Florine Hallez (Le Houlme / FR), Michel Arotçarena (Le Houlme / FR), Sami Bayoudh (Le Houlme / FR), Kaynoush Naraghi (Le Houlme / FR), Florent Dingli (Paris / FR), Damarys Loew (Paris / FR), Célia Jardin (Paris / FR), Soumia Hamada (Paris / FR), Cédric Pionneau (Paris / FR), Martin Technau (Kamp-Lintfort / DE), Solenne Chardonnet (Paris / FR)
Bottom-up approaches are commonly used for proteomics analysis in LC-MS/MS. However, salts present in the digestion buffers can generate ion-suppression that limits peptide detection and greatly impacts proteins identification.
Several AttractSPE®Disks Tips C18 were evaluated for peptide desalting. These SPE Tips, packed with small sorbent beads embedded in a thin and mechanically stable membrane that combines high capacity and small dead volume, are particularly adapted for centrifugation or positive pressure assays.
First, 200µL AttractSPE®Disks Tips C18 with different binding capacities were tested for the manual desalting of 100ng of HeLa digest by centrifugation. Five replicates of each assay were carried out and the nanoLC-MS/MS analyses of peptides were compared to those obtained after direct injection of a non-purified loading solution. This comparison showed that 97% of proteins could be identified after desalting on the C18 tips, with high recovery of all peptides on the whole polarity range. Low variability was obtained for intra- and inter-tips assays (RSD < 3%), highlighting the Tips reliability.
With the aim to automate peptide desalting, the P3S plateform has asked CEM (previously Intavis) to create a custom needle to run home-made StageTips on the DigestPro MSi robot. The program was adapted from manual StageTips protocol and further optimized by playing with liquid aspirate and dispense speeds and volumes. Moreover, P3S has evaluated AttractSPE® C18 Tips compared to home-made StageTips (with Empore C18 SPE disks) for 2µg and 100ng of protein digest. The comparison showed little difference between the two kinds of tips when working with 2µg of peptides but showed a 10% increase in the number of identified proteins with the AttractSPE® C18 Tips when working with 100ng of peptides. Moreover, the peptide intensities were higher with the AttractSPE® C18 Tips. In both cases, 2µg and 100ng starting material, the AttractSPE® C18 Tips performed better in the hydrophilic range, thus demonstrating than this C18 sorbent offers a wider spectrum of interactions compared to home-made StageTips. In addition, the P3S team has optimized the program on the DigestPro MSi robot to reduce the time from 40min down to 10min per sample.
This study has shown that the AttractSPE®Disks Tips C18 can be used both for manual or automated desalting workflows, and are perfectly adapted to single cell-like analysis as well as high peptide amounts, thus covering a wide range of applications.