SP3 (Single-Pot, Solid-Phase, Sample-Preperation) Protocol for ZooMS applications v1

Abstract

Zooarchaeology by Mass Spectromtery (ZooMS) allows for taxonomic identification by the peptide mass fingerprinting of collagen type I. This protocol combines an EDTA based pretreatment, a SP3 (single-pot, solid-phase-enhanced) protein extraction, and a peptide purification (both ZipTips and StageTips have been used and are provided as options) to prepare samples for analysis on a MALDI-TOF-MS. This protocol was developed to see if an SP3 based method would improve extraction efficiency for bone samples with low collagen preservation. However, in preliminary testing it does not perform better than established methods. SP3 for dental calculus has also been used with more success and the protocol for this can be found at dx.doi.org/10.17504/protocols.io.bfgrjjv6. This protocol is based upon the original SP3 protocol published in Hughes et al. (2014) and the developments for palaeoproteomics of bone (Cleland et al., 2018). When using please cite the following three publications the protocol is based upon as well as the DOI for this protocol. Hughes, C. S., Foehr, S., Garfield, D. A., Furlong, E. E., Steinmetz, L. M., & Krijgsveld, J. (2014). Ultrasensitive proteome analysis using paramagnetic bead technology. Molecular Systems Biology, 10, 757. https://doi.org/10.15252/msb.20145625 Hughes, C. S., Moggridge, S., Müller, T., Sorensen, P. H., Morin, G. B., & Krijgsveld, J. (2019). Single-pot, solid-phase-enhanced sample preparation for proteomics experiments. Nature Protocols, 14(1), 68–85. https://doi.org/10.1038/s41596-018-0082-x Cleland, T. P. (2018). Human Bone Paleoproteomics Utilizing the Single-Pot, Solid-Phase-Enhanced Sample Preparation Method to Maximize Detected Proteins and Reduce Humics. Journal of Proteome Research, 17(11), 3976–3983. https://doi.org/10.1021/acs.jproteome.8b00637