Mass Spectrometry Standards

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Horse Myoglobin

Trypsin Digestion of Horse Apomyoglobin
 

Intro:

A trypsin digest of apomyoglobin is easy to make, and useful as a peptide mapping system suitability standard.  Below are a number of points why myoglobin digest is our favorite peptide standard.

1. A trypsin digest of myoglobin is inexpensive to prepare, and stable.
2. A reduction and alkylation is unnecessary, because horse myoglobin lacks cysteine, this is also true for bovine beta casein. The lack of cysteines makes the digest protocol extremely simple for these two proteins.
3. A myoglobin trypsin digest results in relatively few peptides. If you run this standard daily, you soon become familiar with the peptides, and their elution order. With such a simple mixture you can tell at a glance if your mass spectrometer is performing properly.
4. Relatively few large pieces are left after the digest, making a greater percentage of the peaks LC/MS and MS/MS friendly, this is not so true for beta casein.

Note: Myoglobin containing the heme is difficult to digest, apomyoglobin, or myoglobin without the heme, is easy to digest.  Myoglobin with the heme intact is brown in color, because of the iron.  You can acidify the myoglobin to have it release the heme and then purify it away, but it is easier to just buy apomyoglobin.

Materials:

Our vendor of choice for intact myoglobin is Sigma-Aldrich  They sell 60 nmol vials of horse apomyoglobin, Sigma-Aldrich product number A8673-1VL and  A8673-5X1VL . Sigma-Aldrich web reference

Trypsin: Any trypsin will do, we routinely use Promega modified trypsin, but Worthington bulk trypsin works just as well.  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 a 60 nmol vial of horse apomyoglobin 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.  We have not found it necessary to place the digest at 37C.  Trypsin will act slower at ambient temperature, but overnight is more than enough time for the reaction to go to completion.  In addition the elevated temperature, 37C, will accelerate oxidations carbamylations, and deamidations,

Method Notes:

1. Always use apomyoglobin, since the myoglobin with the intact heme does not digest well.
2. 60 nmols of myoglobin is about 1017 ug.
3. The enzyme to substrate ratio in this procedure is approximately 1:50 (20ug/1017ug).
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+).

How it is used as a standard:

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 system suitability standard.  We typically run this standard at 50 to 100 fmols per injection.  If we are doing very sensitive work (silver stained gel protein ID) we will typically run this standard at 5 to 10 fmol.  We routinely bracket our samples with this standard, so we can say, "Our instrument was performing before and after your sample."
3. It is useful as an internal standard.  If you have a difficult, client... we mean project, you can dope the sample with ~5 fmols of the myoglobin standard.  Make sure first that myoglobin will not interfere with the analysis. That is, make sure your client is not looking for myoglobin. You will then learn to relish the moment when you can say, "Where is your protein? There's my protein standard at 5 fmols!"
4. 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 infusion. We have not tried the digest made in Tris for this purpose; the salt might interefere.
5. It is even useful as a MALDI standard after dilution, without desalting.

 

Example of Myoglobin ESI MS Peptide Map

 

 

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