Alternative LC-MS/MS Platforms and Data Acquisition Strategies for Proteomic Genotyping of Human Hair Shafts

Abstract

Protein is a major component of all biological evidence. Proteomic genotyping is the use of genetically variant peptides that contain single amino acid polymorphisms to infer the genotype of matching non-synonymous single nucleotide polymorphisms for the individual who originated the protein sample. This can be used to statistically associate an individual to evidence found at a crime scene. The utility of the inferred genotype increases as the detection of genetically variant peptides increases, which is the direct result of technology transfer to mass spectrometry platforms typically available. Digests of single (2 cm) human hair shafts from three European and two African subjects were analyzed using data dependent acquisition on a Q-Exactive™ Plus Hybrid Quadrupole-Orbitrap™ system, data independent acquisition and a variant of parallel reaction monitoring on a Orbitrap Fusion™ Lumos™ Tribrid™ system, and multiple reaction monitoring on an Agilent 6495 triple quadrupole system. In our hands, average genetically variant peptide detection from a selected 24 genetically variant peptide panel increased from 6.5 ± 1.1 and 3.1 ± 0.8 using data dependent and independent acquisition to 9.5 ± 0.7 and 11.7 ± 1.7 using parallel reaction monitoring and multiple reaction monitoring (p < 0.05). Parallel reaction monitoring resulted in a 1.3-fold increase in detection sensitivity, and multiple reaction monitoring resulted in a 1.6-fold increase in detection sensitivity. This increase in biomarker detection has a functional impact on the statistical association of a protein sample and an individual. Increased biomarker sensitivity, using Markov Chain Monte Carlo modeling, produced a median estimated random match probability of over 1 in 10 trillion from a single hair using targeted proteomics. For parallel reaction monitoring and multiple reaction monitoring, detected genetically variant peptides were validated by the inclusion of stable isotope labeled peptides in each sample, which served also as a detection trigger. This research accomplishes two aims: the demonstration of utility for alternative analytical platforms in proteomic genotyping, and the establishment of validation methods for the evaluation of inferred genotypes. Highlights Test four mass spectrometry configurations to optimize detection of genetically variant peptides Technology transfer of proteomic genotyping assays Improved sensitivity results in higher level of forensic discrimination for human identification using multiple reaction monitoring Graphical Abstract