Validation of a Confirmatory Proteomic Mass Spectrometry Body Fluid Assay

Validation of a Confirmatory Proteomic Mass Spectrometry Body Fluid Assay

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This webinar originally occurred on December 7, 2023
Duration: 1 hour


With increasingly sensitive forensic methods for DNA detection, the source from which a DNA profile is obtained becomes increasingly important. Was an individual’s DNA identified at a crime scene from blood, saliva, semen, or from a touched object? Knowledge of the source of DNA can add important contextual information to investigations as well as evidence presented at trials. Currently, nearly all serology (body fluid) testing is presumptive, meaning the results are not definitive, and consequently do not carry the same scientific and statistical weight as forensic DNA analysis does. To address this issue, the New York City Office of Chief Medical Examiner (NYC OCME) has developed a confirmatory assay for the identification of blood, saliva, and semen using proteins as body fluid markers. Because different body fluids have different functions, they express different proteins that are specific to those functions. For example, in blood the protein hemoglobin carries oxygen, in saliva the protein amylase aids in digestion, and in semen the protein semenogelin functions in reproduction. The presence of these marker proteins, as well as other blood, saliva, and semen marker proteins, can be used to distinguish one body fluid from another. However, in addition to identifying multiple specific markers in each body fluid, an assay must also employ methods that confirm that the markers are actually there. To achieve this, we use mass spectrometry to effectively determine the amino acid sequences of each protein marker, thus confirming their presence in a sample. The assay developed at the NYC OCME uses multiple protein markers for each body fluid. The assay is unbiased, i.e. there is no a priori guessing about what a body fluid might be on a piece of evidence - all samples are treated alike from extraction through analysis, and all body fluid markers are simultaneously checked in each sample. The assay can identify mixtures and determine species. Finally, the assay is fast and inexpensive – mass spectrometers are already found in toxicology laboratories. The NYC OCME Molecular Serology Assay has been accredited by ANAB and approved by the New York State Commission on Forensic Science.

Detailed Learning Objectives

  1. Attendees will gain an understanding of the importance of, and difference between, presumptive and confirmatory tests. 
  2. Attendees will learn how and why specific body fluid marker proteins were selected.  
  3. Attendees will learn why multiple reaction monitoring (MRM) mass spectrometry is a confirmatory test. 


  • Donald Siegel, Ph.D. | Principal Scientist, New York City Office of Chief Medical Examiner

Funding for this Forensic Technology Center of Excellence webinar has been provided by the National Institute of Justice, Office of Justice Programs, U.S. Department of Justice.

Funding for this research has been provided by the National Institute of Justice, Office of Justice Programs, U.S. Department of Justice, Award Number 15PNIJ-21-GG-02712-SLFO. 

The opinions, findings, and conclusions or recommendations expressed in this webinar are those of the presenter(s) and do not necessarily reflect those of the U.S. Department of Justice.

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