Mass Spectrometry Standards

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Bovine Beta Casein

Trypsin Digestion of Bovine Beta Casein
 

Introduction:

A trypsin digest of beta casein is easy to make, and useful as a phosphorylated peptide standard.  Although, some may comment that beta casein is so well phosphorylated that it does not well represent the state of phosphorylation in other proteins. A reduction and alkylation is unnecessary, because bovine beta casein lacks cysteine. The lack of cysteines makes the digest protocol extremely simple for this protein.

Materials:

Our vendor of choice for intact beta casein is Sigma-Aldrich  They sell 250 mg and 1 gm vials of bovine beta casein, Sigma-Aldrich product number C6905-250MG and  C6905-1G . Sigma-Aldrich web reference

Trypsin: Any trypsin will do, we routinely use Promega modified trypsin, but Worthington bulk trypsin works just as well.  We never worry about autolysis products interfering, because even with a 1:20 digest using normal trypsin, the autolysis peaks would be very small.  Trypsin autolysis products will be an issue when you start doing protein ID, where the enzyme to substrate ratio is typically higher.

Protocol:

1. Reconstitute 60 nmol or 1.414 mg of bovine beta casein in 6 ml of 10 mM ammonium bicarbonate (NH₄HCO₃). The ammonium bicarbonate is not adjusted for pH.  Tris base, pH 8, can be substituted for ammonium bicarbonate in this step.
    
2. Add  20ugs of  Promega modified trypsin, and let sit on the bench top, or in a drawer at ambient temperature.  By the next morning, 15-18 hrs, your digest will be ready to use.

Method Notes:

1. 60 nmols of beta casein is about 1414 ug.
2. The wt/wt, enzyme to substrate ratio in this procedure is approximately 1:70 (20ug/1414 ug)
3. The trypsin, enzyme to substrate ratio can range from 1:20 to 1:200 with little effect, the higher the enzyme content the faster and more complete the digest.
4. The final concentration of this digest is 10 pmol/ul
5. We store this digest at -20C, aliquoted, and it seems to be good forever (1yr+).

Usefulness:

1. Add 2ul of this standard to 1 ml of your HPLC solvent A (water, 0.1% formic acid) to get an ESI LC/MS standard that is 20 fmol/ul
2. It is useful as an LC/MS phosphorylation system suitability or phosphorylation test standard.  We typically run this standard at 50 to 100 fmols.
3. Since the resulting standard is at such a high concentration you can often dilute 1:10 or 1:100 for infusion to tune ESI mass spectrometers, without any desalting.  We typically use the digest done in ammonium bicarbonate for this purpose.
4. It is even useful as a MALDI standard after dilution, without desalting.

 

Example of beta casein ESI MS Peptide Map (coming)

 

 

 

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