Analyzing Bone Proteins in Forensic Laboratories Using LC−MS/MS

The application of proteomic analysis in the study of forensic skeletal remains has gained significant interest in improving biological and chronological estimations in medico-legal investigations. It is crucial to maximize throughput and proteome recovery to enhance the applicability of these analyses to forensic casework, while at the same time minimizing interoperator variability and laboratory-induced post-translational protein modifications (PTMs). A recent study compared different workflows for extracting, purifying, and analyzing bone proteins using liquid chromatography with tandem mass spectrometry (LC–MS/MS), including an in-StageTip protocol previously optimized for forensic applications, and two protocols using novel suspension-trap technology (S-Trap) and different lysis solutions. LCGC International discussed this work with Noemi Procopio of the School of Law and Policing and the Research Centre for Field Archaeology and Forensic Taphonomy at the University of Central Lancashire (UK), corresponding author of the paper that resulted from this study.

In your paper (1), you discuss analyzing bone proteins using liquid chromatography with tandem mass spectrometry (LC−MS/MS). What benefits does incorporating this technique bring to the analytical process?

LC–MS/MS is the technique adopted by laboratories dealing with forensic and archaeological skeletal remains for proteomics analyses, mainly because of the high sensitivity and specificity that it offers. Frequently, we must deal with samples which are degraded and that underwent decay processes, so having an analytical technique able to maximize the information obtainable from “difficult” samples is crucial to ensure the success of the analysis. Additionally, it allows us to identify proteins, their post-translational modifications and to obtain relative quantifications that can be correlated with time since death or age at death, to ultimately develop models for age and post-mortem interval prediction based on the proteomics signatures identified in the sample.

What are the challenges in working with bone tissue in forensic analysis that might not occur when working with other body tissues?

Working with bones is challenging, as in contrast with any other soft tissue or biological fluid, the mineralized structure of bones made of a dense matrix of hydroxyapatite (a calcium phosphate mineral) traps the proteins within itself. Therefore, the mineral matrix should be decalcified to release the proteins in solution for subsequent analysis. Moreover, the steps required to prepare a bone sample include reducing samples to a fine powder (in a freeze mill) to improve the efficiency of protein recovery (to increase the surface area in contact with the reagents), a step not required when dealing with soft tissues (where normally a sonicator or a homogenizer are the only instruments needed to prepare the sample). Also, we need to consider that forensic bones are different from fresh bones such as those specimens from bone biopsies or immediately post-mortem during an autopsy–in fact, we deal with bones subjected to taphonomic alterations (post-mortem decay) caused by multiple factors (for example, exposure of the remains to sunrays and ultraviolet (UV) light, cycles of freezing-thawing, of wet-dry, humidity, soil types, microbial erosion, and so forth.) which can make protein extraction more challenging and complex.

You mention that there is increasing interest in the use of the data independent acquisition (DIA) mode compared to the more commonly used data-dependent acquisition (DDA) mode for forensic bone proteomics. Please explain why you think this is the case.

DIA acquisition is starting to gain more interest in the forensic community as it allows for the analysis of all peptides in a mixture, something ideal for forensic samples where often the protein content is limited (due to the reasons explained above) and where less abundant proteins sometimes fail to be detected with DDA approaches. The other advantage is the reproducibility of the analyses, also across different runs, in contrast with DDA analyses (where samples should be run in the same batch to be comparable). Also, quantification is more accurate and reproducible in DIA analyses, another key advantage towards the use of DIA in complex forensic samples.

Briefly state your overall findings in your research.

This research was aimed at improving the existing protocols for bone proteomics in forensic contexts, minimizing the amount of laboratory-induced post-translational modifications and maximizing the reproducibility of the results. This was achieved by testing different extraction procedures (for example, ZipTip protocol in comparison to S-Trap protocol) and by comparing DIA versus DDA analyses using open-source software for fair comparisons. Overall, DIA outperformed DDA data in terms of sensitivity and for the identification of low-abundance proteins, and S-Traps protocol resulted in increased proteome recovery when compared with ZipTips. This will also ensure high reproducibility both within batches (S-Trap protocol requires less manual handling, and consequently operator experience, than ZipTips) and between batches of analyses run over time.

Do your findings correlate with what you had hypothesized or any preconceptions you might have had prior to your work?

We expected DIA to work better than DDA and this was observed in our data. Regarding the comparison of protocols, we did not have any specific preconception.

Were there any particularly unexpected results that stand out from your perspective?

