Hi Krueger,

If you concerned with the isolation of exogenous/ectopically expressed protein, try adding affinity tag to your protein of interest to aid your isolation by affinity chromatography after depleting the major serum proteins like albumin, immunoglobulins, etc using commercially available kits.

I am a bit concerned by what you are doing.

What do you want to do exactly? Do you have antibodies available for this protein? it is a small peptide or a large one?

what you mean stability?what do you really want to monitor?

If your peptide is not interacting with larger proteins you can use Amicon spin filters to isolate low MW plasma constituents.

Hi Krueger,

I suggest first you need to develop a bioanalytical assay for the absolute quantiotation of your small protein in human serum using LC/MS/MS or Lidand Binding assay. In terms of your small protein, maybe LC/MS/MS is top choice without digestion if digestion is not necessary due to its high selectiveity. For ligand binding, you need specific paired antibodies in hand with enough selectivity

Guodong

I would recommend either MS-with TOF or MALDI TOF MS analysis.

If you don't have an antibody to the peptide or a functional assay, then I would agree with Guodong Zhang and Jai Ghosh that mass spectrometry is probably your best option.

Here is one protocol for stability analysis of a peptide in human serum.

Stability assay

300 μL human plasma (Sigma Aldrich) was incubated at 37º C for 30 min. 50 μL of peptide sample (0.3 mM) in 0.1 M phosphate buffer, pH 7.2 was added to the human plasma. The vortexed mixture was incubated at 37º C. Aliquots (30 μL) were taken at 1, 2, 3, 4, 12, 24 and 48 h, quenched with extraction buffer (70 μL) consisting of 50% ACN/H2O, 0.1 M NaCl, and 1% TFA, chilled on ice for 5 min, centrifuged (14000 rpm, 10 min) and analyzed (2 x 20 μL injection) by RP-HPLC and LCMS. Data analysis (n = 3-6) was performed using Prism Version 5 a non-linear fit one-phase decay model.

Muttenthaler M, Araujo AD, Andersson A, Dekan Z, Lewis RJ and Alewood PF. Modulating oxytocin activity and plasma stability by disulfide bond engineering. Journal of Medicinal Chemistry, 2010; 53: 8585-8596.

Thanks everyone. To answer some questions, this protein has been expressed recombinantly (with fusion tag), cleaved, purified, and then bioconjugated to a small peptide. I am essentially looking for potential protein degradation/cleavage over time (mostly the bioconjugation linkage) in human serum. The final protein cannot have an affinity tag on it (this was already used/cleaved prior to obtaining the final product).

Protocols i've read involve a similar one described by markus, with a precipitation step involving methanol. While a small peptide would remain in the soluble portion, i'm worried that my protein might precipitate out with the serum proteins.

Mass spec is ideal, i'm just trying to figure out the best way to clean up the serum/protein solution before running it on the mass spec

Just that habituated to detect the protein that I did not get you want to test the stability :/

Hi Andrew,

Do you have any antibody for this small protein in hand? If yes, try bead-based IP (immuniaffinity extraction) from human serum, after elute using acid and then inject to LC/MS/MS system (In genral, we called IP-LC/MS/MS). In terms of your MW is 8K, after PPT using methanol, I agree with you, most of your target protein in the pellet residue rather than in the supernatant solution after centrifue. If you do not have any antidboy, maybe you can try SPE to extract it from human serum.

Guodong- unfortunately i do not have an antibody on hand. Considering making a small peptide simulating the bioconjugate linkage and testing the stability of this instead, as it and its degradation products will be easier to isolate from the serum

This is a non-trivial problem. You are interested in 'chemical' stability, particularly the conjugate linkage, it seems to me.

Any degradation will elicit a mass shift, and you could use intact mass profiling, provided you a) have enough of your conjugate, and b) can find an ESI-MS with enough sensitivity and resolution. Maybe, as has been suggested, an amicon-type filter would help.

