Kevin M. Legg, PhD & Luciano Arantes, MS & Phillip B. Danielson, PhD

Transcription

1 Confirmatory Blood Identification by Mass Spectrometry: Adapting Proteomics to Forensic Biology Kevin M. Legg, PhD & Luciano Arantes, MS & Phillip B. Danielson, PhD This research was supported in part 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 authors and do not necessarily represent the official position or policies of the US Department of Justice.

2 Criminalistics Workflow: Serological Screening Serological screening is the gatekeeper for DNA/STR testing Submission Serological Analysis Which fluids are present? DNA Analysis Who is present? Positive Result Negative Result

3 Forensic Body Fluid Identification Challenges Faced By Serologists Presumptive (Antibody, Chemical Rxn, Enzyme Activity) and/or time consuming assays (Microscopy) Chemical Rxn Kastle-Meyer Enzyme Activity Acid Phosphatase Antibody Test Kits C T S

4 Serology is Critical Serology can resolve ambiguities that cannot be addressed by DNA alone. Saliva vs. Vaginal Secretions present on bottle. Human blood? Animal?

5 Next Gen Serology: Mass Spectrometry Based Proteomics Project Objectives: 1. Identify Protein Biomarkers for body fluid identification 2. Develop a rapid multi-fluid/multiplex mass spectrometry assay Advantages: Confirmatory detection by mass spectrometry Universal sample preparation & multi-fluid multiplexing Project History: Discovery Phase Identify Candidate Biomarkers Validation Phase Evaluate Biomarker Specificity Assess Robustness, Reliability and Performance Limits

6 Project History: Biomarker Discovery Through Comparative Proteomics

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

8 Project History: Validation of Biomarker Specificity for Target Stains

9 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 Mass Spectrometry (Targeted Ion Assay) 1260 NanoLC Chip Cube & 6520 Q-TOF Rapid method development Cost effective Readily multiplexed

10 Biomarker Specificity Results Population Study 250 samples assessed by Q-TOF 2 proteins/fluid were unique and consistently detected 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 9 NGAL P04279 P61916 P09228 P02808 P07911 P10451 P02790 P01024 P14780 P80188

11 Backlog Method Development: Developing a Blood ID Assay With the Brasilia Police

12 Developing a Blood ID Assay With the Brasilia Police Instituto De Criminalistica Laboratory serves the Brasilia Federal District Quadrupole Time-of-Flight method Chemical: Kastle Meyer Antibody: OBTI & FOB Requirements Human specific Process ~100 samples/week

13 Implementation Plan Blood is most common fluid Agilent 6540 Q-TOF UHPLC Tasks 1 and 2 completed prior to arriving in Brasilia Tasks 3 and 4 completed in 1 week. Training/Familiarization included Laboratory staff completed case sample data acquisition 1 - Protein Selection 2 - Peptide Selection 3 - Reference Standards 4 - Acquisition Method 5 - Case Sample Analysis

14 Protein Selection Protein targets selected from previous validation data Emphasis on high abundance targets More robust for ID in compromised or trace samples 5 blood protein biomarkers selected Protein Target Accession Number Protein Function Alpha 1 Antitrypsin P01009 Protease Inhibitor. Cellular Protection. Apolipoprotein A1 P02647 Component of high-density lipoprotein (HDL). Lipid transportation. Complement C3 P01024 Innate immunity. Complement system activation. Hemoglobin Beta P68871 Oxygen transportation. Hemopexin P02790 Heme scavenger.

15 Peptide Selection I Proteins Peptides Proteins have enormous molecular weight variability Not possible to characterize complex intact mixtures. Statherin (MW = 7,000 Da) Complement C3 (MW = 190,000 Da) Titin (MW = 3,800,000 Da) Trypsin Digestion Cleaves at carboxyl side of Arginine (R) and Lysine (K) Generates MS-Friendly peptides ~ 8-20 AA lengths with multiple basic moieties (positive ESI)

16 Peptide Selection II Hemopexin FASTA file >sp P02790 HEMO_HUMAN Hemopexin OS=Homo sapiens MARVLGAPVALGLWSLCWSLAIATPLPPTSAHGNVAEGETKPDPDVTERCSDGWSFDATT LDDNGTMLFFKGEFVWKSHKWDRELISERWKNFPSPVDAAFRQGHNSVFLIKGDKVWVYP PEKKEKGYPKLLQDEFPGIPSPLDAAVECHRGECQAEGVLFFQGDREWFWDLATGTMKER SWPAVGNCSSALRWLGRYYCFQGNQFLRFDPVRGEVPPRYPRDVRDYFMPCPGRGHGHRN GTGHGNSTHHGPEYMRCSPHLVLSALTSDNHGATYAFSGTHYWRLDTSRDGWHSWPIAHQ WPQGPSAVDAAFSWEEKLYLVQGTQVYVFLTKGGYTLVSGYPKRLEKEVGTPHGIILDSV DAAFICPGSSRLHIMAGRRLWWLDLKSGAQATWTELPWPHEKVDGALCMEKSLGPNSCSA NGPGLYLIHGPNLYCYSDVEKLNAAKALPQPQNVTSLLGCTH Trypsin NFPSPVDAAFR (MW = 1220 Da) YYCFQGNQFLR (MW = 1438 Da) LVQGTQVYVFLTK (MW = 1772 Da)

17 Peptide Selection III Species Specificity BLAST search the Swissprot database for all proteins containing Hemoglobin β peptide GTFATLSELHCDK Human Bonobo Chimp Hemoglobin β peptide is not human specific.

