The author(s) shown below used Federal funding provided by the U.S. Department of Justice to prepare the following resource: Document Title: Development of a Proteomic Assay for Menstrual Blood, Vaginal Fluid and Species Identification Author(s): Donald Siegel, Ph.D. Document Number: 251932 Date Received: August 2018 Award Number: 2010-DN-BX-K192 This resource has not been published by the U.S. Department of Justice. This resource is being made publically available through the Office of Justice Programs’ National Criminal Justice Reference Service. Opinions or points of view expressed are those of the author(s) and do not necessarily reflect the official position or policies of the U.S. Department of Justice. Development of a Proteomic Assay for Menstrual Blood, Vaginal Fluid and Species Identification Final Draft Technical Report NIJ Grant 2010-DN-BX-K192 Principal Investigator: Donald Siegel, Ph.D. Principal Scientist Office of Chief Medical Examiner 421 East 26th Street New York, NY 10016 Tel: 212-323-1434 Fax: 212-323-1560 Email: [email protected] Web: www.nyc.gov/ocme This resource was prepared by the author(s) using Federal funds provided by the U.S. Department of Justice. Opinions or points of view expressed are those of the author(s) and do not necessarily reflect the official position or policies of the U.S. Department of Justice. Final Draft Technical Report NIJ Grant 2010-DN-BX-K192 Development of a Proteomic Assay for Menstrual Blood, Vaginal Fluid and Species Identification TABLE OF CONTENTS ABBREVIATIONS……………………………………………………………………………………………………………………..4 ABSTRACT .................................................................................................................................................................... 6 EXECUTIVE SUMMARY ............................................................................................................................................. 7 SYNOPSIS OF THE PROBLEM ........................................................................................................................................................... 7 PURPOSE ........................................................................................................................................................................................... 8 RESEARCH DESIGN ........................................................................................................................................................................... 8 Menstrual & Venous Blood Collection ........................................................................................................................... 8 Vaginal Fluid Collection ...................................................................................................................................................... 8 Sample Preparation and Processing .............................................................................................................................. 9 FINDINGS ....................................................................................................................................................................................... 12 Menstrual Blood .................................................................................................................................................................. 13 Markers Found in All Women but Also Detected in Other Body Fluids ................................................. 13 Markers Found Only in Menstrual Blood but Not Found in All Women ................................................ 14 Markers Enriched in Menstrual Blood but Detected in Other Body Fluids Not in All Women ..... 14 Vaginal Fluid ........................................................................................................................................................................ 15 Species Identification ........................................................................................................................................................ 16 CONCLUSIONS INCLUDING IMPLICATIONS FOR POLICY AND PRACTICE.................................................................................16 INTRODUCTION ....................................................................................................................................................... 18 STATEMENT OF THE PROBLEM ................................................................................................................................................... 18 Serology Assays ................................................................................................................................................................... 18 Menstrual Blood and Vaginal Fluid Testing ............................................................................................................ 19 LITERATURE REVIEW AND CITATIONS ...................................................................................................................................... 19 Current Serology Methods in Use ................................................................................................................................. 19 Chemical Assays .......................................................................................................................................................... 20 Enzymatic Assays ........................................................................................................................................................ 20 Microscopic Observations ........................................................................................................................................ 20 Immunochemical Assays .......................................................................................................................................... 20 Current Research ................................................................................................................................................................ 21 Messenger RNA Assays ............................................................................................................................................. 21 MicroRNA Assays ........................................................................................................................................................ 21 DNA Methylation Analysis for Forensic Tissue Identification ................................................................... 23 RATIONALE FOR RESEARCH ........................................................................................................................................................ 23 SECTION 1 – MALDI TOF/TOF ANALYSIS OF MENSTRUAL BLOOD ....................................................... 24 BODY FLUID COLLECTION METHODS.......................................................................................................................................... 24 BODY FLUID COLLECTION RESULTS............................................................................................................................................ 25 Length of Periods ................................................................................................................................................................ 25 Volunteer Demographics ................................................................................................................................................. 25 MENSTRUAL BLOOD SAMPLE PREPARATION METHODS ..........................................................................................................26 PROTEIN QUANTITATION RESULTS ............................................................................................................................................ 27 Varying Lengths of Menses ............................................................................................................................................. 27 Five Day Menses Only ........................................................................................................................................................ 31 REDUCTION OF PROTEIN DYNAMIC RANGE ............................................................................................................................... 34 SDS MICROFLUIDIC GEL ELECTROPHORESIS ............................................................................................................................39 PEPTIDE ISOBARIC LABELING FOR MULTIPLEXING AND QUANTIZATION ............................................................................40 How Isobaric Barcoding Works .................................................................................................................................... 41 NANO-HPLC ................................................................................................................................................................................... 42 MASS SPEC ANALYSIS ................................................................................................................................................................... 42 MENSTRUAL BLOOD / VENOUS BLOOD SAMPLE ANALYSIS .....................................................................................................44 2 This resource was prepared by the author(s) using Federal funds provided by the U.S. Department of Justice. Opinions or points of view expressed are those of the author(s) and do not necessarily reflect the official