Tandem mass tag technology is used to individually label different protein samples so they can be combined into a single sample for LC-MS analysis allowing for higher sample throughput, less instrument analysis time, high precision of peptide quantitation among replicates, and fewer missing quantified proteins among different samples.

Relative quantitation compares the amount of an analyte in different samples. Absolute quantitation measures the actual amount of an analyte in a sample using a known standard at different concentrations. Label-free, SILAC and TMT workflows are used for relative quantitation of protein samples. Heavy (i.e. AQUA) peptides are used as standard for absolute quantitation of target proteins.

TMT and TMTpro reagents are different chemicals with different masses, therefore it is not recommended to mix the two tags as the combined samples are significantly more complex resulting in fewer quantified proteins.

Label-free, TMT reagents, and TMTpro reagents can be used with any protein sample type. SILAC and Neucode can only be used with cell lines & model organisms that can be metabolically labeled.

SILAC can be used to multiplex 2 to 3 samples. TMT reagents can be used to combine 6 to 11 samples. TMTpro reagents can be used to measure up to 18 samples simultaneously.

This is most often done through use of a “pool channel” for each multiplex set. This reference channel typically contains an equimolar mixture of all samples which is labeled with one of the TMT tags that is shared among the sets. The pool channel can then be used to normalize relative protein abundance measurements across multiplexed sets.

It is not recommended to add protease inhibitors as they can affect trypsin and trypsin/Lys-C activity.

Both methods provide robust results for immunoprecipitation. Biotin tagged antibodies with streptavidin beads provide lower background but require more upfront sample prep, while protein A/G beads are robust and straightforward in use but result in higher background.

Digestion efficiency is optimized through the proper amount of added enzyme and digestion times, maintaining the correct pH for digestion, and avoiding protease inhibitors and strong denaturants.

Proteome Discoverer

Proteome Discoverer

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No, we will reduce samples with the appropriate reagent and ensure alkylation according to our standardized procedures.

Trypsin or Trypsin/LysC mix are most commonly used for proteomic applications in order to assure reproducibility and complete digestion. Other commonly used enzymes include Chymotrypsin, Pepsin, LysN, AspN, and GluC.

Yes, MS analysis can be used for evaluation of depletion performance. Protein yields after depletion level can be measured using the BCA assay.

There are many strategies to extract Membrane proteins (MPs) from eukaryotic cell lines and tissues, including subcellular fractionation (e.g., sucrose or sorbitol density ultracentrifugation), cationic colloidal silica absorption, and aqueous-polymer two-phase system. Commercially available kits can be also used for membrane protein extraction.