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

Question:

How can I improve label-free quantification of HCP?

I find it difficult to get an accurate label-free quantification of Host Cell Proteins with traditional multiple reaction monitoring using 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 a normal procedure to include an isotope-labeled peptide standard to obtain good HCP data [1].

Another solution is using chemical labeling (like iTRAQ® or SILAC) [2]. Some people even use isotope labeled recombinant proteins. You do this to consider the digestion efficiency of 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 the identification and quantification of HCPs [4].
• Accurate absolute quantification of HCPs is now obtained by adding 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.