Identification and evaluation of potential forensic marker proteins in vaginal fluid by liquid chromatography/mass spectrometry

Abstract

Vaginal fluid is one of the most common body fluids found at crime scenes. Discriminating vaginal fluid from other body fluids is important in forensic science; however, few potential protein markers have been reported to date. Proteomic methods for identifying protein markers have gained attention, although few reports have applied this technology to forensic protein markers. Therefore, to identify characteristic vaginal proteins, we examined various body fluids (nasal secretions, saliva, urine, semen, vaginal fluids, and sweat) using liquid chromatography/electrospray ionization time-of-flight mass spectrometry and peptide mass fingerprinting. We identified three components (average molecular mass values 17,237 ± 2, 18,063 ± 2, and 15,075 ± 1) detectable only in vaginal samples: two human small proline-rich protein 3 (SPRR3) isoforms and a human fatty acid-binding protein 5 (FABP5) with an acetylated (+42) N-terminal region lacking the initiator methionine residue (-131). Using ELISA, these yielded markedly high average values in vaginal fluids. The mass spectra of these proteins were not detected in infant saliva but were detected in the vaginal fluid throughout the menstrual cycle. The results of forensic analysis (detection limit, mixed body fluid samples, casework samples, and blind samples) suggest that these proteins are potential forensic markers. In conclusion, high SPRR3 and FABP5 expression levels, which may be used as potential markers for vaginal fluid identification in forensic science, were detected in vaginal fluids from healthy adults.

Current Methods for Body Fluid Identification Related to Sexual Crime: Focusing on Saliva, Semen, and Vaginal Fluid

“…Methods using microRNA [45,46,48,49] and DNA methylation [52][53][54][55] are also being examined. Furthermore, we have tried to develop novel methods for vaginal fluid identification by GC-MS targeting of 17β-estradiol (E2-17β) and by LC-MS targeting of small proline-rich protein 3 (SPRR3) and fatty acid-binding protein 5 (FABP5) [94,95].…”

A Análise Proteômica No Contexto Forense

“…Eles requerem a integridade estrutural dos marcadores e, além de não serem confirmatórios, apenas presuntivos, outras proteínas ou substâncias podem estar presentes, levando a um resultado falso positivo (STEENDAM et al, 2013;YANG, 2013;LEGG et al, 2014;PATEL et al, 2016;FUJIHARA et al, 2017) e à necessidade de realização de vários testes diferentes para a identificação desses fluidos corporais (YANG, 2013;FUJIHARA et al, 2017). Foram realizadas pesquisas sobre o isolamento e a identificação de biomarcadores de proteínas altamente específicos de manchas biológicas com utilidade forense na identificação de traços de saliva humana, fluido seminal, urina, fluido vaginal, sangue periférico, fluido menstrual (STEENDAM et al, 2013;YANG, 2013;LEGG et al, 2014;IGOH;DOI;SAKURADA, 2015;DAMMEIER et al, 2016;PATEL et al, 2016) e, em menor grau, secreções nasais, fezes, e urina (STEENDAM et al, 2013). Os métodos utilizados com a tecnologia de MS foram capazes de detectar e determinar múltiplos marcadores de proteínas dos diferentes fluidos corporais em um único teste confirmatório (STEENDAM et al, 2013;YANG, 2013;LEGG et al, 2014;DAMMEIER et al, 2016;IGOH;DOI;SAKURA, 2015;PATEL et al, 2016).…”

“…Foram realizadas pesquisas sobre o isolamento e a identificação de biomarcadores de proteínas altamente específicos de manchas biológicas com utilidade forense na identificação de traços de saliva humana, fluido seminal, urina, fluido vaginal, sangue periférico, fluido menstrual (STEENDAM et al, 2013;YANG, 2013;LEGG et al, 2014;IGOH;DOI;SAKURADA, 2015;DAMMEIER et al, 2016;PATEL et al, 2016) e, em menor grau, secreções nasais, fezes, e urina (STEENDAM et al, 2013). Os métodos utilizados com a tecnologia de MS foram capazes de detectar e determinar múltiplos marcadores de proteínas dos diferentes fluidos corporais em um único teste confirmatório (STEENDAM et al, 2013;YANG, 2013;LEGG et al, 2014;DAMMEIER et al, 2016;IGOH;DOI;SAKURA, 2015;PATEL et al, 2016). Além de a análise não ser destrutiva para o DNA, também pode ser usada como exploração (STEENDAM et al, 2013;FUJIHARA et al, 2017), como em uma amostra de 9 anos de idade que foi submetida na época a tratamento químico usado para realçar a visualização do sangue, abrindo, assim, novas oportunidades forenses para a revisão de casos estacionados (PATEL et al, 2016).…”

Development of a Protein Microarray Chip with Enhanced Fluorescence for Identification of Semen and Vaginal Fluid

“…In addition, various methods have been developed for the identification of human semen using protein biomarkers, such as electrophoresis [ 6 ], immunochromatography [ 7 , 9 ], and Raman spectroscopy [ 8 , 10 ]. For the identification of VF, 17 beta-estradiol [ 11 , 12 ], human small proline-rich protein 3 [ 13 ], human fatty acid-binding protein 5 [ 13 ], and cervical protein [ 14 ] have been used as biomarkers, and the electrochemical enzyme-linked immunosorbent assay [ 11 , 12 ] and differential pulse voltammetry [ 11 , 15 ] methods have been used. Although some identification methods using protein biomarkers are commercialized as a pre-screening tool for forensic analysis [ 9 , 16 ], previous methods require a relatively large amount of sample (generally 1.5~2.0 mL) [ 9 , 16 ] to detect a single body fluid.…”