Protein identification

From single gel bands: The gel band is subjected to in-gel proteolytic digestion using our automated DigestPro digestion unit to minimise sample handling. The resulting peptides are analysed by MALDI-Tof MSMS using a Bruker Daltonics UltrafleXtreme 2 mass spectrometer. Database searches are performed using an in-house Mascot server to identify the protein.

Sample submission: The gel slice should be excised as close to the stained band as possible and then placed into an eppendorf tube with water to prevent the gel from drying out. Bands can be sent to the facility at room temperature.

From complex mixtures: We offer two workflows for protein identification from complex samples:

Gel walking: The sample is separated using a 1D SDS-PAGE gel and the gel lane cut into slices. Each slice undergoes in-gel digestion using our DigestPro automated digestion unit to minimise sample handling and the resulting peptides from each gel slice are analysed by acidic RP nano-LC MSMS using our LTQ-Orbitrap Velos or Orbitrap Fusion Tribrid mass spectrometers.

In solution digestion: Alternatively, the sample is subjected to in-solution proteolytic digestion and the resulting peptides fractionated by basic RP chromatography, Strong Anion eXchange (SAX) chromatography or Strong Cation eXchange (SCX) chromatography. Each fraction is then analysed by RP nano-LC MSMS as above.

For each approach, database searches are performed using an in-house Mascot server. The data from each gel slice or LC fraction is pooled to generate a single report listing the proteins identified in the sample.

Please contact us for advice on how to prepare and submit your samples for protein identification analysis.

Edit this page