Use of Protease Inhibitors and Other Reagents
Use of Protease Inhibitors and other Reagents
If samples are quickly kept frozen after cell lysis, protease inhibitors may not be necessary; however, if you must use protease inhibitors during your sample preparation procedures, please use the recommended protease inhibitors below. AVOID inhibitors such as AEBSF and others (see below) that (a) inhibit trypsin or other proteolytic enzymes used to prepare proteins for Mass Spec analysis or (b) modify proteins covalently (as this changes their masses and makes MS identificaiton more difficult).
Protein solutions that will be run on gels MAY be prepared with lysis buffers containing protease inhibitors that do not modify protein masses, since the protease inhibitors will not impact subsequent in-gel digestion and LC/MS/MS analysis (no inhibition of subsequent intentional protein degradation for mass spec prep.
Recommended Protease Inhibitor cocktail IF YOUR SAMPLE REQUIRES PROTEASE INHIBITORS:
PMSF (100 mM), phenanthroline (1 mg/ml), benzamidine HCl (100 mM), pepstatin A (1mg/ml). Add about 1/1000th to 1/500th volume of that stock to your lysis buffers, then ideally remove by dialysis into ammonium bicarbonate (use TEAB instead of ammonium bicarbonate for iTRAQ analysis experiments) before submitting the protein for mass spec analysis (keep solution frozen after dialysis/removal of inhibitors).
Potential Problems with Protease inhibitors: Some protease inhibitors can affect mass spectrometry analyses and should be not be used unless absolutely required by your particular samples and experimental goals (please discuss with Mass Spec Faciity staff).
For example, many protease inhibitor cocktails contain AEBSF (4-(2-aminoethyl)-benzenesulfonylfluoride, a serine-protease inhibitor that both covalently modifies proteins (shifting their peptide masses and therefore changing their detectability by mass spec) AND will inhibit trypsin used in mass spec sample prep. (Adds a mass of 183 preferentially to tyrosines, and also to lysines, histidines, and N-terminal amines). While the trypsin inhibitory effects can be removed by gel separation or ZipTip or other pre-digestion cleanup steps, any covalent modifications on proteins in your sample would still remain.
TLCK and TPCK are also serine-protease inhibitors, so they can inhibit the activity of trypsin, chymotrypsin, and other enzymes.
EDTA should not be used in sample preps with experimental goals of phosphorylation detection when IMAC purification will be used to enrich for phosphopeptides.
Importance of listing all sample treatments, kits, and chemical exposures for proteomic samples:
Both commercial protease inhibitor cocktails and commercial protein preparation kits may contain reagents that derivatize proteins, urea treatment in preparation for trypsin digestion can carbamylate peptides, and some reagents such as detergents can inhibit mass spec ionization and sensitivity. Because these derivatized peptides have different masses, it is crucial that ALL reagents, buffers, kits, and chemical exposures used in sample preparation be listed on your Sample Submission Sheets, so that MS Staff can properly clean up and analyze your samples. Identification efficiency may suffer or even be lost completely if MS staff are not made aware of all reagents and steps your samples have been exposed to.