Baseline drift

[Tina Logonder]

Hello everyone,

I've obtained SEC-SAXS results, and it seems there's been a significant drift in the baseline (chromatogram from SAXS in RAW), although the UV chromatogram appears normal (see attached images). I had three different samples and every sample was measured in duplicates. I'm wondering if these data are still usable for analysis or if repeating the measurements is the only option. If the analysis of these data is feasible, I'm interested in how to proceed with the analysis.

Does anyone happen to know what could be causing this baseline drift? I there anything that I can modify for the next beamtime?

It's also important to mention that zinc ions are present in the buffer (10 uM ZnCl2), as one of the two of my proteins has nine zinc fingers. Could this drift be a result of radiation damage due to the presence of zinc ions, which could accelerate the formation of radicals?

Thank you for your help.

Kind regards,

Tina

[Jesse Hopkins]

Hi Tina,

I'm a bit puzzled by F58, but otherwise everything you're seeing is very indicative of radiation damage to the sample. It all onsets around when the protein is eluting in the UV, and shows a strong upward trend after the peak, which is characteristic of sample getting damaged and stuck on the capillary in the beam and thus continuing to damage. 

It's possible that either REGALS or an integral baseline correction might be able to get something out of the data. However, if this were my data I would definitely collect it again, because even with application of these corrective approaches you're unlikely to get high quality, trustworthy results. 

When collecting again, make sure you do something to minimize the radiation damage. I'd suggest the addition of some glycerol (around 1-5%, I'd start with ~2%) to the buffer, assuming you don't already have it, and/or attenuating the incident x-ray beam. You might also consider adding reducant, and using a buffer that is itself a radical scavenger (e.g. Tris or HEPES instead of Phosphate). Depending on what column you're using (and what's available at the beamline), you might also consider using another column with a higher flow rate (e.g. using a Superdex 10/300 instead of a 5/150 so you can increase the flow rate).

As far as the zinc in the buffer goes, it's hard to say if it's sensitizing the protein. You can see buffer components themselves damage, and based on my personal experience divalent salts do tend to damage much faster than monovalent (e.g. MgCl2 tends to damage in buffers). However, proteins are usually the limiting factor (unless you're measuring in a coflow geometry), and since the onset of your damage coincides with the protein elution it is likely that the protein is damaging. Because damage is very system specific it's hard to say if the zinc materially contributes to the rate of protein damage without running the same experiment without zinc.

I hope that helps. I'm happy to answer further questions/provide advice.

All the best.

- Jese

----

Jesse Hopkins, PhD

Deputy Director

BioCAT, Sector 18

Advanced Photon Source

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[Tina Logonder]

Hi Jese,

Thank you for your quick reply. Thank you for all the recommendations, I should probably have already clarified at the beginning what buffer and column were used.

Column: Superdex 200, increase 10/300

Buffer: 20 mM HEPES pH 7.4 , 150 mM NaCl, 5 % (v/v) glycerol, 0.01 mM ZnCl2, 10 mM β-mercaptoethanol

So unfortunately column that you mentioned, glycerol and HEPES were all already used.

I specifically mentioned the presence of zinc ions in the buffer because, in comparison to other samples processed concurrently, only mine exhibited radiation damage and was the only one with ZnCl2. 

So as far as I understand the option that I can try is beam attenuation and maybe preparing samples without zinc in the buffer for the next beamtime. I will also try REGALS or integral baseline correction.

You also mentioned coflow geometry. Sounds like something worth looking into? But after a quick search seems like SEC-SAXS with coflow geometry is routinely performed only on Australian Synchrotron or did I miss something?

Are there maybe any other possible modifications that I can make regarding buffer composition? I came across radical scavengers (5-methyl uridine, cytidine, cytosine and uridine, sodium nitrate, cysteine, ascorbic acid - Garman and Weik 2021).

Thank you for your advices,

Kind regards,

Tina

petek, 5. april 2024 ob 18:11:20 UTC+2 je oseba Jesse Hopkins napisala:

[Jesse Hopkins]

Hi Tina,

I believe that coflow cells are routinely used at the SAXS/WAXS beamline at the Australian synchrotron (and their new BioSAXS beamline if it's running by now) and the BioCAT beamline at the Advanced Photon Source (my beamline, which is currently unavailable because of the ongoing upgrade of the APS). I believe that there is one at B21 at Diamond, but I don't know if it is routinely available, and there is one in development/available for testing at LiX at NSLS II. There may be others that I'm not aware of as well. If you do have access to such a cell then you would want to run a mismatched buffer sheath, without the zinc (and you'd want to use phosphate instead of HEPES if possible, and maybe remove the glycerol).

If you don't have access to a coflow cell then, given your current buffer composition, your only real option is to reduce the flux density (assuming you need the zinc in the buffer to see whatever it is you're interested in seeing). This would be done either through attenuation or by defocusing the beam at the sample cell. Your beamline scientist wherever this was collected should be able to suggest the best option for that. This will of course reduce your signal to noise, so you may have to increase the protein concentration to compensate.

If you're willing to try other buffer compositions, you could try adding one or more of the radical scavengers you mentioned. However, 5% glycerol tends to be an extremely effective radical scavenger for these types of things (see for example figure 4 in https://www.embl-hamburg.de/biosaxs/reprints/radiation_damage_2015.pdf). Based on recent work I think that sodium nitrate would probably be most effective (https://pubmed.ncbi.nlm.nih.gov/34475280/), but not everything you've listed has been evaluated in a quantitative fashion that I'm aware of. This reference has a few other options: https://pubmed.ncbi.nlm.nih.gov/33021499/

All the best.

- Jesse

----

Jesse Hopkins, PhD

Deputy Director

BioCAT, Sector 18

Advanced Photon Source

To view this discussion on the web visit https://groups.google.com/d/msgid/bioxtas_raw/e08422d8-795e-4be4-ab77-edc3dcc9b981n%40googlegroups.com.

[Tina Logonder]

Thank you, Jese, for all the recommendations.

petek, 5. april 2024 ob 23:45:26 UTC+2 je oseba Jesse Hopkins napisala: