Proteomics > Which service should I request? > Proteome Identification and Quantification > Protein / Peptide Identification
Proteomics > Which service should I request? > Characterization of Purified Biomolecules > Confirmation of protein identity (same as Protein / Peptide Identification)

Protein / Peptide Identification

General description

Mass spectrometry (MS) can be used for the identification of proteins/peptides in complex mixtures or in purified samples (e.g., peptide mapping). This service allows, among others, to identify proteins from gel bands and/or from purified fractions, to detect the quality and the complexity of a protein sample. A database containing all the protein FASTA sequences of the studies organism is required to perform these analyses.

Please note, if you are interested in:

• identification of interaction partners -> check our Interaction Proteomics page. Replicates and proper experimental design are required for this analysis.
• confirmation of a protein by determination of its mass -> you should select the Service Type Characterization of Purified Biomolecules and select the service accordingly

Workflow

Based on the biological question and on the type of sample, a typical workflow for processing the samples prior to MS and data analyses consists of:

1. Protein digestion

The first step for the identification of unknown proteins is protein digestion using proteases (e.g. trypsin). We can process samples in the following forms:

Sample form	Examples of digestion protocols	Comments
Solution	in solution /TCA precipitation, microwave-assisted	Whenever possible, use volatile buffers. Limit the amount of SDS and avoid CHAPS/Tween.
Gel bands	regular trypsin digestion from Coomassie Blue stained gel bands (whenever possible avoid silver staining)	please cut the bands carefully, avoiding large excess of "empty" gel. Provide them in a tube w/o any liquid
Dry	resolubilization + in-solution digestion	please provide the estimated concentration and mention potential salts
Beads (ideally magnetic ones)	on-beads digestion / SP3 digestion, microwave-assisted	Please note that IgG/Protein A/.. would be extensively identified in your samples. Just cover the beads with Tris / Ammonium bicarbonate
Tissues/cell pellets	Lysis + PreOmics iST protocol	only upon agreement.

Depending on the type of samples:

• Protein mixtures. Minimal recommended amount: 10 µg (less material can be used)
• Confirmation of protein identity ("purified" protein). Minimal recommended amount: 50 – 250 fmol (optimal amount: 1 pmol)

2. LC-MS analysis

At FGCZ, the default LC-MS approach for protein identification is based on:

• data acquisition on one of our Q-Exactive systems (Thermo, see Planetorbitrapusing Data Dependent Acquisition (DDA - also named Shotgun proteomics).
  • In the case of experiments that require higher sensitivity, the data would be acquired on newer generation instruments such as the Orbitrap Lumos(Thermo)
  • See all out instruments in our Instrument Intranet page)

3. Data analysis

For the identification of proteins, the information within the acquired raw files is extracted and matched against protein FASTA databases using search engines.
Some important points:

• if a protein is not in a database, it cannot be identified using the standard approaches
• for common organisms (e.g., human, mouse, ...) the databases are well-annotated and reviewed. We mainly use Uniprot databases.
• examples of search engines are MSFragger (through FragPipe), MaxQuant, Mascot, PEAKS, Byonic.
  • For most experiment we use FragPipe
  • For user-specific sequences, we normally use PEAKS or Byonic, adding the custom sequence to the database of the organism