Technology

Mass Spectrometry Based Proteomics

Proteomics aims to achieve a global view of biological samples at the protein level, similar to genomics and transcriptomics that examine the DNA and RNA levels, resectively. Examination of the proteins, which are the active entities in the cells is much closer to the cellular phenotype than genes and transcripts and is therefore expected to better represent the cell state. Beyond the analysis of cellular proteomes, proteomics can be applied to the analysis of protein modifications (e.g. phosphorylations, acetylations), protein interactions (with other proteins, DNA, RNA and small molecules) and the proteomes of non-cellular samples such as body fluids.

Mass spectrometry-based proteomics is the main approach that enables a global view of proteomes. In ‘shot-gun’ proteomics, which is the principal proteomics technique, proteins are digested into peptides, which are then analyzed by liquid chromatography (LC) coupled to the mass spectrometer (MS). The MS performs two types of scans: In the MS scans the peptide mass to charge ratio (m/z) and its intensity are determined, and in MS/MS scans, precursor peptides are selected, fragmented and the m/z of the fragments is determined. A typical LC-MS/MS run takes 2-4 hours, and is composed of thousands of cycles; each composed of one MS scan followed by 10-20 MS/MS scans. Following the mass spectrometric analysis, computational analysis of the raw data reveals the identity and quantitative changes in thousands of proteins and is the basis for further bioinformatic, systems biology, and biological research.

In our lab

Our laboratory is equipped with the Q-Exactive mass spectrometer [1], which was introduced in June 2011 by Thermo Scientific. It is based on the Orbitrap technology, providing high resolution and high mass accuracy, combined with high speed and high dynamic range. For peptide separation, we use the EASY-nLC1000 ultra high performance liquid chromatography (UHPLC) system from Thermo [2]. The LC and MS are connected through the EASY-Spray nano electrospray source with a heated 50 cm columns with 2um C18 beads (Dionex). Data analysis is performed in the MaxQuant environment with the Andromeda search engine  [3].

Quantitative Proteomics Using SILAC

Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) is a type of metabolic labeling technology that enables accurate quantification in proteomic experiments [4]. In SILAC, cells are cultured in the presence of heavy amino acids, typically lysine and arginine, which are synthesized with 13C and 15N. For labeling, heavy or light amino acids are added to growth medium deprived of these amino acids and supplemented with dialyzed serum. After 5-10 passages the cells are fully labeled and are ready for the experiment. Following the experiment, cells or lysates of two to three differentially labeled samples are combined and proteins are digested and analyzed together. In the MS scans peptides appear as a pairs (or triplets in the case of triple-SILAC), which can be very accurately quantified.

 References

  1. Michalski A, Damoc E, Hauschild JP, Lange O, Wieghaus A, Makarov A, Nagaraj N, Cox J, Mann M, Horning S: Mass spectrometry-based proteomics using Q Exactive, a high-performance benchtop quadrupole Orbitrap mass spectrometerMolecular & cellular proteomics : MCP 2011, 10(9):M111 011015.
  2. Nagaraj N, Kulak NA, Cox J, Neuhauser N, Mayr K, Hoerning O, Vorm O, Mann M: System-wide perturbation analysis with nearly complete coverage of the yeast proteome by single-shot ultra HPLC runs on a bench top Orbitrap. Molecular & cellular proteomics : MCP 2012, 11(3):M111 013722.
  3. Cox J, Mann M: MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nature biotechnology 2008, 26(12):1367-1372.
  4. Ong SE, Blagoev B, Kratchmarova I, Kristensen DB, Steen H, Pandey A, Mann M: Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics. Molecular & cellular proteomics : MCP 2002, 1(5):376-386.