Direct coupling of an automated 2-dimensional microcolumn affinity chromatography-capillary HPLC system with mass spectrometry for biomolecule analysis

Microcolumn liquid chromatography: instrumentation, detection and applications

“…multidimensional chromatography. Microcolumn LC has been interfaced with thin-layer chromatography applying infrared detection [220], conventional high-performance liquid chromatography [2211, microcolumn LC [222][223][224], gas chromatography [225][226][227][228][229][230][231], supercritical fluid chromatography [232] and capillary electrophoresis [233]. The coupling of individual separation techniques increases the total peak capacity of the chromatographic system, which is equal to the product of the peak capacities of the individual dimensions.…”

The Expanding Role of HPLC in Drug Discovery

“…The technique that has been used most widely for directly assessing protein-small molecule and protein-protein interactions is affinity chromatography-mass spectrometry. Kassel et al [16] presented one of the first papers coupling affinity chromatography with mass spectrometry. In their work, a two-dimensional LC/LC/MS method was developed to assess protein-ligand binding.…”

Protein Identification and Characterization by Mass Spectrometry

“…This approach provides a high-sensitivity system that complements the extensively utilized two-dimensional TLC mapping procedure (Watts et al, 1996). Specific capture of tyrosine-phosphorylated peptides has been achieved using an antiphosphotyrosine column in-line with an LC-MS (Kassel et al, 1995). The specificity of the system was demonstrated, but the sensitivity of the approach was not determined.…”

“…The specificity of the system was demonstrated, but the sensitivity of the approach was not determined. Kassel et al (1995) also produced a more specific affinity column by immobilizing a recombinant SH2 domain to the first column, thus allowing capture of only tyrosine phosphopeptides containing the specific sequence motif recognized by this domain. Amankwa et al (1995) developed an "enzyme reactor" system employing a broad-specificity tyrosine phosphatase on-line prior to either capillary zone electrophoresis (CZE)-MS or LC-MS.…”