Monitoring stability by loss of starting material can be challenging - can you really discriminate 100% starting material (no degradation, no losses) and 95% starting material (potentially, 5% degradation, or just 5% noise in the system). An assay based on appearance of products can be more sensitive, since these start, by definition, from zero.

I presume you are making the protein heterologously. Why not synthesise it in heavy form (using13C amino acids in the medium)? This can then be used as an in vitro reference, added after the serum incubation step, to provide a reference against which you can measure the loss of the protein through degradation.

A final word of caution. Please do not rush your samples onto a nanoflow reversed phase column, prior to MS. Sample preparation is everything here. Plasma or serum can be a real problem because of the huge asymmetry in protein abundance!

Another approach, inspired by the comment of Rob Beynon, would be to use a 15N isotopically labelled peptide (cost of ~€30/L of culture) monitored by 1H-15N HSQC using NMR which will only detect the 15N labelled protein.

You will not need to separate your sample and with the exception of the need to add 5% D2O, the method is non destructive so you can couple it with another one. You may also be able to simultaneously assess the conjugated and the cleaved forms.

Potential pitfalls are, low sensitivity ( about 50-100uM detection limit) and detection problems if your protein is associating with membranes or large biomolecules.

If you have antibody for the protein or peptide, the easiest way ia western blotting. You can get idea about the MW, degredation. Though quantification is not so accurate with this method.

Depending of what applications and parameters that are of interest the most suitable methods varies but it is probably a combination of two or more.

If you are intersted in kinetics and affinity you can use a biosensor to determine variations in kinitic rate constans. The advantage of monitoring the kinetics is that you will pick up small variations very early. Prefearbly use a biosensor that can run both sera and purified assays. To my knowledge the two best are Fortébio and Attana.

Not sure of primary goal-- you say you want to isolate it and also that you want to test its stability in serum. Does that mean you want to know how long you can leave it in serum before isolating it? Get it purified ASAP, don’t give other components this substrate to play with. Standard use of ammonium sulfate precipitation to treat serum before processing works well for antibodies and large serum proteins; not personally familiar for smaller polypeptides, but if you can separate the big proteins (especially albumin) away from your target this way, the two-step process would bring you most of the way home. My experience in 2-D electrophoresis of serum proteins was always to pretreat on an AffiGel-Blue column to remove albumin—so voluminous and an interfering busybody. Just make sure it wasn’t sequestering your target, though!

Also test the best storage buffer and conditions for the isolate (some buffers may be oxidative, etc.). Know the isoelectric point and use that as a gentle means of manipulation and testing for change; methanol would be harsh—can’t you precipitate all the protein then dialyze your small PP out some other way? That said, some purified polypeptides are unhappy alone, and do well with an innocuous carrier to keep them company—cures various problems such as sticking to labware or precipitation problems. That storage buffer is crucial to stability.

Regarding stability: I ran a project monitoring large protein stability in storage over time (two years), testing buffers and temperatures. Main parameter for “stability” was enzyme activity; we also tested breakdown products—what are you getting in a dialysate? The pH often changes. Dilute solutions deteriorated more rapidly than concentrates under the same conditions. In electrophoresis (1-D) the bands get blurry with deterioration; you may also find poorer binding with specific antibody if the antigenic site has been affected. One test I wouldn't skip is IEP.

It depends of your disponibility of tools, instruments, resources, etc. I think the best one is to measure the protein activity. Other way, circular dicroism could be a good option.

DSC offers an unusual method for characterizing proteins in serum. See attached paper. I have pdfs of 3 other related papers. Let me know if you are interested and I will send them. [email protected]

Affinity purification is a better tool provided the affinity ligand is known. Other wise pass the mixture through 5 KDa and 10 KDa cut off columns. You will get enough protein to evaluate through PAGE with 15% resolving. Alternatively, after passing through the columns the fraction can be subjected to HPLC/ MS

I understand the idea is monitoring the protein stability in human serum, why so many comments on isolation and purification? It is not necessary (I think).

If the problem is isolation, use affinity chromatography, although ultrafiltration through between 5 and 10 kDa and diafiltration is the easier way if yo have not the right column for chromatography.