u/bluemooninvestor — reddlx

Looking for a general all-purpose lipid extraction protocol for broad coverage in LC-MS/MS

Hello everyone,

I am trying to get started in lipidomics. I have done some proteomics but totally new in lipidomics.

Can someone suggest a general all-purpose lipid extraction protocol for broad coverage in LC-MS/MS. I don't have any specific lipid type in mind. Want to do untargeted data aquision after labelling with some isotope labeled Glucose. A broad coverage protocol would be great.

I have come across the Folch method, MTBE based protocols and many others, but there are so many variants.

Just wanted to start with a solid proven method with decent coverage. Any suggestions please.

Oh yes. I am working on adherent cancer cell line grown on plates.

u/bluemooninvestor — 4 days ago

▲ 6 r/massspectrometry

BEH Amide vs BEH HILIC for C13 Glucose based Metabolomics

Hi everyone,

I am doing C13 Glucose based Metabolomics in mammalian cell line samples. I am doing the extraction and all and sending samples for acquisition. I have option to outsource samples to two vendors:

Vendor A has BEH Amide column but charges twice as Vendor B who has BEH HILIC column. Rest equipment is similar.

I know BEH amide is better but is it worth spending twice for BEH amide column over BEH HILIC? How much is the quality difference?

Want to use the data for flux analysis. Untargeted data acquisition.

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u/bluemooninvestor — 6 days ago

▲ 4 r/proteomics+1 crossposts

Hello everyone,

I have been using Pierce Desalting Spin columns for peptide cleanup, and it required 300 uL of 50% ACN to elute.

Now, I have to use the Pierce C18 columns. Despite both of them being small spin columns, the C18 column protocol says that only 20 uL of 70% ACN should be used for elution.

My question is, isn't 20 uL a very small volume? Why is there such a huge difference in elution volume between the two spin columns (Desalting v C18 one)? Lastly, is there any general understanding in the proteomics community to use a larger elution volume with the Pierce C18 spin columns.

The protocol also says one may use 70% ACN with 0.1% TFA, but no idea why that is not the standard elution solution for this setup.

I understand that most hardcore proteomics labs would probably not be using Pierce C18 spin columns, but this is what I am using as a part-time proteomics guy. I don't have any other C18 setup, and I will send the cleaned-samples to a mass-spec facility.

And advice on the Pierce C18 spin columns is greatly appreciated.

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u/bluemooninvestor — 16 days ago

▲ 42 r/labrats

Hello everyone,

When you are doing western blot for 10-15 proteins and showing common loading control (actin) for these 10-15 proteins, how are you doing it?

Of course you are cutting the blot but are you stripping and reprobing it too? Or are you just stripping and reprobing it 10-15 times without cutting it? I have read that stripping makes the quanitation less accurate.

Or are you just showing loading control in a different blot and proteins are not necessarily from the same blot (that would be wrong I guess?)

I can't fathom how to show 15 proteins with one loading control. What am I missing? I am doing regular gels, not precast gradient ones.

Edit: If one is using multiple gels, then showing loading control fror one of the blots isn't ideal, is it? But it that how people do it?

Examples :

Extended figure 7 of this nature paper

https://pubmed.ncbi.nlm.nih.gov/34707288/

Fig 6 Science paper

https://pubmed.ncbi.nlm.nih.gov/37917749/

u/bluemooninvestor — 22 days ago

▲ 26 r/proteomics

Recently I have run into Lip-ms, and there are quite a few high impact papers describing the technique. But there are really few papers who have actually used it for doing science.

I think it is the same for several other techniques like proximity-labelling MS, certain advanced variants of thermal proteome profiling etc.

Why do you think it is like this? If these techniques are so great, why aren't they being used for actual science on a much greater scale? Or is my assumption wrong?

Excited to listen to your views.

For perspective, I am a cancer biologist trying to do omics.

reddit.com

u/bluemooninvestor — 23 days ago

▲ 2 r/proteomics

I have submitted a paper to JPR for the first time. It has been three weeks and the status is still "Editorial Review".

Does the manuscript status change to Peer Review or something like that when it's under actual review? Or does it mean it's still with the editor only?

reddit.com

u/bluemooninvestor — 28 days ago