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