posted on 2023-11-27, 13:06authored byZheng Zhang, William E. Wallace, Guanghui Wang, Meghan C. Burke, Yi Liu, Sergey L. Sheetlin, Stephen E. Stein
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 in 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 3 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 an increased number
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.