Dear scientists ! I know this kind of looks like a very general and basic question but here is my problem: I have been working for few months already to covalently bind NHS derivated small molecules to amine groups of lysine residues of proteins (45 KDa). The small molecules being hydrophobic, I first dilute them in DMSO and then react overnight with the protein in PBS (room temperature). At all time of reaction, from beginning till end, the solution is limpid with no precipitation or aggregation. My protein bears 20 lysine residues, so 20 positions to bind. As it is hard to define the equivalents to use for such reactions (small molecules being 10 times smaller than the protein) I used a range of equivalents varrying from 50 to 200. I am expected to obtain proteins conjugated with 1 to 20 small molecules.
1) By SDS I am able to see an increase of molecular weight of my conjugated proteins but no trace of any unconjugated ones.
2) Mass spectra analysis showed a shift of charge of the conjugated proteins but from calculations, the best case seems to show that a maximum of 2 to 3 molecules bound per protein (instead of 20 as I would have expected).
Meaning the conjugation ration is extremely poor. Has anyone else faced the same problem ? If reaction obviously works, how can I increase the conjugation rate ? Can I react for more time ? Also, what would be the technique to see the portion of unconjugated protein ?
Any comment, suggestion, questions would be highly appreciated.
Thanks to all of you !
@Françoise
Hope the following information helps (see below):
• RE: "What would be the technique to see the portion of unconjugated protein?"
Capillary electrophoresis (CE, ICE) and isoelectric focusing (IEF) may provide better sensitivity, resolution, and quantification than traditional SDS-PAGE with densitometry. CE and IEF are standard techniques in biopharma for analyzing complex mixtures of bioconjugates such as ADCs (see references below). It may help to check what CE and IEF instruments are available at your institution or by external contract labs.
"Development and Characterization of Multi-Platform Antibody Drug Conjugates"
http://c.ymcdn.com/sites/www.casss.org/resource/resmgr/WCBP_Speaker_Slides/2014_WCBP_Jason_Starkey.pdf
"Analytical methods for physicochemical characterization of antibody drug conjugates"
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3092617/
"Conjugation site heterogeneity causes variable electrostatic properties in Fc conjugates"
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3713463/
• RE: "If reaction obviously works, how can I increase the conjugation rate?"
Although your protein may have 20 lysines (and an uncapped N-terminus?), not all of them will have the same reactivity. At neutral pH, some of the lysines may form ammonium salt bridges with acidic side chains (Glu and Asp). If your protein can tolerate pH 8.5–9.5 phosphate or carbonate buffer, then more of the lysines should be deprotonated and reactive. Spin-column or dialysis can be used for buffer exchange to yield a product solution at physiological pH. Continue using DMSO (or acetonitrile) cosolvent for the hypdrophobic NHS ester, but using more than 50 molar equivalents of the NHS ester may not be beneficial. Also, overnight should be more than enough reaction time. NOTE: Over-labeling may affect the biological activity of your protein.
Differential lysine reactivity in bioconjugation of proteins/peptides to small molecules has been reported by industrial and academic researchers (see below):
"Development of a Potential High-Throughput Workflow to Characterize Sites of Bioconjugation by Immuno-Affinity Capture Coupled to MALDI-TOF Mass Spectrometry"
http://pubs.acs.org/doi/abs/10.1021/bc300413c
"Chemical Re-engineering of Chlorotoxin Improves Bioconjugation Properties for Tumor Imaging and Targeted Therapy"
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3086956/
The suggestion to try iCIEF as analytical tool is a good one, since it tends to be less sensitive to differences that MS can experience related to ionization efficiency. The change in net charge can be seen as "ladder" of different pI species that reflects each number of conjugated species, including those that have no bound small molecule. If you examine the literature about lysine conjugation, you will find that typically one gets a distribution (either Gaussian or Poisson). If you integrate the peak areas from IEF or iCIEF, you can calculate the average conjugation level. Remember that each level (for example n=1) corresponds to a mixture itself, made up of all the different species containing only one bound molecule. To get at this "micro heterogeneity" requires a different technique, such as peptide mapping (either with or without MS detection).
The other point to consider is that NHS esters are hydrolytically labile. At neutral pH, you have two competing reactions, conjugation to lysines versus hydrolysis of the ester leading to a non-reactive free acid product. Therefore longer reaction times may have no benefit since your reagent is no longer available. I recommend doing a pH study, you might find that a lower pH (where the reaction is slower but the NHS is more stable) gives better outcome. Or maybe higher pH where reaction is faster (but NHS half-life is shorter) is better.
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