What is SILAC technique?
SILAC (stable isotope labeling by amino acids in cell culture) is a technique based on mass spectrometry that detects differences in protein abundance among samples using non-radioactive isotopic labeling.
How does SILAC mass spec work?
In SILAC experiments, proteins are metabolically labeled by culturing cells in media containing normal and heavy isotope amino acids. This makes proteins from the light and heavy cells distinguishable by mass spectrometry (MS) after the cell lysates are mixed and the proteins separated and/or enriched.
Why is SILAC used?
SILAC (1) is used to quantify protein expression differences in up to 2 or 3 samples from cells.
How is SILAC quantitative?
SILAC is based on direct addition of selected stable isotope amino acids into the cell culture medium, allowing superior quantitative analysis of the cellular proteome compared to other labeling methods.
What is triple SILAC?
Triple SILAC quantitative proteomic analysis reveals differential abundance of cell signaling proteins between normal and lung cancer-derived exosomes. J Proteomics.
How does isotope labeling work?
Isotopic labeling (or isotopic labelling) is a technique used to track the passage of an isotope (an atom with a detectable variation in neutron count) through a reaction, metabolic pathway, or cell. The reactant is ‘labeled’ by replacing specific atoms by their isotope.
Is SILAC quantitative?
Conclusion. The SILAC-based quantitative proteomic techniques, including the spike-in SILAC and triple-SILAC, are successfully applied to identify secreted protein changes in mimic tumor microenvironment in vitro.
How does Tandem Mass Tag work?
Isobaric tags for relative and absolute quantitation (iTRAQ) or Tandem Mass Tags (TMT) is a quantitation method in which peptide N-terminus and side chain amines are covalently labeled with tags of varying masses. Up to 16 different samples or treatments can be analyzed using this technique.
Why is SILAC quantitative?
SILAC is known to be a very accurate and precise quantitative method,13 likely because it allows the mixing of differentially labeled samples early in the experimental workflow, reducing variable sample losses from each experimental step.
What are Labelled amino acids?
Stable Isotope Labeling by/with Amino acids in Cell culture (SILAC) is a technique based on mass spectrometry that detects differences in protein abundance among samples using non-radioactive isotopic labeling. It is a popular method for quantitative proteomics.
How do you identify isotopes?
The relative abundance of each isotope can be determined using mass spectrometry. A mass spectrometer ionizes atoms and molecules with a high-energy electron beam and then deflects the ions through a magnetic field based on their mass-to-charge ratios ( m / z m/z m/z ).
What is the difference between iTRAQ and TMT?
iTRAQ (isobaric tagging for relative and absolute quantification) is available in 4-plex and 8-plex formats, while TMT (tandem mass tags) is available in 2-plex, 6-plex and (since recently) 10-plex formats.
Why do we label TMT?
Among those, stable isotope labeling of peptides using isobaric reagents such as tandem mass tags (TMTs) enables multiplexing of up to 11 samples (2). Each of these 11 tags can be used to label primary amines in peptide digests via the reaction with the NHS ester-based reactive group.
What is super SILAC?
Specifically, the super-SILAC technique uses a mixture of SILAC-labeled cells as a spike-in standard for accurate quantification of unlabeled samples, thereby enabling quantification of human tissue samples.
How does isotope analysis work?
Stable isotopic analysis looks at the isotopes—atoms with extra or missing neutrons—of different elements. Unlike unstable isotopes such as carbon-14, which degrades over time, stable isotopes never decay. There are over 250 known stable isotopes, and 80 of the periodic table’s first 82 elements have them.