Using LC–MS/MS in Analyzing Bones for PMI Estimation: An Interview with Noemi Procopio, Part III

As part of “From Sample to Verdict,” LCGC International sat down with Noemi Procopio, who is the Principal Investigator of the "Forens-OMICS" team and a Senior Research Fellow in Forensic Science at the University of Central Lancashire, to talk about her team’s work (1,2). In Part II of our conversation with Procopio, she discussed post-mortem interval (PMI) estimation and how bone proteomics can be used to accurately estimate PMI. In Part III, Procopio discusses how her team analyzes bone samples for PMI estimation.

Will Wetzel: Can you walk us through the workflow of analyzing a bone sample for PMI estimation, from sample collection to LC–MS/MS data interpretation?

Noemi Procopio: The protocols that we follow for proteins and metabolites are similar, despite destruction being slightly different for the two. Starting from sample collection, we collect our samples in the United States from different human taponomy facilities. We collect bone chunks, ideally or bone powder, and then the bone chunks are converted into bone powder in a freezer meal. We are milling the bones in liquid nitrogen so that we avoid overheating and damaging the proteins and metabolites in all the molecules.

After we collect, we transform the chunks into powder, that's when we weight the right amount of bone powder that is needed, which for our protocols for proteins is 25 milligrams. For metabolites is 50 milligrams. So still a tiny, tiny portion. We don't need to do massive invasive samplings. We just need a tiny little sample. And with that, the protocol splits a little bit for proteins and metabolites. For proteins, we do an extraction, which includes decalcification. A weak acid helps decalcify the bone.

Then, for the bone pellet, we apply something like guanidine hydrochloride that can further solubilize our proteins in the pellet, and then we perform some buffer exchanges to get rid of this buffer and place proteins in a buffer that is more friendly for trypsin digestion, such as ammonium acetate or bicarbonate. We do that using some NWCO filters or alternatively, we can extract proteins using these new devices called S traps, where you have a C18 membrane at the bottom of the column.

The column is like a filter that can trap your proteins, and then you can just wash your sample and put that in the buffer that you want to use for the subsequent steps. Then, we do trypsin digestion overnight or for a couple of hours, depending on the protocols. This process chops proteins into smaller peptides. And then the proteins get eluted. The idea is to concentrate and purify the peptides, and after they are purified in and eluted in 50% acetonitrile, normally, they are evaporated, and then they are ready to be resuspended and injected into the LC–MS/MS for metabolites, just a different extraction.

We use a combination of water, methanol, and acetonitrile, and that's allowing us to get all the polar compounds in solution. Then, we just perform some washings as well before evaporating the samples in a speed vacuum to make them ready to be resuspended and injected. So, it's just the protocol of the extraction. It's a bit faster with metabolites in that you didn't need to do the digestion. It's a bit more straightforward for lipids. Similarly, you can get the lipid fraction using a combined extraction. When you also extract metabolites, you can also extract lipids in the same extraction. So that's also allowing us to maximize the amount of information from a tiny little sample.

References

1. Procopio, N. Forens-omics: How a Multi-omic Approach Can Reveal the Mysteries of the Postmortem Interval. The Pathologist 2024, 6. Available at: https://thepathologist.com/issues/2024/articles/nov/forens-omics/ (accessed 2025-07-24)
2. Procopio, N.; Bonicelli, A. From Flesh to Bones: Multi-omics Approaches in Forensic Science. Prot. Sys. Biol. 2024, 24 (12–13), 2200355. DOI: 10.1002/pmic.202200335