An Improved Sample Preparation Method for Protein and Peptide Identification from Human Hair

Published

November 27, 2023

Author(s)

Zheng Zhang, William E. Wallace, Guanghui Wang, Meghan Burke Harris, Yi Liu, Sergey Sheetlin, Stephen E. Stein

Abstract

A fast and sensitive Direct Extraction (DE) method developed in our group can efficiently extract proteins in 30 min from a 5 cm-long hair strand. Previously we coupled DE to downstream analysis using gel electrophoresis followed by in-gel digestion, which can be time-consuming. In searching for a better alternative, we found that a combination of DE with a bead-based method (SP3) can lead to significant improvements in protein discovery from the human hair. Since SP3 is designed for general applications, we optimized it to process hair proteins following DE and compared it to several other in-solution-digestion methods. Of particular concern are genetically variant peptides (GVPs), which can be used for human identification in forensic analysis. Here we demonstrated improved GVP discovery with the DE and SP3 workflow which was three times faster than the previous in-gel digestion method and required significantly less instrument time depending on the number of gel slices processed. Additionally, it led to increased numbers of identified proteins and GVPs. Among the tested in-solution digestion methods, DE combined with SP3 showed the highest sequence coverage with higher abundances of the identified peptides. This provides a significantly enhanced means for identifying proteins and GVPs in human hair.

Citation

ACS Journal of Proteome Research

Volume

23

Issue

1

Pub Type

Journals

Download Paper

https://doi.org/10.1021/acs.jproteome.3c00627

Local Download

Keywords

SP3 method, variant peptide discovery, protein spectrum, reproducibility, trace detection

Citation

Zhang, Z. , Wallace, W. , Wang, G. , Burke Harris, M. , Liu, Y. , Sheetlin, S. and Stein, S. (2023), An Improved Sample Preparation Method for Protein and Peptide Identification from Human Hair, ACS Journal of Proteome Research, [online], https://doi.org/10.1021/acs.jproteome.3c00627, https://tsapps.nist.gov/publication/get_pdf.cfm?pub_id=956612 (Accessed October 10, 2025)