Mass spectrometry

Protein Analysis by Mass spectrometry

The facility offers liquid chromatography-mass spectrometry (LC-MS) measurements for protein identification, characterization of posttranslational modifications, protein quantification and the respective data interpretation. Most methodologies are based on the analysis of peptides generated from enzymatic digestion of the original protein mixtures. To keep the technology platforms state-of-the-art, the corresponding methods are constantly improved, and novel protocols are developed and established.

Our portfolio contains the following types of analysis. Also, various combinations of these are possible. If additional applications are needed, please contact the facility to discuss possible solutions.

  • Identification of gel-separated proteins from coomassie or silver-stained gels or of purified proteins in solution (sample preparation guidelines). The aim of the analysis can range from a simple identification to a complete sequence characterization from N- to C-terminus (such as limited proteolysis experiments or analysis of truncated protein versions) or a comprehensive characterization of post-translational modifications (PTMs). A dedicated analysis of histone modifications, requiring special sample preparation methodologies, can also be offered. In all cases label-free quantification on MS1 level is applied which can be used to perform relative quantification of detected proteins and PTMs from different conditions.

  • Analysis of affinity-purified protein complexes (AP-MS)
    The aim is the identification of the bait protein and its binding partners, often including a modification analysis. We also analyse samples derived from enzyme-catalyzed proximity labeling experiments and samples of immunopurified chromatin-interacting proteins crosslinked by formaldehyde. (cross-linked species are not analysed). Distinguishing potential interactors from unspecifically binding proteins requires the analysis of appropriate controls. Label-free quantification on MS1 level is applied to perform relative quantification of detected proteins and PTMs. Statistical significance of differentially abundant proteins and PTMs can be computed if at least 3 (biological) replicates are provided. We strongly recommend performing pilot experiments to optimize conditions before submitting biological replicates.

  • System-wide protein identification and label-free relative protein quantification
    For this type of analysis diverse types of samples can be submitted. For cell pellets of cultured cells with appr 10-100 µg protein content we provide an easy to use sample preparation kit, for other sample types and amounts different protein extraction methods are performed. After the proteolytic digest a maximum of 1 µg of the unfractionated sample can be directly analysed by LC-MS/MS, where up to 4000 proteins with at least 2 peptides/protein can be identified and relatively quantified by label-free quantification on MS1 level. In case, a protein of interest cannot be identified and/or quantified by this untargeted so-called shotgun approach, a more sensitive and targeted method called parallel reaction monitoring (PRM, see below) can be applied to the unfractionated samples.

  • Consistent monitoring and label-free relative quantification of multiple target proteins in complex samples by targeted analysis using parallel reaction monitoring (PRM)
    Here, instead of trying to sequence all peptides present in the sample, only peptides derived from a protein of interest are targeted for identification in a complex sample. For each protein of interest several (5-10) proteotypic peptides have to be chosen, either from data recorded earlier, from databases, or from in-silico digests. Up to 100 peptides derived from 10-20 proteins of interest can be targeted per MS-run. The recorded fragment ions in the MS/MS spectra are used for identification by database searching and for quantification which is at least 10 times more sensitive as compared to peptide-based quantification in MS1 mode.

  • System-wide protein identification and TMT-based relative protein quantification
    In case an extensive proteome coverage is required and/or many biological conditions are compared to each other on a system-wide level, we perform isotopic labeling using the isobaric tag system Tandem Mass Tag (TMT), which allows the multiplexing of up to 16 different channels. After the digest, samples are differentially labelled, mixed and appr 200 µg of the mix is fractionated by strong cation exchange chromatography to reduce the sample complexity. Approximately 50 SCX fractions are analysed by LC-MS/MS. Here, up to 7500 proteins can be identified and relatively quantified. Ratio compression is an intrinsic problem of isobaric tagging, leading to the underestimation of relative abundance changes. On the other hand, this method offers highest coverage of the analysed proteome.

  • System-wide identification and TMT-based relative quantification of post-translational modifications
    We have established a workflow for the analysis of phosphorylated and acetylated peptides, which can be performed in cooperation with the customer. It follows the TMT workflow described for the proteome quantification including additional PTM-specific enrichment steps.

  • Intact protein mass spectrometry (in cooperation with the MFPL mass spectrometry facility).
    Purified intact proteins in solution are analysed by MS to accurately determine their mass. The main applications are performed under denaturing conditions with the aim to control for the quality of expressed proteins or to check for possible modifications. For more details click here.

  • Depending on the analytical question, different peptide fragmentation methods can be applied (HCD, CID, ETD, EThcD).

 

All analyses are performed using high resolution and high mass accuracy LC-MS and LC-MS/MS.

Bioinformatic data analysis and data interpretation

  • Protein identification is performed using MS Amanda (Dorfer et al) or Mascot (Perkins et al) within the Proteome Discoverer framework.
  • Post-translational modifications are localised using ptmRS (Taus et al).
  • Proteins from label-free samples are quantified using apQuant (Doblmann et al), whereas TMT-labelled samples are quantified using the Thermo Fisher Reporter Ions Quantifier Node or IMP-Hyperplex. Statistical significance of differentially abundant proteins is determined using limma (Ritchie et al). This requires at least three replicates per condition.
  • Results are exported in a user-friendly Microsoft Excel spreadsheet format using MS2Go, which assembles and structures the data (developed in house).