HPLC-Chip/MS technology in proteomic profiling.
HPLC-chip/MS is a novel nanoflow analytical technology conducted on a microfabricated chip that allows for highly efficient HPLC separation and superior sensitive MS detection of complex proteomic mixtures. This is possible through on-chip preconcentration and separation with fluidic connection made automatically in a leak-tight fashion. Minimum precolumn and postcolumn peak dispersion and uncompromised ease of use result in compounds eluting in bands of only a few nanoliters. The chip is fabricated out of bio-inert polyimide-containing channels and integrated chip structures, such as an electrospray emitter, columns, and frits manufactured by laser ablation technology. Meanwhile, a variety of HPLC-chips differing in design and stationary phase are commercially available, which provide a comprehensive solution for applications in proteomics, glycomics, biomarker, and pharmaceutical discovery. The HPLC-chip can also be easily integrated into a multidimensional separation workflow where different orthogonal separation techniques are combined to solve a highly complex separation problems. In this chapter, we describe in detail the methodological chip usage and functionality and its application in the elucidation of the protein profile of human nucleoli.
Vollmer
Martin
M
Agilent Technologies, Hewlett Packard-Strasse 8, 76337, Waldbronn, Germany. martin_vollmer@agilent.com
van de Goor
Tom
T
D002851:Chromatography, High Pressure Liquid / Q000379:methods; D002852:Chromatography, Ion Exchange; D006367:HeLa Cells; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods; D036103:Nanotechnology / Q000379:methods; D009687:Nuclear Proteins / Q000302:isolation & purification; D040081:Protein Array Analysis / Q000379:methods*; D020543:Proteome / Q000302:isolation & purification; D040901:Proteomics / Q000295:instrumentation / Q000379:methods*
D009687:Nuclear Proteins; D020543:Proteome