Nothing comes to mind at this point.

What were the major challenges you encountered in your work?

This work was done to improve an already existing method, and therefore it was quite straightforward in terms of work to be done in the laboratory.

What best practices can you recommend in this type of analysis for both instrument parameters and data analysis?

Regarding instrument parameters, we recommend using high-sensitivity mass spectrometers (such as the Exploris 480 Quadrupole-Orbitrap Mass Spectrometer) and the classic C18 columns for proteomics. For data analysis, we believe that both DDA and DIA acquisition modes can offer very good results, however DIA outperforms when data are analyzed with open-access software (such as DIA-NN) in contrast with DDA open-access software (such as MaxQuant). For our routine analyses, we are using both DIA and DDA approaches, but we tend to use other software (such as Progenesis QI for Proteomics and Mascot) for the DDA data analysis.

Can you please summarize the feedback that you have received from others regarding this work?

The forensic community showed a great interest in this protocol–amongst colleagues, there is the perception that extractions using the S-Trap technology are preferrable to in-Stage-Tips for improved reproducibility and decreased inter-operator variability. The same approach is also used for other tissues by others working on forensic samples, and results seems to be equally good. For DIA analyses, we are not the first reporting the improved outcomes of this acquisition mode in contrast with DDA, making DIA analyses already highly supported by the forensic community.

Do you believe that the method can be adapted to perhaps other tissues, bodily fluids, or perhaps skin or muscle?

Yes, the only variation is the lack of a decalcification step, needed only for bone tissue. S-Trap manufacturers have published protocols for soft tissues; we had to adapt their protocols, however, due to the lack specific recommendations for dealing with mineralized bone samples.

What are the next steps in this research?

This research has reached its main goal, which was the development of the improved assay for dealing with forensic bones for forensic applications, and we are already using our updated protocol on daily bases when working on skeletal forensic samples. We will consider new steps when products that have the potential to further streamline and optimize the extraction procedure become available on the market.

References

1. Gent, L.; Chiappetta, M. E.; Hesketh, S.; Palmowski, P.; Porter, A.; Bonicelli, A.; Schwalbe, E. C.; Procopio, N. Bone Proteomics Method Optimization for Forensic Investigations. J. Proteome Res. 2024, 23 (5), 1844–1858. DOI: 10.1021/acs.jproteome.4c00151

Noemi Procopio is a UKRI Future Leaders Fellow, and the Principal Investigator of the "ForensOMICS" Team at the University of Central Lancashire. She was appointed at UCLan in 2022 as Senior Research Fellow in Forensic Taphonomy after working at Northumbria University for three years as Lecturer in Forensic Science, and then as Senior Research Fellow in Forensic Science. She graduated with first class honors in Biotechnology at the University of Turin with a thesis titled “Forensic Genetics: Analysis of Samples ‘Low Template DNA.’” Following her BSc, she obtained a two-year MSc degree (first class honors) in Molecular Biotechnology at the same university, with a thesis titled “Body Fluids Mixture Analysis From Immunochromatographic Cards” under the supervision of Sarah Gino. She then joined the University of Manchester to start her PhD in Forensic Proteomics under the supervision of Michael Buckley. Her thesis was titled “Proteome Taphonomy, Biomolecular Investigations into the Process of Decay for Forensic and Archaeological Applications.” In 2019, she was awarded with the prestigious UKRI Future Leaders Fellowship with the project titled "’Forens-OMICS’: A Cross Disciplinary Implementation of Omics Sciences to in vivo and Post-Mortem Ageing Investigations for Forensic Applications.” The aim of her project is to investigate how biomolecules, and in particular proteins, metabolites, and DNA, can survive and decay after death to identify new biomarkers in human bones capable of revealing information on the chronological age and the post-mortem interval (PMI) of the individuals. She is also investigating on the use of metabarcoding analyses to study microbial populations for individual identification and PMI estimation purposes. Part of her research also involves the application of proteomic analyses to archaeological human remains to investigate on lifestyles and health conditions of past populations. She is fellow of the American Academy of Forensic Science, member of the Italian Academy of Forensic Sciences and of the International Society for Forensic Genetics. Recently, she has been appointed as Visiting Professor at the University of Eastern Piedmont in Novara (Italy) and as Visiting Scholar at the University of Texas State. In 2022, she was awarded the Excellence Award in Biology and Forensic Genetics at the “Excellence, Investigation and Forensic Awards, 4th Edition,” held in Milan, Italy. Photo couresy of Noemi Procopio.