Protein/Peptide Mass Spectrometry Analysis

Protein Identification by Proteolysis followed by Mass Spectrometry and Database searching

Sample handling:

This procedure is sensitive to protein contamination from extraneous sources such as containers, reagents, and your hands and clothing. To avoid poor results it is important to keep everything that comes in contact with the sample as clean as possible. This requires the use of properly cleaned containers and wearing lab coats and gloves when handling samples. All reagents must be of high quality, as free of contaminants as possible, and freshly prepared.

Sample submission:

Samples can be submitted either as gel slices or as liquid samples. For the best results samples should be composed of as few proteins as possible, i.e. single band or spot from a gel, or a single protein in solution. Liquid samples should be as free of salts, detergents, buffers and non-volatile components as possible. See the table below for a list of components to avoid. Samples blotted to PVDF membranes can also be analyzed, but generally the sensitivity will be lower. Samples from Western blots will generally not yield useful results.

Liquid samples: Submit at least 10 picomol in no more than approximately 20 microliters

Gel pieces: Submit at least 50 picomol of protein. It is not necessary to completely destain the gel piece

Database searching:

Please provide any additional information on the protein sample, such as apparent molecular weight, name of organism, etc. This will facilitate the database search for protein identification.

Intact Protein Analysis by Mass Spectrometry

Sample handling:

To avoid losses from adsorption to surfaces, use clean polypropylene tubes. Avoid polystyrene, glass or autoclaved containers.

Use only freshly prepared, high purity reagents and solvents. Mass spectrometers are sensitive to ionic contaminants from any source including buffers, detergents, bacteria, molds, etc.

Wear gloves and lab coats when handling and transferring samples. Proteins from your hands can contaminate your samples.

Sample submission:

Protein samples for intact mass analysis should be dry or in solution and as concentrated as possible (at least 2.5 microliter @ 15 pmol/microliter for MALDI-Tof and 10 microliter @ 1 pmol/microliter for LC/MS-ESI-Q-Tof analysis). Intact mass analysis cannot be performed on proteins from gels or blots. If possible, submit samples in 200 microliter microtubes in order to maximize sample recovery.

Protein samples should be as free of salts, detergents, buffers, and non-volatile components as possible. Volatile solvents are generally acceptable.

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@illinois.edu