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Mass Spectrometry Sample Preparation


By far the most common contaminant in mass spectrometry is keratin. This protein comes from skin, hair and dust. It’s everywhere. It’s on everything. It will be in your sample.

However, the cleaner your sample, the more likely it is that your protein of interest (along with any PTMs that you may be interested in!) will be detected. So, always wear gloves. Never touch your tubes with bare hands. Wash everything (using soap and water) that will come into contact with your protein(s): e.g. gel boxes, glass plates, coomassie/silver staining containers. Use a fresh box of pipette tips, a fresh bag/box of eppendorf tubes, fresh blades, etc. Consider a small separate stock of “keratin-free” chemicals – you never know how many labmates have had their bare hands stuck in the Tris bottle!

Mass Spec-incompatible compounds

Some chemical compounds can also cause problems during MS detection.
  1. Detergents: design your isolation procedure so that you can get rid of SDS, Triton X-100, NP-40, Tween, CHAPS, etc., before MS.
  2. PEG: this compound and related polymers are observed very well by MS, and will likely obliterate the signal for your protein.
  3. DMSO, DMF, glycerol: to be avoided. These compounds are highly viscous, can be observed in spectra, and/or can interact with tubing and other machine parts.
  4. Acetic acid/plastic polymers: while acetic acid is often used in MS protocols (see below), it is imperative that you don’t contaminate your stock glacial acetic acid bottle with plastic. Don’t put plastic pipettes or pipette tips into the stock bottle; every time you do this, a little bit of plastic is released into the solution. Plastic polymers can thus easily build up to a point where we will begin to see them in your sample. Instead, measure out what you need with a clean glass graduated cylinder. You can also pour from the stock bottle into a separate container, and pipette from this – but dump this out after removing what you need.


We usually use about 1ug of trypsin to digest your protein sample prior to mass spectrometric analysis. Some commonly used compounds can kill trypsin activity.
Compound Allowable concentrations
SDS 0.1%
Guanidine HCl 0
2-mercaptoethanol 0
OG/NP40 2%
Urea 4M
Acetonitrile 40%

Volatile Buffers

Avoid salts and non-volatile buffers in your final purification steps, if possible. In most cases, we will simply dry your sample down in our speed-vac, and resuspend it in 50mM ammonium bicarbonate pH 8.3 for an overnight tryptic digestion. If your sample contains non-volatile salts or buffers, they will still be there. Besides interfering with the protease, these compounds can also suppress ionization of your peptides, affect chromatography, and form adducts that interfere with peptide detection. Consider using the following volatile buffers in your final purification step(s).

Triethylammonium phosphate pH 3
Ammonium formate pH 3-5
Pyridinium formate pH 3-6
Ammonium acetate (-pH with acetic acid) pH 4-6
Pyridinium acetate pH 4-6
Trimethylammonium formate pH 5-6
Triethylammonium acetate pH 6-7
Ammonium bicarbonate pH 8
Ammonium carbonate pH 8-10

Some acids and bases are also volatile. These are great, e.g. for protein elution from a solid support/column.

Volatile Acids   Volatile Bases
TFA   Ammonia
Acetic acid   Methylamine
Formic acid   Ethylamine
Carbonic acid   Triethylamine

Standard Lab Protocols

In Gel Tryptic Digest
TAP-tag Protein Purification
GST-tag Protein Purification and PreScission Protease Cleavage
Yeast - Transformation by Electroporation
Yeast - Lithium Acetate Transformation
Yeast - Extraction of Plasmid DNA
Yeast - Extraction of Genomic DNA
Formulation of Tris-Glycine Gels