Manual Microcolumn Chromatography for Sample Cleanup Before Mass Spectrometry

摘要

Protein biochemistry has benefited greatly from the increasing availability of both matrix-assisted laser desorption ionization (MALDI-) and electrospray ionization mass spectrometers (ESI-MS) due to their high sensitivity and tolerance to some commonly used buffers However, analysis of biologically derived samples at sub-pmol levels often provides serious challenges, e.g., although MALDI-MS is more robust with regard to many buffers and other contaminants found in biologically derived samples than ESI-MS, many of these compounds result in reduced signal intensity and poorer quality spectra. Sample cleanup protocols have used simple rinsing of the crystallized sample (1), novel preparation of the metal surface with matrix (11,12) or other materials(4,5), or they have used a separate surface for cleanup/crystallization before attachment to the MALDI-MS probe (3,13). However, another property of MALDI-MS that is not alleviated by these approaches is that of ionization suppression in peptide mixtures (2), To overcome this problem, the mixture has to be partially fractionated. In ESI-MS, this can be overcome by using capillary reverse-phase, high-performance liquid chromatography (RP-HPLC) before on-line mass analysis.