msb-quantworks

SILAC

This service is designed for the quantitative and qualitative analysis of cell based systems. The results from this experiment provide a catalogue of the proteins present in all samples and a statistical analysis reflecting the changes in the protein levels observed across the samples, along with associated pathway information.

Stable isotope labeling with amino acids in cell culture (SILAC) is a quantitative proteomics technique that involves the incorporation of a label into proteins in vitro prior to analysis by LC/MS/MS. Cells are labeled during the culturing process using media containing light or heavy amino acids, the heavy amino acids have stable isotope atoms incorporated e.g. lysine (13C6H1215N2O) or arginine (13C6H1215N4O2). The labeled amino acids are used in protein synthesis, after several passages the labeled residues have been fully incorporated into the proteins. The labeled amino acids are equivalent to their unlabeled counterparts and their presence has no impact on the biological system.

Cells from light and heavy cultures can be lysed and mixed to create a population of both heavy and light labeled pairs of proteins. The pooled sample is then processed using our GeLC/MS approach. The GeLC/MS technique uses an offline first dimension SDS-PAGE protein level separation to reduce the complexity of samples prior to an online second dimension reverse phase peptide level separation. The protein sample resolved on the SDS gel is divided into a number of segments which are digested with trypsin prior to the online analysis. The number of segments dictates the depth of coverage and the time to analyze a single sample. Digested samples are analyzed by LC/MS/MS and protein identification data are collated into a non-redundant list per gel lane. Protein differential expression is calculated as a function of the peak areas of the light and heavy peptides detected in the sample.

The steps involved in a chemical labeling experiment are:

  • Sample preparation – cell lysis
  • Label incorporation analysis – a screen to ensure full incorporation of labeled amino acids into the system. Data are evaluated with MaxQuant.
  • SDS-PAGE – Invitrogen Novex mini-gels, stained with colloidal coomassie.
  • Segmentation – 10, 20 or 40 segments. The number of segments is dictated by project objectives, sample type, complexity and budget. The default number of segments for this service is 40.
  • Sample digestion – robotic reduction and alkylation followed by trypsin digestion.
  • Nano LC/MS/MS – 250 nL/min liquid chromatography to yield high sensitivity; interfaced to state-of-the-art Orbitrap Velos Pro or Q Exactive tandem mass spectrometer.
  • Data analysis – analysis using MaxQuant with quantitation and normalization based on integrated peak areas. Protein identification is performed with Mascot. Statistical assessment of significant differences and optional pathway analysis.
  • Report generation – PDF report, Excel and Scaffold files. Data rationalized and presented to ensure clarity and the ability to understand the results.

Key features of a metabolic experiment are:

  • High throughput.
  • Relatively low cost as multiple samples are pooled
  • Sample mixing early in the sample processing workflow improves quantitation accuracy
  • Multiplexing enables 2 or 3 samples in a single experiment

For more information on Thermo Scientific Pierce SILAC labeling reagents, visit www.thermoscientific.com/pierce.