u/Active_Tomorrow_1798

I am working on proteomics from animal tissue and have been trying to detect a specific protein of interest (~380 kDa) using bottom-up LC-MS/MS on a Bruker timsTOF Flex with diaPASEF acquisition. Despite consistently identifying 8000+ protein groups per run, my protein of interest is completely absent from every analysis. I would appreciate any troubleshooting suggestions from people who have dealt with large, low-abundance proteins.

Sample and workflow:

• Fresh frozen animal tissue homogenised using a Dounce homogeniser, followed by probe sonication
• Lysis in 8M Urea/Tris pH 8
• Trypsin + LysC double digestion overnight at 37°C
• Attempted SDS-based dissociation as an alternative lysis approach
• ~200 µg protein input loaded onto EvoTips for LC-MS/MS

What I know about the protein:

• Approximately 380 kDa molecular weight
• Periciliary localisation in specialised tissue
• Low predicted abundance based on transcriptomic data
• Large number of predicted tryptic peptides but many may fall outside optimal detection range

What I suspect may be causing the issue:

1. Incomplete solubilisation - the protein may not be fully extracted from its native periciliary matrix environment despite urea and SDS treatment
2. Incomplete tryptic digestion - at 380 kDa, even partial digestion would generate very large peptides outside the 300-1800 m/z detection window
3. Ion suppression from highly abundant proteins competing for ionisation
4. The protein may simply be below detection threshold in bulk tissue without enrichment

What I have already tried:

• Dounce homogenisation alone
• Probe sonication in urea buffer
• SDS dissociation followed by urea buffer exchange

My specific questions:

1. Is protein size itself a limiting factor for bottom-up proteomics detection, or is it purely an abundance and digestion efficiency issue?
2. For low-abundance large proteins in specialised tissues, would immunoprecipitation or proximity labelling-based enrichment before digest be worth pursuing?
3. Should I consider an alternative digestion strategy such as adding Asp-N or Glu-C in combination with trypsin to increase peptide coverage across the protein sequence?
4. Would fractionating the digest by high-pH reverse phase before LC-MS/MS realistically improve detection sensitivity enough to pull out a single low-abundance protein from a complex background?
5. Has anyone had success detecting large periciliary or extracellular matrix proteins from retinal tissue specifically?

Any suggestions on systematic optimisation steps to rule out each possibility without going in the wrong direction would be greatly appreciated.

Thanks in advance.