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Mass Spectrometry Unit

Samples submission: Guidelines for Proteomics sample submission/prep

PROTEIN ID

Samples submitted for mass spectrometry analysis can come in:

• gel slices
• liquid solutions

PROTEIN ID/PTM DETERMINATION FROM GELS:

1. Reduce contamination from keratin other proteins. Here's how:
   • Tips to avoid keratin contamination in gel and sample preparation - Feb 08 [.ppt, 3.7 Mb] Contamination, primarily in the form of proteins present on the fingers and hair of investigators, lab dust should be avoided.
   • Always wear gloves when handling samples
   • Never touch gels directly with bare fingers or leave gels exposed to dust in the lab atmosphere. This includes any apparatus involved in the preparation of gels or any surfaces that come into contact with gels. For example, use ethanol-soaked kimwipes to clean working area/aparatus.
   • Tubes, pipette tips and other supplies that come into contact with the sample should come from keratin-free, clean closed plastic bags/boxes prior to use.
   • Hair out of the way to reduce keratin contamination (keratins are omnipresent ) & use unopened containers, or ones that have been rinsed several times w/EtOH/H2O.
2. Use MS-compatible gel staining procedures (external link)
   It is strongly recommended that you use Coomassie blue (or Colloidal Coomassie blue) stains rather than Silver. If you can not visualize bands with Coomasie try to scale up your isolation to increase the amount of protein. Silver stain, with its lower detection limit may not contain sufficient protein for MS detection. Ag ions (a) inhibit trypsin digestion by binding at the active site of trypsin and chymotrypsin. (b) in the MS process, they induce ion suppression & background interference. If you do want to use silver stain make sure to use the MS Facility Ag stain protocol (external link)

PROTEIN ID/PTM DETERMINATION FROM LIQUIDS:

Reduce contamination from keratin & other proteins. Contamination, primarily in the form of proteins present on the fingers and hair of investigators as well as dust in the lab should be avoided. Some general recommendations:

• always wear gloves when handling samples
• the buffer should contain no urea, no detergents
• see Protein precipitation protocols for cleaning samples

PROTEIN INTACT MOLECULAR WEIGHT DETERMINATION:

Samples should be in liquid form. Some general recommendations:

• need a minimum of 20 uL of protein, at a concentration of at least 5 nanogram/uL
• the buffer should contain no urea, no detergents
• see Protein precipitation protocols for cleaning/concentrating a protein sample for mass spec submission

References/Suggested literature:

1. Ag stain: Shevchenko et al. (1996) Analytical Chemistry, 68:850-858
2. Detergents: Funk et al. Threshold values for detergents in protein and peptide samples for mass spectrometry Rapid Commun. Mass Spectrom. 2005; 19: 2986-2988