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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.
FSF Emerging Forensic Scientist Award
Oral Presentation
Core Project ObjectivesCore 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
ProteinProtein--Based Body Fluid IDBased 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)
Biomarker Discovery through
Comparative Proteomics
Comparative Proteomics: 2DComparative Proteomics: 2D--HPLCHPLC
Biomarker Discovery Strategy: • Body Fluid Proteome Mapping
• Differential Analysis by in-house software
• Biomarker Identification by nano-electrospray ionization mass spectrometry.
1st Dimension (pH)
2nd Dimension
(Hydrophobicity)
Individual Proteins
Validation of Biomarker Specificity for Target Stains
Biomarker Specificity and PerformanceBiomarker 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 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37
Semen
Saliva
Urine
Vaginal and Menstrual
Peripheral Blood
Epidid Smg-2
PAP
Smg-1
PSA
Smg-1
Smg-2
EpididPSA PAP
GlycoPAP
PSA
Glyco
CYS-D CYS-D Cys-SACYS-SA CYS-SA His-1 Stath
SubmSubmMuc5B
Muc5B
Muc5B
OsteoUromo Osteo OsteoUromo
Uromo
Muc5B Muc5B
Muc5B
Cornu Cornu
IgGFc IgGFcIgGFcLy6
Ly6 MMP9
Neut NeutSupra Supra
Hemβ Hemβ
HemoComp Comp
Hemo
Instrument Run Time (min.)
Multiplex Assay for Biomarker SpecificityMultiplex 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
TargetedTargeted--Ion Assay: Seminal Fluid SampleIon Assay: Seminal Fluid Sample
Raw Assay Data
• Only seminal fluid
biomarkers detected.
• No non-seminal fluid
biomarkers detected.
Database Search
Results
• Only seminal fluid
biomarkers
detected.
• No non-seminal fluid
biomarkers detected
Q-TOF MS
Biomarker Specificity ResultsBiomarker Specificity Results
Population Study
• 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 Semenogelin I/II P04279
Epididymal secretory protein E1 P61916
Saliva Cystatin_SA P09228
Statherin P02808
Urine Uromodulin P07911
Osteopontin P10451
Peripheral Blood Hemopexin P02790
Complement C3 P01024
Menstrual Blood and Vaginal Fluid
Matrix Metalloproteinase P14780
NGAL P80188
CaseworkCasework--Type SamplesType Samples
Casework Sample Results
• Substrates, Mixtures, Environmental/Chemical Insult, etc.
• Co-extraction and analysis of DNA and protein performed successfully
Seminal Fluid Urine Saliva Vaginal Fluid Peripheral
Blood
PSA
PA
P
SMG
-II
SMG
-I
NP
C2
PA
EP
UM
OD
OP
N
SMR
3B
CST
2
CST
5
STA
TH
HTN
1
MU
C5
B
CO
RN
NG
AL
LYP
D3
FCG
BP
MM
P9
SBSN
HB
B
HP
X
C3
Semen + Spermicide
Pblood + Blue Star
Pblood + Soil
Saliva + Tabacco
Urine + Coffee
Green indicates protein identification
Environmental/Chemical Insult Studies (representative samples)
Casework Type Sample: Semen/Saliva MixtureCasework Type Sample: Semen/Saliva Mixture
Locate Stain (ALS) Extract
Quantifiler ® Duo
Mass Spec Results (seminal fluid/saliva mixture)
Mixed STR Profile with Identifiler® Plus
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.
Developing an Assay
that is Viable for
Forensic Lab Use
Instrument Platform Options 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
Six-fluid multiplex assay on a nanoflow QQQ
• 16 Proteins, 53 peptides, 160 transitions
2x the throughput compared to Q-TOF assay
Developing Developing an Assay for an Assay for Forensic Lab UseForensic Lab Use
SMG
PSA
PAP
3 nL seminal fluid
PAP
PSA
SMG
Peptide from
questioned sample
Synthetic peptide
standard
Introducing Controls and StandardsIntroducing Controls and Standards
Internal Standards
Confirms biomarker
detection in the
questioned sample.
Absolute Quantitation (AQUA) internal standards
• Identical chemical composition
Matching response ratio and retention time
• Mass shift via stable isotope incorporation (15N and 13C)
Internal Positive Control • Confirms successful target protein processing (i.e., digestion,
ionization and detection)
• Non-Human control proteins are processed with each sample
Add 1:1 Ratio of Control protein and Control standard
Questioned Sample
Internal Controls and StandardsInternal Controls and Standards
Successful Processing Processing Failure
1:1 Response ≠ 1:1 Response
In Progress: Developmental ValidationIn 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
per DNA QAS, SWGDAM, SWGTOX Guidelines
In progress for
6 body fluids
Completed
Estimated
Completion: 2014
FSF Emerging Forensic Scientist Award
Oral Presentation
AcknowledgementsAcknowledgements
NMS Labs/CFSRE/Arcadia
Dr. Christian Westring
Dr. Barry Logan
Ms. Heather Mazzanti, MSFS
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