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.
The following techniques, which are partly used in combination, are offered. If additional applications are needed please contact the facility to discuss possible solutions.
- Identification of proteins from coomassie or silver-stained gels (sample preparation guidelines).
- Identification of proteins in solution or bound to antibody-coupled beads (sample preparation guidelines).
- Identification, localization and relative quantification of post-translational modifications, such as phosphorylation, acetylation, methylation or ubiquitination.
- Analysis of histone modifications (tryptic digests after propionylation of lysine side chains).
- Analysis of phosphopeptide enriched samples (enrichment via TiO2).
- Identification and relative quantification of potential protein interaction partners or PTMs in affinity purified protein complexes.
- Proteome-wide identification and relative quantification of proteins and PTMs from complex protein mixtures, such as whole cell lysates. To reduce the sample complexity, proteolytic digests are in many cases separated by SCX chromatography using offline fraction collection prior to LC-MS/MS analyses.
- Relative quantification of proteins or PTMs:
- Label-free quantification is performed either on the MS1-level or on the MS2-level for samples which do not require fractionation after the digest.
- Quantification using isotopic labeling (iTRAQ, TMT) is applied for high complex protein mixtures, which require fractionation.
- Absolute quantification of selected proteins is performed using isotopically labeled internal standard peptides applying an in house developed strategy (EtEP-strategy, Holzmann et al., 2009) via selected reaction monitoring.
- 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.
- 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.
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).