Review: Direct Identification of Forensic Bodily Fluids Using Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry

Emily C. Lennert





bodily fluid, blood, semen, saliva, urine, serology, matrix-assisted laser desorption, MALDI, time of flight, TOF, mass spectrometry, MS

Article Reviewed

  1. Kamanna, S.; Henry, J.; Voelcker, N. H.; Linacre, A.; Kirkbride, K. P. Direct identification of forensic bodily fluids using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. International Journal of Mass Spectrometry. 2016, 397-398, 18-26.


The opinions expressed in this review are an interpretation of the research presented in the article. These opinions are those of the summation author and do not necessarily represent the position of the University of Central Florida or of the authors of the original article.


Bodily fluids may be encountered at a crime scene, and the type of fluid must be identified, e.g. blood, urine, saliva, or semen. Several presumptive tests exist for each fluid type; however, these tests are often prone to false positive or false negative results. For example, luminol is a common presumptive test for blood detection; however, materials such as bleach or those containing copper may produce false positive reactions. Therefore, a screening method that is rapid and precise is desired.

Matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) is a mass spectrometric technique capable of detecting protein biomarkers in biological samples. MALDI works by ionizing the applied matrix surrounding the sample using a laser. This gentle ionization creates ions with minimal fragmentation, allowing for the detection of large molecule such as proteins. Previous research has indicated that MALDI-TOFMS may be used to accurately and reproducibly detect hemoglobin in blood, α-amylase in saliva, and prostate specific antigen (PSA) and semenogelin protein markers in semen. The authors of this study sought to apply this method for protein biomarker identification for a forensic purpose, and to develop an appropriate and practical forensic analysis method.

Recent bodily fluid samples were obtained from volunteers: blood, saliva, urine, and semen were collected. Aged samples of fabrics stained with various bodily fluids, such as blood, were obtained from proficiency test kits provided by Collaborative Testing Services (CTS) Incorporated. Four aged test kits were obtained. Samples were dated 2004, 2011, 2012, and 2014, i.e. approximately 11, 4, 3, and 1 year(s) old, respectively, with respect to when the study was conducted.

“Top-Down” Analysis of Bodily Fluid Mixtures

Bodily fluid mixtures were prepared for “top down”, i.e. intact, protein analysis. A mixture containing 2 μL of diluted blood in water, 3 μL of undiluted semen, and 5 μL of undiluted saliva was prepared. Then, 1 μL of this blood/semen/saliva mixture was combined with 1 μL of a MALDI matrix solution, sinapic acid (SA) in 50% acetonitrile/water with 0.1% trifluoroacetic acid. Additionally, urine samples were prepared by mixing 1 μL of urine with 1 μL of a MALDI matrix, α-cyano-4-hydrocinnamic acid (CHCA) in 50% acetonitrile/water with 0.1% trifluoroacetic acid. Samples were then analyzed by depositing 2 μL of each solution onto an individual polished steel plate. Analysis of the samples allowed for detection of all target proteins in the samples. Target proteins are listed in Table 1, below.

Table 1. Target proteins for each body matrix sample

Body Matrix Target Protein
Blood Hemoglobin-β
Saliva α-amylase I
α-amylase II B
Pancreatic α-amylase
Semen Semenogelin I
Semenogelin II
Prostate Specific Antigen (PSA)
Urine Uromodulin

“Bottom-Up” Identification of Bodily Fluids

Samples were prepared for “bottom up” identification by protein digestion with trypsin. Protein digestion breaks the proteins in each sample into fragments, creating a characteristic fragmentation pattern or “fingerprint”. This pattern is known as the peptide mass fingerprint (PMF) and may be used to identify the protein. This is a form of confirmatory identification. After sample digestion, each digested sample was mixed with α-CHCA matrix and placed on a steel plate for analysis. PMF results were used to identify each protein based on its unique “fingerprint” by comparison to a protein database. Each target protein was successfully identified based on its PMF in the samples.

Analysis of Aged Samples

Aged samples were examined to evaluate the stability of target proteins in samples. Samples originated from proficiency test kits and varied in age from 1, 3, 4, and 11 years old. Each test kit contained four fabric samples, each sample was stained with various bodily fluids to simulate possible real-world evidence. Each sample was extracted, and SA matrix was applied to the extract for MALDI-TOFMS analysis. Hemoglobin proteins were successfully identified in samples up to 11 years old. For confirmation of identification, samples were subjected to trypsin digestion and analyzed. Hemoglobin proteins were identified based on PMF, i.e. a confirmatory identification was obtained. This indicated that hemoglobin proteins maintain stability over long periods of time on fabric substrates.

Analysis of Bodily Fluids Deposited on Fabric

Recently obtained bodily fluids were deposited on several types of fabric: nylon, cotton, and cellulose acetate. After deposition, samples were allowed to dry. Then, fibers were removed from the fabrics for direct analysis. The fibers were placed in SA matrix on ITO-coated slides and analyzed. Blood and saliva produced positive results for the target proteins on all fabric types. However, semen produced variable results, depending on the fabric type. PSA, semenogelin I, and semenogelin II ionized well, i.e. produced a good instrumental signal, on nylon, but ionized less well on cellulose acetate. On cotton, these proteins were detected only in trace amounts. This indicates that the substrate may have a significant effect on the analysis of bodily fluid samples by this direct approach.

Scientific Highlights

  • “Top down” screening of bodily fluids by MALDI-TOFMS allowed for the rapid identification of bodily fluids based on target proteins.
  • “Bottom up” analysis using trypsin digestion allows for confirmatory identification of the sample based on the PMF of the proteins present in the sample.
  • Hemoglobin proteins were detectable on samples aged 11 years, indicating this method may be applicable to aged samples.
  • Fabric type, i.e. substrate, may significantly impact the analysis of proteins by the direct analysis approach demonstrated in the study.


When a bodily fluid is encountered at a crime scene, the type of bodily fluid must be identified for subsequent analysis.

Potential Conclusions

MALDI-TOFMS may provide a method of identifying bodily fluids based on the proteins present in the sample.