In Mass Spectrometric Peptide Mapping, the protein/antibody is cleaved into smaller peptides using a specific protease. The peptides are analyzed by mass spectrometry and the observed peptides correlated to the protein/antibody amino acid sequence.
The identified peptides in the peptide map thus confirms the specific amino acid sequences covered by the peptide map, as well as the identity of the protein/antibody. Peptide mapping using a single protease will typically give a sequence coverage of 30-90%. By using two or more proteases this coverage can be increased to 95-100%.
Detailed characterization of a protein/antibody requires careful investigation of the protein/antibody sequence to select the best proteases, and choice of nano-flow LC-MS/MS or standard flow UV LC MS/MS to observe the peptides. The difference between the two techniques is related to the amount of material needed for the analysis – nano-flow can be conducted on low microgram protein/antibody whereas the high-flow analysis requires at least 20 microgram per analysis. The high-flow analysis include a UV-trace of the chromatogram.
The peptide mapping using nanoLC-MS/MS is performed on a Dionex 3000 system running at 300 nl/min equipped with C18 capilary column connected to a high-resolution Q-TOF instrument (Bruker Impact HD). The instrument is running either with a short or long gradient, and the Q-TOF set to data dependent analysis mode (DDA), switching between MS and MS/MS mode.
The analysis provide a Base Peak Chromatogram, and all MS/MS data is collected for peptides with mass/charge from 200-1600 Da.
High-flow LC-MS/MS with UV
The peptide mapping using high-flow LC-MS/MS is performed on a Agilent 1200 system running at 0.2 ml/min equipped with C18 column connected to a high-resolution Q-TOF instrument (Bruker Impact HD). The instrument is running either with a short or long gradient, and the Q-TOF set to data dependent analysis mode (DDA), switching between MS and MS/MS mode.
The analysis provides both a UV- and a Base peak chromatogram, and all MS/MS data is collected for peptides with mass/charge from 200-1600 Da.
Protein/Antibody analysis by mass spectrometry requires a pure protein/antibody in sufficient amounts to obtain good data. It is important that samples are prepared in a clean laboratory to avoid contamination with human keratin.
The samples can be submitted in liquid or lyophilized form.
Purify the protein/antibody
- The chromatographic purity should be >90%
- Avoid detergents and keep buffer concentration at a minimum. LC-ESI MS can be done on samples containing small amounts of salts, urea or detergent, but the best result are obtained with low buffer strength in volatile solvents without detergents
- Minimum sample amount: ~ 10-50 microgram protein or ~ 0.5 milligram antibody.
Put the antibody sample into microcentrifuge tube
- Use a Eppendorf Safe Lock tube supplied, or similar tubes from your lab
- Lyophilize or speed vac the antibody to a solid sample to ensure stability during shipment
- Alternatively, freeze or refrigerate to +4 oC for cold shipment of the liquid sample