Development and Testing of a Mass Spectrometry Based Assay for the Identification of Biological Stains. Kevin Legg

Transcription

1 Development and Testing of a Mass Spectrometry Based Assay for the Identification of Biological Stains Kevin Legg This project was supported by DNA R&D Grants 2006-DN-BX-K001, 2009-DN-BX-K165, 2011-CD-BX-0205, and 2012-DN-BX-K035 awarded by the National Institute of Justice, Office of Justice Programs, US Department of Justice. Points of view in this presentation are those of the author and do not necessarily represent the official position or policies of the US Department of Justice.

2 FSF Emerging Forensic Scientist Award Oral Presentation

3 Core Project Objectives Challenges faced by Serologists Presumptive Results (e.g., Salivary Amylase) Laborious Assays (e.g., Microscopy) No Reliable Tests Available (e.g., vaginal fluid) Objective: To identify and quantitate Protein Biomarkers for reliable body fluid identification. Deliverable: A confirmatory multiplex assay for six human body fluids that can be used for casework. Saliva Seminal Fluid Vaginal Fluid Peripheral Blood Menstrual Fluid Urine

4 Protein-Based Body Fluid ID Advantages Body Fluids are Protein Rich Direct detection of High-Specificity biomarkers by mass spectrometry Assays can be Multiplexed Minimally Consumptive of Evidence Assay Development Discovery Phase Identify Candidate Biomarkers Validation Phase Evaluate Biomarker Specificity Assess Robustness, Reliability and Performance Limits (in progress)

5 Biomarker Discovery through Comparative Proteomics

6 Comparative Proteomics: 2D-HPLC Biomarker Discovery Strategy: Body Fluid Proteome Mapping Differential Analysis by in-house software Biomarker Identification by nanoelectrospray ionization mass spectrometry. 2 nd Dimension (Hydrophobicity) Individual Proteins 1 st Dimension (ph)

7 Validation of Biomarker Specificity for Target Stains

8 Biomarker Specificity and Performance Biomarker Validation Samples from 50 humans/fluid Assess specificity and consistency of detection Evaluate performance on casework-type samples Validation Assay Strategies 2D HPLC Slow (36 hrs / sample) Expensive ($1500 / sample) Antibodies Expensive Exhaustive characterization with presumptive results Mass Spectrometry (Targeted Ion Assay) Rapid method development Cost effective Readily multiplexed

9 Multiplex Assay for Biomarker Specificity Targeted-Ion Validation assay simultaneously scans for the presence of six target body fluids in single run 23 unique biomarkers using 53 diagnostic peptide targets Semen Epidid Smg-2 Smg-1 Smg-1 PSA PAP PSA Epidid PSA PAP PAP Saliva CYS-D CYS-D CYS-SA CYS-SA His-1 Cys-SA Stath Smg-2 Glyco Glyco Muc5B Muc5B Muc5B Urine Uromo Osteo Uromo Osteo Osteo Subm Subm Uromo Vaginal and Menstrual Supra IgGFc Supra Ly6 Neut IgGFc Neut IgGFc Muc5B Ly6 Cornu Cornu Muc5B Muc5B MMP9 Peripheral Blood Hemβ Hemo Hemβ Comp Hemo Comp Instrument Run Time (min.)

10 Targeted-Ion Assay: Seminal Fluid Sample Raw Assay Data Only seminal fluid biomarkers detected. No non-seminal fluid biomarkers detected. Q-TOF MS Database Search Results Only seminal fluid biomarkers detected. No non-seminal fluid biomarkers detected

11 Population Study Biomarker Specificity Results 250 samples assessed by Q-TOF targeted-ion assay 2 proteins for each fluid were both unique and consistently detected Menstrual Blood and some male urine samples are challenging. Body Fluid Protein Biomarker (examples) Accession Number Seminal Fluid Saliva Urine Peripheral Blood Menstrual Blood and Vaginal Fluid Semenogelin I/II Epididymal secretory protein E1 Cystatin_SA Statherin Uromodulin Osteopontin Hemopexin Complement C3 Matrix Metalloproteinase NGAL P04279 P61916 P09228 P02808 P07911 P10451 P02790 P01024 P14780 P80188

12 PSA PAP SMG-II SMG-I NPC2 PAEP UMOD OPN SMR3B CST2 CST5 STATH HTN1 MUC5B CORN NGAL LYPD3 FCGBP MMP9 SBSN HBB HPX C3 Casework-Type Samples Casework Sample Results Substrates, Mixtures, Environmental/Chemical Insult, etc. Co-extraction and analysis of DNA and protein performed successfully Environmental/Chemical Insult Studies (representative samples) Seminal Fluid Urine Saliva Vaginal Fluid Peripheral Blood Semen + Spermicide Pblood + Blue Star Pblood + Soil Saliva + Tabacco Urine + Coffee Green indicates protein identification

13 Casework Type Sample: Semen/Saliva Mixture 10ul of saliva/seminal fluid mixture onto a cotton sheet. Q-TOF reveals the presence of both seminal fluid and salivary biomarkers. DNA analysis reveals a mixed DNA profile. Mixed STR Profile with Identifiler Plus Locate Stain (ALS) Quantifiler Duo Extract Mass Spec Results (seminal fluid/saliva mixture)

14 Developing an Assay that is Viable for Forensic Lab Use

15 Instrument Platform Options Limitations to a Q-TOF Assay Too slow for caseworking labs ( 45 min/run) Less than optimal assay sensitivity Lack of internal positive controls and reference standards Multiple Reaction Monitoring (MRM) on a Triple Quadrupole (QQQ) Faster and user friendly Lower capital cost More sensitive and selective Multiplex capability with quantitation

16 Developing an Assay for Forensic Lab Use Six-fluid multiplex assay on a nanoflow QQQ 16 Proteins, 53 peptides, 160 transitions 2x the throughput compared to Q-TOF assay PSA 3 nl seminal fluid PSA SMG PAP PAP SMG

17 Introducing Controls and Standards Absolute Quantitation (AQUA) internal standards Identical chemical composition Matching response ratio and retention time Mass shift via stable isotope incorporation ( 15 N and 13 C) Internal Standards Synthetic peptide standard Confirms biomarker detection in the questioned sample. Peptide from questioned sample

18 Internal Controls and Standards Internal Positive Control Confirms successful target protein processing (i.e., digestion, ionization and detection) Non-Human control proteins are processed with each sample Questioned Sample Add 1:1 Ratio of Control protein and Control standard Successful Processing Processing Failure 1:1 Response 1:1 Response

19 In Progress: Developmental Validation Combination of analytical and DNA/serology requirements 1. Population Studies 2. Biomarker Characterization 3. Species Specificity 4. Sensitivity/LLOD 5. Reproducibility & Repeatability 6. Carryover Studies 7. Stability 8. Mixture Studies 9. Additional casework type samples Completed In progress for 6 body fluids Estimated Completion: 2014 per DNA QAS, SWGDAM, SWGTOX Guidelines

20 FSF Emerging Forensic Scientist Award Oral Presentation

21 NMS Labs/CFSRE/Arcadia Dr. Christian Westring Dr. Barry Logan Ms. Heather Mazzanti, MSFS Acknowledgements Forensic Practitioners Dr. Patricia Speck (UTHSC) Colorado Bureau of Investigation National Science Foundation Major Research Instrumentation Program National Jewish Health Dr. Nichole Reisdorph Dr. Rick Reisdorph National Institute of Justice Minh Nguyen and Chad Ernst Lois Tully University of Denver Dustin Gilbert Brett Freidman Katie Leeway