Fast methods based on mass spectrometry for peptide identification. Application to sex determination of human remains in tooth enamel

Abstract

Two new fast methods are proposed for the first time to assign biological sex to prehistoric human remains. Both methods are supported on sexually dimorphic amelogenin protein fragments (AMELX and AMELY) analysis in tooth enamel. The first one is based on flow injection analysis, electrospray ionization and high-resolution mass spectrometry (FIA-ESI-HRMS) with a run time of three minutes per sample. The second method is based on tandem mass spectrometry (FIA-ESI-MS/MS) with a low-resolution mass spectrometer. In this case, run time is one minute per sample.When FIA-ESI-HRMS was used, two accurate mass-to-charge ratios, corresponding to the diprotonated ions of the molecular species [M + 2H]2+of both peptides, and 6 MS/MS transitions, 3 characteristics of peptide specific to the Y isoform of amelogenin and 3 to the X, were selected for identification purposes. In the FIA-ESI-MS/MS method, 6 MS/MS transitions, 3 characteristics of peptide specific to the Y isoform of amelogenin and 3 to the X, were measured. In both cases, no separation step is carried out once the sample is injected into the system.The two methods were applied to a set of 16 tooth samples from prehistoric human remains. The results obtained in the sex determination with the rapid methods were confirmed using a liquid chromatographic based method (LC-HRMS). Results were in complete agreement among methods.A very important increase of sample throughput was obtained with the new proposed methods. When the LC-HRMS method was used, time between sample injections was 101.4 min (run time, 100 min; time require for injection, 1.4 min). When the FIA-ESI-HRMS and FIA-ESI-MS/MS methods were used, in this time (101.4 min) it was possible to analyze 23 and 48 samples, respectively. Moreover, the possibility to assign sex using low-resolution mass spectrometers means that a greater number of laboratories could perform AMEL analysis because the cost of the instrumentation is reduced.