Protein/Peptide Mass Spectrometry Analysis

Mass Spectrometry of Protein Digests (Peptide Mass Fingerprinting)

Sample from in-solution digests:
10 pmol of protein/peptide or more is required in solution or lyophilized
Protein in solution: as concentrated as possible; total volume 10-20 ml

Sample from in-gel digests:
A minimum of 50-100 pmol of protein subjected to digestion
Submitted peptide extract should not contain organic solvents (e.g. acetonitrile)

Please follow the Guidelines for MS Sample Preparation

Analysis is performed mostly by Matrix Assisted Laser Desorption Ionization (MALDI) or by Electrospray Ionization (ESI) technique.

For Sample Submission please download Protein Services Request Form. 

*FOR SERVICE FEES, please contact the facility at 217-333-4695 or email proteinsciences.uiuc.edu.

Turn-Around time: 1-3 working days

MALDI mass spectrum of a peptide mass map obtained from tryptic in-gel digest.

 
Mass Spectrometry Methods

Mass spectrometry measures gaseous ions (cations or anions) in a high vacuum environment. Different mass spectrometry methods are usually classified based on the way that gas phase ions are generated (i.e., ionization methods). The ionization techniques are important because biological molecules such as proteins, peptides, and oligonucleotides are not volatile by nature. Modern used ionization methods for these macromolecule analysis are Electrospray Ionization (ESI) and Matrix-assisted Laser Desorption Ionization (MALDI).

• Electrospray Ionization
  In ESI, a high voltage is applied to a liquid flow of sample containing solvent. The high potential disrupts the solvent into charged droplet, which will eventually result in gaseous ions during this process. A number of solvent can be used for dissolving samples in ESI, including CH3CN, CH3OH, CHCl3, H₂O, and mixtures of these solvents. The routine solvent system used in this laboratory is 50/50 CH3CN/H₂O. About 0.1% HCOOH is normally added to the sample to help increase ion production. Different from most other mass spectrometry methods, ESI generates predominantly multiply charged ions due to multiple protonation. For example, a protein with a mass 9,990 Da and 10+ charges will show up as mass 1,000 in the mass spectrum (i.e., the mass to charge ratio, (9,990+10)/10). There are computer programs that can do the calculation and generate a transformed spectrum on the real mass scale (for the above example, the transformed spectrum will show a component at 9,990 Da). The mass range for this technique exceeds 100 kDa. It typically requires 10 - 50 pmol of samples. The mass accuracy is normally 0.01% of the protein molecular weight. Non-volatile salts, buffers and detergent are harmful to this technique. Samples should be submitted in water or mixtures of water with acetonitrile and methanol.
  

• Matrix-assisted Laser Desorption Ionization
  MALDI uses laser as the energy source to generate gaseous ions. The operation requires the sample to be mixed and co-crystallized with a matrix The laser energy is absorbed by the matrix molecules and transferred to the analyte molecules imbedded in the matrix crystal, which results in the ion formation. The matrices are normally small organic molecules with strong absorption at the particular laser wavelength.  MALDI usually generate molecular ions with 1+ to 4+ charges. It is good for direct mixture analysis without HPLC separation. MALDI is also very sensitive and typically requires 1-10 pmol of samples. The mass accuracy is 0.01-0.05% of the protein molecular weight using the current time-of-flight mass spectrometer. It can tolerate moderate amount of salt. Excessive amount of salt is still harmful. Any contaminant or solvent that prevents the formation of matrix-analyte crystal will result in no spectrum.

Peptide Synthesis & Purification | Protein Sequence Analysis 
 2D Gel Electrophoresis

Protein Sciences Facility
Peter Yau, Ph.D - Director
315 Noyes Laboratory, 505 South Mathews Avenue, Urbana, IL 61801
Phone: (217) 333-4695     FAX: (217) 244-1142      
Email: proteinsciences@uiuc.edu