On-line capillary column immobilised metal affinity chromatography/electrospray ionisation mass spectrometry for the selective analysis of histidine-containing peptides.
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.
School of Biomedical and Natural Sciences, Interdisciplinary Biomedical Research Centre, Nottingham Trent University, Nottingham, UK.
Gushinder
G
Kaur-Atwal
Daniel J
DJ
Weston
Philip S
PS
Green
Susan
S
Crosland
Philip L R
PL
Bonner
Colin S
CS
Creaser
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002846:Chromatography, Affinity / Q000379:methods*; D006639:Histidine / Q000032:analysis*; D007098:Imino Acids / Q000737:chemistry; D008969:Molecular Sequence Data; D009862:Online Systems; D010455:Peptides / Q000032:analysis*; D012710:Serum Albumin, Bovine / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D053719:Tandem Mass Spectrometry
D007098:Imino Acids; D010455:Peptides; D012710:Serum Albumin, Bovine; D006639:Histidine; C008109:iminodiacetic acid