Need advice on how to define buffer and sample region in SEC-SAXS

[XiaoLan Yao]

Hi Jesse,

I am processing some SEC-SAXS data. The data are not ideal and I would greatly appreciate your advice regarding buffer subtraction and where to choose sample region or regions. I attached two screenshots here, one is unsubtracted with buffer range auto selected by RAW, the other is subtracted with sample auto selected by RAW. My questions are the following:

1) The baseline seems to be gradually shifting upward, especially at later times. What is the best way to choose buffer region for subtraction in this case? 

2) The Subtracted plot is generated using RAW auto selected buffer region (as shown in the unsubtracted screen shot). The Rg in this plot does not show an obviously flat region, it drifts downward from the beginning to the end of the peak. What might cause this and  What is the best way to define the sample region in this case? 

Thanks!

[Jesse Hopkins]

Hi,

It looks to me like there’s several issues with this dataset. You can see a drifting baseline pretty clearly. However, the baseline looks okay prior to first elution peak, so this may indicate some damaged sample that stuck on the capillary (there are other possibilities as well, depending on the sample cell in use). The second issue is that you have two very poorly resolved peaks. I’d have to know what column you were using and what the elution volume for the first peak is,, but given the broad range of Rg across that peak I wonder if it’s a void or near void peak with either large nonspecific aggregate or several large unresolved species in it. The second peak shows a distinct shoulder, indicating some separation of at least two species, but there’s still a range of Rgs and no baseline resolved separation.

Do you know how big (either Rg or MW) you expect your sample to be? Which peak seems like it’s what you’re interested in, the first or second?

As for ways to approach this, given the lack of resolution you will almost certainly have to use a deconvolution on the dataset. Given the baseline drift, you’ll probably need to use REGALS (https://bioxtas-raw.readthedocs.io/en/latest/tutorial/s2_regals.html). For REGALS I would subtract from buffer before the peak and have at least one baseline component as well as the elution components. However, if you wanted to try EFA first (https://bioxtas-raw.readthedocs.io/en/latest/tutorial/s2_efa.html) which is easier, I’d recommend picking two equal sized buffer regions equally spaced from the peak on either side of the peak of interest. For example, for the second peak which is  centered around 550 or so, you could use one buffer region at 200 and one at 750, and then do EFA over just the range of that second peak, which might work.

Hope that helps. Let me know if you have further questions.

All the best.

- Jesse

----

Jesse Hopkins, PhD

Director

BioCAT, Sector 18

Advanced Photon Source

Thanks!<Unsubtracted.png><Subtracted.png>

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[XiaoLan Yao]

Hi Jesse,

Thank you very much for the detailed reply. This is very helpful. To answer your question, we collected SEC-SAXS-MALS-DLS data at APS with a Wyatt 030S5 300 Å column years ago. Based on the frame number of the first peak vs the last frame number (303/966, ~ 31%), it does look like the first peak is at the void volume. This protein is predicted to have a large disordered region and an elongated shape, I wonder if these factors contributed to the early elution volume? We only used GE SD200 column in our lab, it always elutes off after the void, so maybe a different column at APS would have worked better? Based on the SEC-MALS data, the elution volume at the center of the Rayleigh Ratio peak is 7.4 ml out of 20.6 ml column volume, which is ~ 36%. I wonder if the difference of peak volume between the MALS vs SAXS  measurements is yet another indication of heterogeneity? There is also a 2^nd peak in the SEC-MAS data, but at a much later elution volume compared to the SEC-SAXS data (screenshot attached).

Based on MALS, the main peak has a MW of 275 kDa (screenshot attached). I am interested in the first peak. I suspect the 2^nd one is degradation. 

Thanks!

Xiaolan

[Jesse Hopkins]

Hi Xiaolan,

What beam line did you collect the data at (apologies if it was mine and I’m forgetting, I think you took one of our workshops, but don’t remember if you collected data)? Do you know that the SAXS data collection was triggered simultaneous to the start of the MALS? And furthermore that it ended at the same time as the MALS? Otherwise the difference in % elution isn’t relevant. You should check the exposure time of the SAXS data and the flow rate of the column and convert both datasets to be plotted vs. time. Then if you triggered at the same time you can directly compare the position of the peaks.

If the first peak is running near the void of the Wyatt column and poorly resolved, then yes, you probably do want to redo the data collection on a different column. I’d have to see the elution traces from your superdex 200 to say for sure whether it’s the right column or not. If you’re interested in collecting the data again, my beamline at the APS has a wide variety of columns available for SEC-MALS-SAXS, including S200s. You can find more info here:

https://www.bio.aps.anl.gov/pages/how-to-design-saxs-exp.html

Also, your emails are getting caught in the mailing-list’s spam filter, and it doesn’t notify me for a couple of days, so sorry for the slow response. If you want to discuss further/faster you could consider directly emailing me, preferably at jhop...@illinoistech.edu.

All the best.

- Jesse

----
Jesse Hopkins, PhD
Director
BioCAT, Sector 18
Advanced Photon Source

On Aug 13, 2025, at 2:50 PM, XiaoLan Yao <yao...@umsystem.edu> wrote:

Hi Jesse,

Thank you very much for the detailed reply. This is very helpful. To answer your question, we collected SEC-SAXS-MALS-DLS data at APS with a Wyatt 030S5 300Å column years ago. Based on the frame number of the first peak vs the last frame number (303/966, ~ 31%), it does look like the first peak is at the void volume. This protein is predicted to have a large disordered region and an elongated shape, I wonder if these factors contributed to the early elution volume? We only used GE SD200 column in our lab, it always elutes off after the void, so maybe a different column at APS would have worked better? Based on the SEC-MALS data, the elution volume at the center of the Rayleigh Ratio peak is 7.4 ml out of 20.6 ml column volume, which is ~ 36%. I wonder if the difference of peak volume between the MALS vs SAXS  measurements is yet another indication of heterogeneity? There is also a 2^nd peak in the SEC-MAS data, but at a much later elution volume compared to the SEC-SAXS data (screenshot attached).

Based on MALS, the main peak has a MW of 275 kDa (screenshot attached). I am interested in the first peak. I suspect the 2^nd one is degradation. 

Thanks!

Xiaolan

<SEC-MALS.png><MW.png>

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<SEC-MALS.png><MW.png>