How about protein A or G resins...The melon gel works as flow-through mode of purification, protein A is bind & elute mode (orthogonal strategies). Without knowing about a cost comparison, this application works well and recovery would be satisfying. If you investigate the IgG CDR domains (fab) nsmol kit is ready to use and a promising tool as a front-end approach.

Thanks ismail. i am looking for simple extraction of igG from human serum. i need to run every day 30 samples.

We should know more regarding the analysis purpose to recommend several prep options. Protein A/G is efficient and can be used as either a well plate format for multichannel sample processing or as an online column providing analytical separation or trap functionality. There are many options to integrate the resin which is a single and easy step of IgG extraction. Affinity must be always the first option for higher selectivity, especially for a matrix-like serum/plasma. Here are some points that need to be enlightened;

What is your instrumental setup (high res/low res, MALDI, ESI, LC, or CE equipped) and sample prep strategy (Does the enzymatic cleavage take place (assuming 30 samples per day this is inapplicable)?

What would be the acquisition purpose (quantification, epitope binding clarification, discovery)?

To suggest a versatile workflow we should at least know how you are able to collect data (bottom-up/top-down/ MRM quantotypic peptide integrating/middle-up, native/intact, etc..?

My aim is to extract the IgG from human serum, followed by TCEP reduction, and quantify the light chain by using an LC-Q exactive mass spectrometer.

Here is a good paper below describing the kappa and lambda ratio differentiation covering IgG light chain isolation from serum. This used melon gel and you may switch it to protein A&G ( I prefer protein G to pull out efficiently all subclasses (1,2,3,4)). 96-Well plate formats or spin columns would be highly cost-efficient at sample prep you can combine resin with.

Article Phenotyping Polyclonal Kappa and Lambda Light Chain Molecula...

You may use SEC or C4-C8 columns for chromatography part which enables the separation of heavy and light chains. SEC would be highly preferable due to the online desalting (removal of any small molecules) purpose that keeps your orbitrap source clean and reproducible.

Good luck..

Hi ismail, Thank you very much for your information.

I run the 10 samples with TCEP reduction. I noticed that RT and m/z were shifted from sample to sample (light chain and heavy chain). Here, my question is: is it possible to quantify light chain absolutely?

You should omit the chromatographic molecular interaction-based separation for total light chain quantification. I mean hydrophobic interaction chromatography, electrostatic interaction (IEX, mainly CEX) chromatography, and reversed-phase would not be convenient. This is why I suggested SEC. Possibly, heavy chain and light chain fractions of different serum samples bear varying epitope binding sequences (peptide mapping differentiation of the CDR regions), and this feature presents different hydrophobicities and/or PTMs, stereoselectivities (configurational modifications-binding pockets) which can be observed as shifted retention times. Light chains (including kappa and lambda, IgG subclasses) can be quantified by the MALDI-TOF system without the need of chromatographic resolution. Because all heavy chains, and both kappa and lambda chains give signals at distinguished mass ranges and a low number of charge variants at MALDI (ESI gives multiply charged) will ease the assessment. You can also differentiate the kappa and lambda variants.

On the other hand, intact mode SEC-ESI-MS analysis may give accurate, comparable, and reproducible results in terms of total light chain quantification. It can provide satisfying resolution between heavy and light chains by masses and thereby, total light chain quantification can be done by integrating the kappa and lambda chains' responses. Here is an example of this suggestion;

Article Analysis of Reduced Monoclonal Antibodies Using Size Exclusi...

This will enable (I guess) Total light chains because SEC probably cannot separate closely MW variants, but by analyzing the single peak as light chains you may observe the kappa and lambda transitions at the mass spectrum.

If you are not interested in the deconvoluted MW and/or charge distribution or peptide sequence of the light chains of IgGs, and even kappa and lamba separate quantification is not the case you are targeting, in this scenario, LC-SEC-UV method development may be considered primarily since it is cheaper and easier to optimize for total quantification purposes in comparison to MS. There are lots of white papers regarding this mode of application.

There are also non-mass spec-based methodologies typically used in clinical laboratories for Serum Free Light Chain (FLC) assay;

such as;

serum immunoassays (Freelite, Seralite, Diazyme)

Immunofixation electrophoresis

Immunoturbidimetric and immunonephelometric assays...

Good luck...İEA

Thank you very much ismail.

Hi ismail,

The pattern of multiply charged ions varies between samples; in that case, what is the best method to collect the peak area for quantitative studies (TIC, EIC, SIM, PRM or DIA).

As my thoughts, forming product ions whether as PRM, SRM, or DIA/DDA acquisition is not necessary. Selecting SIM or EIC may also be prone to the loss of other abundant ions that belong to kappa or lambda fractions. Filtering a single ion from a bunch of ions may divide the sensitivity and since you indicated charge distribution is not constant as an expected situation, this strategy may lead you to filter the inconvenient ion for some light chain variants. As total light chain quantification is the thing we aimed herein we should include all candidates into the single pot (mean TIC). Otherwise, irreproducible and inaccurate results may be reported. Therefore I prefer to choose the integration of total ions of per light chain variant....

The RT and m/z vary between samples. Is it good to compare all the samples to one standard for quantification? What is the internal standard I may use for this study?

Thanks!!

IS spiking will not give absolute quantification. Spiked-in peptides cannot be implemented because you are not going through signature peptide acquisition (digestion period and cost limitation of your project). Protein spiking is also useless and non-comparative at all...

Any IS can be used to check the instrument performance only.

A known amount of human IgG as a standard can be used for external calibration but I am not sure how this would be creditable enough.

Here is another and more simple option. Without reducing the Ab, just quantify the IgG after isolating it from serum using protein G (Like titer analysis). Considering each IgG presents 2 light chains, double the concentration of the quantified IgG gives roughly the concentration of light chains..You may get the ratio of kappa and lambda by mass spectrum interrogation.

A more selective, accurate, and easy-to-use but costly strategy is combining the kappa and lambda affinity LC resins (Thermo capture select series) with protein G tandemly and just quantifying the purified light chains by simple BCA/Bradford protein assays. By doing this, you can separately quantify free and IgG-conjugated light chains as well...

Good luck with your assay...