Solid phase extraction-liquid chromatography (SPE-LC) interface for automated peptide separation and identification by tandem mass spectrometry

Abstract

Reversed-phase solid phase extraction (SPE) is a simple and widely used technique for desalting and concentration of peptide and protein samples prior to mass spectrometry analysis. Often, SPE sample preparation is done manually and the samples eluted, dried and reconstituted into 96-well titer plates for subsequent LC–MS/MS analysis. To reduce the number of sample handling stages and increase throughput, we developed a robotic system to interface off-line SPE to LC–ESI-MS/MS. Samples were manually loaded onto disposable SPE tips that subsequently were connected in-line with a capillary chromatography column. Peptides were recovered from the SPE column and separated on the RP-LC column using isocratic elution conditions and analysed by electrospray tandem mass spectrometry. Peptide mixtures eluted within approximately 5 min, with individual peptide peak resolution of ∼7 s (FWHM), making the SPE-LC suited for analysis of medium complex samples (3–12 protein components). For optimum performance, the isocratic flow rate was reduced to 30 nL/min, producing nanoelectrospray like conditions which ensure high ionisation efficiency and sensitivity. Using a modified autosampler for mounting and disposing of the SPE tips, the SPE-LC–MS/MS system could analyse six samples per hour, and up to 192 SPE tips in one batch. The relatively high sample throughput, medium separation power and high sensitivity makes the automated SPE-LC–MS/MS setup attractive for proteomics experiments as demonstrated by the identification of the components of simple protein mixtures and of proteins recovered from 2DE gels.