Common protein contaminants in mass spectrometric protein ID

Mass spectrometry analysis of protein
Alphalyse
Jun 3. 2016

Questions:

Why was Keratin identified in my 1D gel band?

  1. Is the keratin determined by the protein identification service in my 1D gel band contamination? Or is it a relevant human protein from the sample?
  2. How is it possible that only Keratin was identified in the sample although the protein band was clearly visible?
  3. Does this mean that keratin was the only protein in the sample? Or is the identification of other proteins impossible when even small amounts of keratin are present?
  • The size of my protein and band cut from the gel was 50-55kDa. Keratin is 66kDa, so how did Keratin get into the sample?
  • Is there even the slightest possibility that something happened in your lab?

These are just a few things that come to my mind as I’m thinking about what might have gone wrong. Please help me with these concerns before I start the whole process once again.

Answer:

Keratin identified in a gel band might be a natural protein purified from the cells. But more frequently, it is a contamination that occurred somewhere during protein purification and sample processing. Keratin is present in all dust in the lab, from dead skin cells from humans and small pieces from your woolen sweater [1, 2].

If the keratin contamination occurs before the gel electrophoresis, we observe it as protein bands in the gel around 55 kDa and 65 kDa. When identifying this keratin, we see a high score and good sequence coverage and often identify several keratin types. Since your gel band was observed at Mw 55 kDa, it is either contamination that happened before the electrophoresis or the keratin was purified from the cells [1, 2].

Keratin contamination can also occur after the electrophoresis during gel staining or gel scanning and spot excision if dust comes in contact with the sample. In such cases, the keratin amount is usually lower, and you only observe a few keratin peptides in the mass spectra. It is still possible to identify the main protein component in the gel band [1-4].

At Alphalyse, we take extreme care to work in a clean, dust-free environment. We handle all samples in 96-well plates together with quality control standards. We rarely observe contamination from our lab.

The best advice to avoid keratin contamination is to prevent dust in the lab in general and always work with gloves. You should clean the electrophoresis and staining equipment and keep them free of dust. Also, remember to close the lids on pipette tips.

References:

[1]          Hodge et al.: “Cleaning up the masses: Exclusion lists to reduce contamination with HPLC-MS/MS,” Journal of Proteomics2013

[2]          Keller et al.: “Interferences and contaminants encountered in modern mass spectrometry,” Analytica Chimica Acta, 2008

[3]          Rappsilber et al.: “Large-Scale Proteomic Analysis of the Human Spliceosome,” Genome Research, 2002

[4]          The Common Repository of Adventitious Proteins, cRAP, is a list of proteins commonly found in proteomics experiments present either by accident or through contamination of protein samples.

More posts about Database Searching & Protein Identification
  • What does the Mascot Score mean?

  • Eppendorf tubes with protein

    Mass spectrometric identification of proteins from Western blots

  • Mass spectrometry analysis of protein

    Common protein contaminants in mass spectrometric protein ID

  • Protein molecular weight calculator

    GPMAW lite – Protein Sequence Calculator (and much more)

  • Speedy and detailed protein ID of SDS gel bands

About Alphalyse

Alphalyse uses expertise in the field of protein chemistry and bioinformatics, combined with top-of-the-line mass spectrometry equipment, to provide protein analysis services globally.

Alphalyse aims to deliver high quality data and customer service to all of our clients in a fast and convenient way.

www.alphalyse.com

Newsletter Subscribe to The Alphalyse Newsletter and get more
information on protein analysis methods and applications
© 2021 Alphalyse, Inc.