Proteomics

• What is the most appropriate analytical method for isolating and analyzing proteins and peptides from your cell cultures, tissues, or biofluids?
• How can the proteins of interest be enriched or purified?
• What technology should be used for digestion, separation (including fractionation if necessary), and mass spectrometric (MS) analysis of your samples?
• What is the best method for quantifying the proteins in your sample: multiplexing using tandem mass tags (TMT), metabolic labeling using stable isotopes (SILAC), open profiling using data-independent acquisition (DIA), or "label-free" quantification (LFQ)?

Proteomic analyses are typically performed using nano-liquid chromatography systems and mass spectrometric detection with time-of-flight (ToF) or Orbitrap mass spectrometers, depending on the nature of the study, the complexity of the samples, and the desired quantification and identification parameters. For these projects, the facility has state-of-the-art mass spectrometers with ion mobility separation (Bruker tims-ToF Pro, Thermo Orbitrap Exploris 480 with FAIMS).

In addition to LC-MS-based analyses, the Core Facility offers proteomics technologies for single-cell proteomics (Fluidigm CyToF Helios (in collaboration with CF Flow Cytometry)) and spatial proteomics (Bruker Rapiflex for MALDI imaging MS and Fluidigm Hyperion for laser ablation ICP-MS, in collaboration with CF Imaging).

The particular challenge of proteomics experiments is the mostly individual question and the higher complexity of the proteome (isoforms, post-translational modifications), as well as the fact that no amplification method is available for proteins. Thus, the analytical detection limit is determined on the one hand by the performance of the instruments and on the other hand by the dynamic range of the biological sample and its matrix. The best possible sample preparation and design of the experiment is therefore crucial for successful performance.

If you are interested in integrating proteomic analyses into your research project, please contact the Proteomics Unit at an early stage to plan the project accordingly.

Services

• Protein identification and characterization
  can be applied in a wide variety of biological materials (cell lysate, excised gel band, body fluids, immune precipitate, biological fraction, tissue sample, etc.). Depending on the complexity of the sample (a few proteins versus whole proteome) and the research question (global listing of contained proteins versus specific identification of target proteins), different methods of sample preparation (lysis, enzymatic digestion, clean-up etc.), separation (chromatography with different separation columns, length, gradients etc.) and mass spectrometry are established and applied. The parameters of MS analysis can be adjusted to take into account, for example, post-translational modifications of proteins.

• Protein quantification
  – TMT: Multiplexing using Tandem Mass Tags allows highly accurate quantification of up to 18 samples simultaneously in a multiplex analysis. The measurement principle is based on isobaric labeling reagents that provide a uniform signal of all samples at the peptide level, but allow quantification of individual samples after fragmentation. To increase the coverage of the proteome (more proteins identified and quantified) the sample can additionally be fractionated offline. From cell cultures, typically between 6,000 and 10,000 proteins can be quantified simultaneously using this method. For studies with more than 18 samples, multiple runs can be combined using internal pooled standards.
  – SILAC: Metabolic labeling using stable isotopes allows labeling of proteins in vivo (e.g., through the cell culture medium) and then tracking the signals in mass spectrometry. The method is suitable for quantification of newly synthesized proteins (pulse chase) or assignment to different cells (source tracking).
  – DIA: Data-independent Acquisition refers to a method for quantitative profiling of biological samples. In this method, a spectral library is usually created in advance through intensive characterization of the biological sample in focus. This library is then used to assign mass signals. This method combines the advantages of label-free quantification with the good coverage of the proteome and the low number of missing values, which can usually only be achieved with multiplex methods.
  – LFQ: Label-free quantification is the standard mode of quantitative mass spectrometry. In LFQ, samples are measured separately in the so-called Data-Dependent Acquisition (DDA) mode and then the chromatograms and mass signals are compared. The challenge here is to obtain data profiles that are as complete as possible. This method does not require cost-intensive isotope-doped labeling reagents and offers high flexibility for studies with open sample numbers.

   

• Spatial and Single-Cell Proteomics
  Recent developments in proteomics allow increasingly sensitive and accurate identification and quantification of mass signals. Using MALDI imaging, image data of the proteome can be generated on tissue sections. CyTOF technology enables targeted analysis of proteins using metal-labeled antibodies at the single cell level.

• Sample preparation and method development
  Depending on the problem, the project effort ranges from the application of standard protocols to the adaptation of such protocols to the development of new methods. In most cases, enrichments/depletions or adaptations of enzymatic digestion conditions are necessary. The choice of a suitable lysis buffer is absolutely critical for the successful solubilization of the proteins in focus - if the proteins are not reliably brought into solution, detection is not possible. Sample preparation therefore represents a central component of proteomics projects.