Proteome Quantitation (iTRAQ, TMT, Label-Free)

iTRAQ

iTRAQ technology coupled with 1D or 2D LC-MS/MS for multiplexing 4-8 samples simultaneously in a single experiment is available. iTRAQ enables relative and absolute quantitation of proteins and peptides by labelling samples with isotope encoded reporter ions. Differential expression of proteins of interest can be determined.

The protein sample is acetone precipitated, digested and labelled. Four (or eight) different samples can be labelled with iTRAQ reagents. Labelled peptides are first separated by High pH reverse phase chromatography with fraction concatenation. Each concatenated fraction is further analysed by LC-MS/MS. Results are compared using a specialised software.

Request Form: iTRAQ Analysis

For more information please refer to PI publications:

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TMT

Tandem mass tag (TMT) technology coupled with 1D or 2D LC-MS/MS for multiplexing 10 samples simultaneously in a single experiment is available. TMT enables relative and absolute quantitation of proteins and peptides by labelling samples with isobaric chemical tags providing multiplexing capabilities for relative quantitative proteomics analysis. Differential expression of proteins of interest can be determined.

As with iTRAQ labelling, the protein sample is precipitated, digested and labelled.Once labelled, all samples are mixed and analysed in a single LC-MS experiment. Because the isobaric tags possess the same chemical properties, all peptides from different TMT-labelled samples co-elute during LC separation. Once the peptides enter the mass spectrometer, they are detected simultaneously as a single and indistinguishable precursor ion peak.

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Label-Free

Our label-free quantitation service offers relative quantitation of hundreds to thousands of proteins across sample sets. This technique uses data dependent mass spectrometry to sequence and compare the abundance of 6-25 amino acid chains between two or more biological samples.

When compared to label-based quantitation (TMT, iTRAQ, etc.) label-free quantitation typically has greater dynamic range and greater depth of analysis. While data is typically presented as a relative quantitation, a number of techniques aimed at estimating the absolute abundance of each protein within a sample can also be performed. This analysis allows estimates of the composition of protein samples i.e. what fraction of the total each protein species accounts for.

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Frequently Asked Questions

What amount of protein is required?

The optimal amount for an iTRAQ experiment is 50 µg tagged protein. Losses during sample cleanup can be considerable and are sample specific. We therefore recommend that 200-500µg of sample is provided; greater amounts will improve sensitivity for low abundant proteins.

What type of samples can be analysed?

Cell lysates, secreted protein samples, but not radioactive samples.

# Note: secreted samples must be in serum/plasma free media.

What is the maximum volume of sample?

The sample volume must be less than 250 µL.

Can I use protease inhibitors?

Yes, protease inhibitors will minimise protein degradation.

Is it important that the proteome you are looking at is in currently available databases?

Yes, as proteome analysis will not work on samples from an unknown genome.

 

Please refer to our FAQs brochure for more details.

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