Hi Naga,

why it needs to be intact?

I would recommend to do the absolute quantification with SRM using a labeled peptide, with a specific AA sequence for your light chain to spike in a known amount of it and compare the intensities of the peptides/fragments directly.

Best,

Murat

Hi Murat,

Clinical laboratories not accepting enzymatic digestion of proteins. So i am looking for intact light chain quantification.

Thanks!

Best,

Naga

If you already have a purified sample than you can analyse the content of your antibody in total using a standard protein assay like Bradford or BCA. The reemaining calculations can be done by simple stoichiometry ofeavy to light chain proportions.

I am a little confused about your statement that clinical laboratories are not accepting enzymatic digestion since I know colleagues doing clinical studies with MRM LC-Ms analysis with digested samples.

The best way for this is to use the MALDI-TOF MS configuration. For ESI top-down or native-MS-like approach is needed for the analysis of either heavy or light chains. You may combine SEC and native analysis for ESI-LCMS or gas phase fragmentation of antibody light chain to select SRM-like produced peptide for quantification. ESI produces multiple charge states for large molecules therefore occurring charge envelopes reduce the precursor intensity if the top-down quantification is aimed. MALDI gives reduced charge states thus either the identification or quantification approach would be more easy/more practical if the sample is not a protein complex but a purified antibody.

Rather than the selection of MS or acquisition technique, it is more important to choose the sample prep strategy herein. Garbage in garbage out for any MS system. What is your consideration about the sample prep and what is your sample matrix? how would you purify/clean, reduce, and fractionate your sample? It is more critical to assess prior to MS detection. Otherwise many interfering compounds, and protein peptides make your quantification worse and that is why it is recommended to use MRM and signature proteolytic peptide identification is more appropriate to perform absolute quantification...

Hi Murat,

I need to run 100 samples everyday, so i could not digest 100 samples and wait for long hours.

Thanks!

Best,

Naga