r/proteomics

So we're using mouse brain tissue for TurboID. We do a streptavidin pulldown, followed by SP3-based sample prep, trypsin digestion, peptide resuspension in formic acid, and then DDA/DIA LC-MS/MS.

We previously ran essentially the same workflow successfully, but this batch gave extremely poor MS data. The proteomics core tried multiple acquisitions, including increasing the amount injected, but all experimental samples only gave ~20 proteins identified; controls gave even fewer.

The important thing is that the instrument itself appears fine, and the chromatograms/TIC showed very low overall ion signal rather than a normal signal with poor identification. The core does not think this is an instrument issue or obvious ion suppression problem.

We've measured protein concentration at multiple stages:

• Protein before pulldown: sufficient
• Protein after pulldown: sufficient
• After SP3 + trypsin digestion + resuspension: measurable peptide amounts

We used the Pierce Quantitative Colorimetric Peptide Assay after digestion/resuspension. Interestingly, the previous successful run had only ~6.3 µg peptide (180 ng/µL × 35 µL), so this doesn't seem to simply be a case of insufficient peptide quantity.

SP3 was performed using the same protocol as the successful run, and all samples were processed identically. We used the same trypsin lot as before, although the core is planning to test a fresh trypsin lot in the next experiment.

There were some biological differences between the successful and current batches. My main question: What could cause a large discrepancy between a reasonable peptide concentration measured after SP3/digestion and extremely low LC-MS/TIC signal, especially when the same workflow previously worked with even less peptide?

Would you suspect peptide composition/quality, digestion, SP3 recovery, some brain-derived interfering material, or something specific to the TurboID/pulldown samples? What additional QC or comparison would you look at?

Basically I am treating my cells with a drug, which I expect to crosslink certain target proteins. However, it's not a very stable drug so it's side chains and all might degrade, leading to difference in expected and actual mass in the crosslink. Basically even if I expect the crosslinker to be 300 Da, it might even be 260Da, so I am in the dark.

Is there any way to do open-search (Fragpipe like) for crosslinked peptides. I can constrain it with the knowledge that crosslinks will happen between cysteine residues.

Thanks

Hello, we have been using Rigaku ultima IV diffractometer in our lab for several years. Every now and then we come across communication problems with PC and instruments. And the service people changed instruments mother board once, PCle boards three times which costs a lot.

We have the same problem again. The XG property shows cannot generate x-rays, check generator. We have tried trouble shooting the the serial ports also.

Please help me diagnose and solve this problem. (Also how do I approach if the problem is board cannot be to communicate with chiller, which we cannot not see in pc)