18 Peptide Selection III Species Specificity BLAST search the Swissprot database for all proteins containing peptide Complement C3 peptide AAVYHHFISDGVR Human Complement C3 peptide is human specific.

19 Peptide Selection IV Final Target List Protein Accession Peptide m/z Alpha 1 Antitrypsin Apolipoprotein A1 Complement C3 Hemoglobin Beta Hemopexin P01009 P02647 P01024 P68871 P02790 DTEEEDFHVDQVTTVK FNKPFVFLMIEQNTK LSITGTYDLK SVLGQLGITK EQLGPVTQEFWDNLEK LLDNWDSVTSTFSK DYVSQFEGSALGK VSFLSALEEYTK ATEHLSTLSEK TELRPGETLNVNFLLR SNLDEDIIAEENIVSR SSLSVPYVIVPLK IHWESASLLR TGLQEVEVK SAVTALWGK VNVDEVGGEALGR GTFATLSELHCDK EFTPPVQAAYQK LLVVYPWTQR LYLVQGTQVYVFLTK YYC[+57.0]FQGNQFLR NFPSPVDAAFR GGYTLVSGYPK

20 Custom Peptide Library Acquisition Method I Reference Standards Cost effective (~$15/peptide) but crude purity (50% purity) Qualitative confirmation of biomarker ID Populate PCDL spectral library & retention time parameters Hemopexin NFPSPVDAAFR [M] = m/z =

21 Acquisition Method I Reference Standards Populating Personal Compound Database Library (PCDL) Manager

22 Acquisition Method II Directed MS/MS Mode Restrict fragmentation to ions present in preferred table 6540 Q-TOF and AJS Conditions 1290 LC Conditions Parameter Value Polarity Positive Capillary Voltage 3500 V Drying gas 300 C, 10 l/min Nebulizer 30 psi sheath gas 300 C, 11 l/min Acquisition Rate 6 (MS) and 3 (MS/MS) spectra/second Acquisition Range m/z (MS) and m/z (MS/MS) Collision Energy Using static formula based off precursor m/z 3.1*(m/z) *(m/z) Internal Refrence Mass and m/z Precursor Selection 2 precursors/cycle Active Exclusion After 2 Spectrim Release After.05 minutes Parameter Value Column 2.1 x 150 mm RRHD Eclipse Plus C18 Column termperature 50 C Mobile Phase A: H2O.1%FA B: ACN.1% FA Flow rate.5 ml/min Gradient Time %B initial Stop time 15 min Post time 3 min Injection volume 20 ul, 10 ug peptide per injection

23 Data Analysis Masshunter + PCDL Library Proteomics software not required Compound List Masshunter ID Parameters Accurate mass < 10 ppm RT.2 minutes DB Score > 65 Accurate Mass & Retention Time MS/MS Library search

24 Backlog Casework Sample Analysis:

25 Casework Sample Analyses 30 suspected bloodstains selected from casework Most of them related to homicides or attempted murders Presumptive tests results were compared to Protein-based Human Blood ID by Mass Spectrometry SOME OF THE SAMPLES AFTER STAIN EXTRACTION FROM SWAB OR GAUZE

26 Case Sample Results I Presumptive Confirmatory

27 S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11 S12 S13 S14 S15 S16 S17 S18 S19 S20 S21 S22 S23 S24 S25 S26 S27 S28 S29 S30 N UMBER OF PEPTIDES Case Sample Results II Protein-based Human Blood ID Results Hemopexin A1AT C3 Apo A-1 Hemo beta EVIDENCE

28 Details: Sample 18 & 23 Attempted Murder

29 Details: Sample 18 & 23 Attempted Murder S18: Sample collected from the floor S23: blood splattered plastic bottle

30 Details: Sample 18 & 23 Attempted Murder S18: Sample collected from the floor S18 S18 Presumptive Blood Tests EVIDENCE KASTLE-MEYER OBTI FOB S18 NEG NEG NEG S23 POS POS POS Mass Spectrometry PEPTIDES FOUND RELATED TO Hemo beta Apo A-1 A1AT Hemopexin POS NEG POS POS POS POS POS NEG

31 Details: Sample 18 Attempted Murder Serological screening is the gatekeeper for DNA/STR testing Based only on presumptive test results, sample 18 & 27 would have not been sent to DNA analysis Sample 18 was sent to DNA analysis but did not yield a DNA profile Hemoglobin, A1AT, Hemopexin identified via mass spec Proteins likely remain viable longer than DNA

32 Summary MS method outperformed presumptive tests Human blood was confirmed in all samples Correctly identified false negative results from presumptive tests With added benefits of: Reliable and confirmatory stain ID Batch processing potential Robotics ready

33 Future Directions Department of Defense Grant Award Rapid Innovation Fund Development and deployment Fully automated six fluid multiplex workflow Saliva, Seminal fluid Urine Vaginal fluid Menstrual blood, Peripheral blood

34 Acknowledgements Instituto De Criminalistica (Brasilia, Brazil) Luciano Arantes Eduardo Ramalho Rodrigo Santos Agilent Technologies Andre Santos Tom Gluodenis NMS Labs/Center for Forensic Sci. Dr. Christian Westring Dr. Barry Logan Heather McKiernan University of Denver Danielson Laboratory National Institute of Justice Minh Nguyen and Chad Ernst