Simple way to perform label-free quantification of Host Cell Proteins

1000 proteins quantified in e. coli mock lysate - HCP
Alphalyse
Jun 25. 2018

Question:

 

I find it difficult to get an accurate label-free quantification of Host Cell Proteins with LC-MS. It also seems that my data is difficult to reproduce. – Even if I perform multiple LC-MS runs for each sample.

Is there anything I can do to improve on the quantification and the reproducibility of the data?

 

Answer:

 

For quantitative proteomics experiments it has been normal procedure to include an isotope labeled peptide standard to obtain good HCP data [1].

Another solution is the use of chemical labelling (like iTRAQ® or SILAC) [2]. Some people even use isotope labeled recombinant proteins. You do this to consider the digest efficiency on a full length protein [3].

All of the above methods are time-consuming – and they are not always possible to use [2].

So what can you do instead?

 

With the new SWATH® LC-MS technology, label-free quantification of HCPs is no longer a problem

The reasons for this are:

  • SWATH® analysis is a data-independent acquisition method. This results in very reproducible MS data – both for identification and quantification of HCPs [4].
  • Accurate absolute quantification of HCPs is now obtained by the addition of standard proteins. They are added in known amounts and are digested within the sample.
  • The SWATH® LC-MS is running at robust microflow rates. Therefore, there is no longer a problem with time-consuming nanoflow HPLC or carry-over.

 

Label-free quantification of thousands of proteins

In order to test the new LC-MS method, we applied SWATH® label free quantification to an E. coli cell lysate. With this method we could identify and quantify more than 1.000 proteins, up to a total sum of 969.158 ppm. The expected sum is 1.000.000 ppm – see graph below.

 

1000 proteins quantified in e. coli mock lysate - HCP

 

The analysis is so robust and reproducible that the standard deviation for individual proteins is below 20% in repeat experiments.

So to sum it up:
With the SWATH® technique it is now much easier to quantify proteins reproducibly. And in addition, it is done without isotope labeling.

 

More information on analysis of HCP:

The SWATH® technology is a form of data-independent analysis (DIA). You can learn more about the technology here: “What is SWATH”

Find out more on the identification and quantification of Host Cell Protein on the Alphalyse website: www.alphalyse.com/hcp

 

References: 

[1]          Zhu-Shimoni et al: “Host Cell Protein Testing by ELISAs and the Use of Orthogonal Methods“, Biotechnology and Bioengineering2014

[2]          Tscheliessnig et al: “Host cell protein analysis in therapeutic protein bioprocessing – methods and applications.“, Biotechnology Journal, 2013

[3]          Bracewell et al: “The Future of Host Cell Protein (HCP) Identification During Process Development and Manufacturing Linked to a Risk-Based Management for Their Control“, Biotechnology and Bioengineering2015

[4]          Heissel et al: “Evaluation of spectral libraries and sample preparation for DIA-LC-MS analysis of host cell proteins: A case study of a bacterially expressed recombinant biopharmaceutical protein.“, Protein Expression and Purification, 2018

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.

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