Modular stop and go extraction tips with stacked disks for parallel and multidimensional peptide fractionation in proteomics

Proteome complexity necessitates protein or peptide separation prior to analysis. We previously described a pipet-tip based peptide micropurification system named StageTips (STop and Go Extraction Tips), which consists of a very small disk of membrane-embedded separation material. Here, we extend this approach in several dimensions by stacking disks containing reversed phase (C-18) and strong cation exchange (SCX) materials. Multidimensional fractionation as well as desalting, filtration, and concentration prior to mass spectrometry in single or tandem columns is described. C-18-SCX-C-18 stacked disks significantly improved protein identification by LC-MS/MS for an E. coli protein digest and by MALDI-MS for a 12 standard protein digest. Sequential fractionation based on C-18- followed by SCX material was also developed. This multidimensional fractionation approach was expanded to parallel sample preparation by incorporating C-18-SCX-StageTips into a 96-well plate (StagePlate). Fractions were collected into other C-18-StagePlates and desalted and eluted in parallel to sample well plates or MALDI targets. This approach is suitable for high throughput protein identification for moderately complex, low abundance samples using automated nanoelectrospray-MS/MS or MALDI-MS.