Intact protein mass spectrometry

Abstract

Intact protein mass spectrometry can be used as a technique to validate the successful purification and verify the mass of a recombinant protein. While not as high resolution as other forms of mass spectrometry (resolution ~ 1 Da), the version of intact protein mass spectrometry described in this protocol is sufficient to check for the presence of truncated products, amino acid substitutions or oxidations, and post-translational modifications. (This detailed protocol was written based on the work published in the following paper and might need to be modified for your specific protein of interest: Horst, B.G. et al. Allosteric activation of the nitric oxide receptor soluble guanylate cyclase mapped by cryo-electron microscopy. eLife, 8, e50634 (2019), https://elifesciences.org/articles/50634)

Materials and Reagents

• Purified Protein of Interest (POI)

• Buffer A (see recipes and Note 1)

• Vivaspin 500 spin concentrator (Sartorius)

• 0.22 µm spin filter (Millipore Sigma)

• Formic acid (FA, Millipore Sigma)

• Water (HPLC Grade, Millipore Sigma)

• Acetonitrile (HPLC Grade, Millipore Sigma)

Equipment

• Pipettes

• Centrifuge

• 1200 HPLC System (Agilent)

• 6224 Time-of-Flight (TOF) Mass Spectrometer (Agilent)

• ProSwift Column (Thermo Scientific)

Procedure

1. Purify your protein of interest (POI) to greater than 95% homogeneity.

2. Exchange your POI into Buffer by pipetting the POI into a Vivaspin 500 spin concentrator and diluting the volume to 500 µL. (See Note 2)

3. Concentrate the protein to under 100 µL by spinning in a centrifuge at the recommended angular velocity.

4. Repeat steps 2 and 3 two more times for a total of three dilution and concentrations. Final volume should be approximately 50 µL

5. Filter the POI through the spin filter.

6. Measure the protein concentration and adjust so the final protein concentration is between 1-5 µM

7. Separate samples using an Agilent 1200 HPLC system using a conditioned ProSwift Column. Elute proteins from the column over 12 minutes by gradient elution from 95% water + 0.1% FA to 95% acetonitrile + 0.1% FA

8. Analyze intact proteins using an Agilent 6224 time-of-flight mass spectrometer with a Turbospray ion source in positive ion mode.

Data Analysis

Protein masses were deconvoluted using the Agilent Masshunter B.0.5 software suite and the Maximum Entropy algorithm.

Recipes

Buffer: 25 mM Hepes, pH 7.5, 25 mM NaCl (See Note 1)

Notes

1. Buffer concentration will depend on the stability of the POI; however phosphates and borates should be avoided as they are not soluble in acetonitrile.

2. Ensure the MWCO is at least three times smaller than the molecular weight of your protein to avoid sample loss. For example, for a 10 kDa protein, use a 3,000 MWCO filter or smaller.

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