A standard data interpretation for each data set is provided. If requested, we also offer additional bioinformatic services, subject to additional costs.
For gel samples, we recommend any Coomassie or fluorescence dye over silver staining. In case you must use silver staining, please make sure that a mass spectrometry compatible silver staining protocol or kit is used. Please consult Silver_Staining_Destaining.pdf (PDF, 113KB) for more detailed explanations. You must submit any protein samples in a gel or on PVDF membrane according to our guidelines: PMSCF_Guidelines.pdf.
For in-solution samples, we request from you an accurate estimate of protein concentration and buffer composition. This also applies to affinity pull-down beads (see below). Our preferred solubilization buffer is 8M urea in 100mM TRIS/HCl pH 8 (optionally with protease or phosphatase inhibitor cocktails).
For identification and quantification, proteins are generally reduced, alkylated and digested according to our internal standard operating procedure. Peptides are analyzed by nanoLC-MS/MS with a LC gradient length adapted to the expected complexity of the sample. MS/MS spectra are searched with Easyprot, MaxQuant/Andromeda, and/or Trans-Proteomic Pipeline (TPP) tools against an appropriate non-redundant database and its reversed decoy. For relative or absolute proteome quantification we offer non-targeted and targeted nanoLC-MS/MS workflows (DDA, DIA, PRM, SIM) with label-free or stable isotope labeled samples. Please enquire for more details.
We also offer a direct protein digest workup on affinity pulldown beads. The method is similar to the liquid phase approach. We request that you provide us with an estimate of the protein amount (as accurate as possible) bound to the beads (at least in form of the amount of antibody used), and that beads are free from any detergents or glycerol.
We have a very efficient and fast protein extraction protocol for dilute protein samples, like conditioned cell medium. Please use serum-free cell media for such experiments. The extracted proteins are separated by SDS-PAGE and fractions (slices) are analyzed as described under Fractionation. Alternatively, we can offer a protein concentration approach based on functionalized disk membranes with the possibilit to fractionate peptides after digestion of proteins.
PTM characterization: We offer phosphopeptide analysis with a validated protocol. Liquid phase samples are reduced, alkylated and digested. Phosphopeptides are enriched fully automatically with Fe-NTA cartridges on an Assay-Map BRAVO liquid handling station. The enrichment protocol can be extended using TiO2 spheres for in-depth phosphoproteomics, if wished for. We can offer several data-dependent nanoLC-MS/MS acquisition modes using multistage HCD/CID activation (Fusion Lumos), ETD with/without HCD assistance (Fusion Lumos), or HCD (timsTOF instruments). Our preferred mode is HCD [ref. Koch et al. PloS ONE 4: e4552]. Of course, we can analyse any other (MS-friendly) post-translational modification and we operate open MS search algorithms for the identification of non-expected PTMs. Please enquire [e.g. ref. Greganova et al. PloS ONE 5: e9486].
For molecular mass determination of intact proteins, we request a pure protein sample in a MS-compatible buffer like ammonium-acetate for static nano-ESI. In case of a more complex or contaminated sample, the intact mass determination is made by micro- or nano-flow LC-MS. The latest orbitrap technology allows measuring proteins with molecular weights exceeding 150 kDa.
We can offer a variety of small-scale protein/peptide fractionation methods based on chromatographic principles, e.g. iso-electric focusing, ion-exchange, reversed phase.