Protein Preparation

Protein extraction is an important step in any proteomics experiment. It often starts with cell lysis and cell fractionation, followed by specific enrichment and/or isolation of a particular protein of interest (affinity purification), or removal of interfering or contaminating substances (i.e. immunodepletion).

Many techniques are available for the disruption of cells, including physical and detergent-based methods. Historically, physical lysis has been the method of choice for cell disruption; however, physical methods often require expensive equipment (french prss, homogenizer, cryogrinding).
Detergent-based lysis is more popular due to ease of use, low cost and efficient protocols. However, many detergents interfere with the downstream LC-MS analysis. A list of MS compatible detergents can be found on our protein digestion page.

Overviews by Life Technologies (former Pierce, now Thermo)

Sample Preparation for Mass Spectrometry page

Mass Spectrometry Sample Preparation Handbook page
Physical cell lysis page
Detergent based cell lysis page

Protein Extraction from Tissues and Plants

Cell culture related protocols

  • Protocols for cell lysis and subcellular fractionation go to page

Protein preparation from Body Fluids
blood, plasma, urine, CSF etc

Isotopic labelling strategies
SILAC (stable isotope labeling using amino acids in cell culture) is a metabolic labelling technique for comprehensive identification, characterization and quantification of proteins by LC-MS/MS. Isotopically labeled amino acids (typically Lys and Arg) are incorporated in to proteins during cell culture.

  • ThermoPierce SILAC Isotope Labeling Reagents and Kits go to page
  • Sigma SILAC Isotope Labeling Reagents and Kits go to page
  • Mann lab SILAC protocol go to page

  • Planet Orbitrap SILAC workflow page

There are various chemical labeling strategies for concurrent peptide identification and multiplexed proteomics quantitation by mass spectrometry. Most quantitative proteomics reagents incorporate stable isotopes into the isobaric tag portion of the reagents and are used to label at the protein or peptide level:

  • Sigma iTRAQ (Isobaric Tags for Relative and Absolute Quantification) go to page
    Amine-reactive, 8-plex reagents

  • ThermoPierce TMT Overview (Tandem Mass Tag) Reagents go to page
    Amine-reactive, 6-plex Tandem Mass Tag Reagents go to page
    Amine-reactive, 10-plex Tandem Mass Tag Reagents go to page
    Cysteine-Reactive, 6-plex Tandem Mass Tag Reagents go to page
    Carbonyl-reactive, 6-plex aminoxyTMT Reagents go to page

  • Planet Orbitrap TMT Overview (Tandem Mass Tag) Reagents page

Crosslinking strategies

  • ThermoPierce Protein Interaction Crosslinking for Mass Spectrometry go to page
  • ThermoPierce Crosslinking Protein Interaction Analysis go to page
  • ThermoPierce MS cleavable DSBU (Disuccinimidyl Dibutyric Urea) go to page
  • ThermoPierce Crosslinking Mass-tagged crosslinkers go to page
  • Creative Molecules crosslinking reagents go to page
  • Cell surface crosslinking reagent go to page [Nature protocol in Ref 1]
  • Soft ware (crosslinking) tools from the Aebersold lab go to page

References

  1. Ligand-based receptor identification on living cells and tissues using TRICEPS. Frei AP, Moest H, Novy K, Wollscheid B. Nat Protoc. 2013 Jul;8(7):1321-36. doi: 10.1038/nprot.2013.072. Epub 2013 Jun 13. link