Gel-Based Protein Sequencing

Application	Service	Description
Gel-based sequencing	Label free TMTcalibrator TMT LC-MS2 TMT LC-MS3	Identification of proteins from discrete regions in SDS-PAGE and non-denaturing gels Part of unbiased discovery or enrichment of target proteins prior to SRM or PRM analysis Can be used for biotherapeutic analysis

Advantages

• Detection and quantification of proteins from a 1D or 2D gel band
• Human, rodent, monkey, pig and others all covered
• Compatibility with a wide variety of sample matrices, including tissue, serum, cell lysates, CSF, exosomes, etc.
• Includes full computational proteomic analysis and bioinformatics 

Protein Identification

Proteome Sciences offers this service to enable the identification of proteins in a gel band or 2-dimensional  gel spot. Generally this is designed for laboratories that have already run gels, but we can also run standard SDS-PAGE  and non-denaturing 1- and 2-dimensional gels if required.

For the greatest sensitivity towards your target you should cut the band close to the expected molecular weight region.  Please note; gels should only be stained with mass spectrometry-compatible stains. Typical gel band dimensions will be ~5mm x 2mm. We can get an identification with as little as 1ng of protein on gel.   Generally we would use a Label Free approach and a Orbitrap Mass Spectrometer, but for multiple samples where more precise quantification is required, different samples can be TMT labelled.

We can provide similar services for isolated proteins in solution e.g. following LC or capillary electrophoresis. We recommend submitting samples at a protein concentration of ≥1µg/µL in 10µL of buffer. As some detergents and other buffers can affect the performance of the Mass Spectrometer or interfere with the assay, please call/email so we can discuss before you plan sample preparation

Steps Involved

• Sample preparation: In-gel digestion with trypsin or another more specific protease according to assay needs
• Mass Spectrometry analysis: Digested samples are analysed with 0.5hr LC/MS2 runs
• Data analysis: Protein identification is performed with Proteome Discoverer

The turnaround time is typically 2-3 weeks.

Deliverables

• Report generation: will be PowerPoint/Word document listing the proteins detected and the raw MS data 
• An Excel workbook containing the detailed qualitative and quantitative protein identification data.

What’s included	Incoming sample QC Protein capture/elution, TMT labelling unless label free analysis Mass spectrometry-based peptide and protein ID Bioinformatics Report discussing the proteins sequenced in the form of a PowerPoint presentation An Excel based QuantSheet detailing qualitative and quantitative protein identification data.
Material required	This is sample and assay specific and will be detailed in the agreed Statement of Work - please contact us to discuss your specific needs so we can start the Statement of Work preparation
Typical turnaround time	Generally 2 to 3 weeks from receipt of samples and completion of incoming sample QC