Q: What are common contaminants that could affect my protein sequencing results?

A: Typical contaminants that we see affecting samples are non-target proteins, and non-target antibodies [1, 2, 3].

Protein Contaminants

The most common protein contaminants include keratin and serum albumin [1, 2, 3]. Other Mass Spectrometry (MS) protein contaminants identified in research labs worldwide by a recent Human Proteome Organization study are casein and E. coli proteins [3]. Keratin is an epidermal structural protein that is found on the outer layer of skin, hair, nails and eventually in dust [1, 3]. Keratin is especially difficult because its size ranges between 40 – 70 kDa for both human and bovine keratin [4] – a common contaminant of cell culture reagents. This size can be confounded with the 50 kDa heavy chain of antibody samples sent for protein sequencing. As such, an antibody sample may look perfect in gel, but still have keratin contamination. Bovine serum albumin (BSA) is commonly used during routine life science experimentation, from protein quantification, Western blotting to even PCR [1, 3]. Casein is a milk-based protein that will also be present during Western blotting [3]. Finally, E. coli proteins might occasionally be pulled down during purification experiments of the target recombinant protein and/or antibody [3].

Here are some ways that you can avoid introducing these protein contaminants to the protein sample you wish to sequence [1]:

  • Maintain a clean lab area, free of dust
  • Make sure your tip boxes are closed, and if possible, use disposable, filter pipette tips
  • Routinely clean your electrophoresis, staining, microcentrifuge and speed-vac equipment to avoid introducing contaminant particles through contact or aerosol
  • Always wear gloves
  • Use only High-Performance LC (HPLC)-grade reagents
  • Try to perform protein preparation under laminar flow hoods, or in clean, dust-free, low-air turbulence environments

Luckily, these protein contaminants do not typically affect the spectra as they have distinct, recognizable signatures compared to the target proteins or antibodies [1, 2, 3]. Thus, they can be quickly identified in an initial MS run and often can be separated from your sample using standard purification methods that include HPLC [1, 2, 3]. Furthermore, we take pride in our lab practice and care. We keep a dust-free, clean environment and routinely clean our equipment with the appropriate solutions. We rarely come across contamination. However, we are highly equipped to face challenges. Our MS- and machine-learning-based protein sequencing assay is sensitive, robust and standardized. Our research core scientists have rich, diverse experience in protein chemistry, proteomics and in setting up MS labs national and internationally. We are confident we can pick up and account for potential contaminants. If you are concerned about protein contaminants, please feel free to reach out to us to discuss your protein sequencing project prior to embarking on it.

So why bother with these contaminants? Most of us don’t consider things such as BSA contaminants as they’re often buffer components. The problem, actually, lies in abundance, especially if their abundance drastically exceeds that of your target protein. Through the removal process, the yield of your target protein might decrease. If higher concentration of contaminants is detected, a higher quantity of your target protein may be required. Stay tuned for the next mAb Mondays’ Q&A, where we tackle protein sample quantity and purity!

Antibody Contaminants

If antibodies other than your target antibody are present in your sample, it might be difficult to discern between them with a reducing SDS-PAGE acrylamide gel because the banding pattern of each antibody would be overlaid on top of one another. HPLC may be used to separate these additional antibodies, but they have to be sufficiently different in mass, charge and other properties. Once in the mass spectrometer, the digested contaminant and target antibodies might also be difficult to tell apart from one another. In the past, we have been able to sequence target antibodies from animals’ ascites fluid among the animal’s polyclonal background. Though sometimes manageable, oligoclonal samples are challenging. Please contact us if you suspect antibody contaminants in the antibody sample you wish to sequence.

 

Maria Rosales Gerpe, Ph.D
Scientific Writer
Rapid Novor, Inc.

 

References

[1]K. Hodge, S. T. Have, L. Hutton and A. I. Lamond, “Cleaning up the masses: exclusion lists to reduce contamination with HPLC-MS/MS,” J Proteomics, vol. 88, pp. 92-103, 2013.
[2]J. J. Pitt, “Principles and Applications of Liquid Chromatography-Mass Spectrometry in Clinical Biochemistry,” Clin Biochem Rev, vol. 30, no. 1, pp. 19-34, 2009.
[3]A. W. Bell, E. W. Deutsch, C. E. Au, R. E. Kearney, R. Beavis, S. Sechi, T. Nilsson, J. J. Bergeron and HUPO Test Sample Working Group, “A HUPO test sample study reveals common problems in mass spectrometry-based proteomics,” Nat Methods, vol. 6, no. 6, pp. 423-30, 2009.
[4]H. Bragulla and D. G. Homberger, “Structure and functions of keratin proteins in simple, stratified, keratinized and cornified epithelia,” J Anat, vol. 214, no. 4, pp. 516-559, 2009.