Multiple Reaction Monitoring Tandem Mass Spectrometry Approach for the Identification of Biological Fluids at Crime Scene Investigations

Knowledge of the nature of biofluids at a crime scene is just as important as DNA test to link the nature of the biofluid, the criminal act, and the dynamics of the crime. Identification of methods currently used for each biological fluid (blood, semen, saliva, urine) suffer from several limitations including instability of assayed biomolecules, and low selectivity and specificity; as an example of the latter issue, it is not possible to discriminate between alpha-amylase 1 (present in saliva) and alpha-amylase 2 (present in semen and vaginal secretion. In this context, the aim of the work has been to provide a predictive protein signature characteristic of each biofluid by the recognition of specific peptides unique for each protein in a single analysis. A panel of four protein biomarkers for blood, four for saliva, five for semen, and two for urine has been monitored has been monitored by using a single multiple reaction monitoring (MRM)-based method targeting concomitantly 46 different peptides. Then, The optimized method allows four biological matrices to be identified when present on their own or in 50:50 mixture with another biofluid. Finally, a valid strategy combining both DNA analysis and liquid chromatographic-tandem mass spectrometric multiple reaction monitoring (LC-MS-MRM) identification of biofluids on the same sample has been demonstrated to be particularly effective in forensic investigation of real trace evidence collected at a crime scene

Systematic Nucleo-Cytoplasmic Trafficking of Proteins Following Exposure of MCF7 Breast Cancer Cells to Estradiol

We have used a proteomics subcellular spatial razor approach to look at changes in total protein abundance and in protein distribution between the nucleus and cytoplasm following exposure of MCF7 breast cancer cells to estradiol. The dominant response of MCF7 cells to estrogen stimulation involves dynamic changes in protein subcellular spatial distribution rather than changes in total protein abundance. Of the 3604 quantitatively monitored proteins, only about 2% show substantial changes in total abundance (>2-fold), whereas about 20% of the proteins show substantial changes in local abundance and/or redistribution of their subcellular location, with up to 16-fold changes in their local concentration in the nucleus or the cytoplasm. We propose that dynamic redistribution of the subcellular location of multiple proteins in response to stimuli is a fundamental characteristic of cells and suggest that perturbation of cellular spatial control may be an important feature of cancer

Quantitative determination of l-Asp, d-Asp and NMDA in samples from mouse prefrontal cortex by MRM LC-MSMS analysis.

<p>Samples were obtained from brain of three individual animals from each group (<i>DDO</i>^+/+, wild type mouse; <i>DDO</i>^-/-, D-aspartate oxidase (DDO) knockout mouse). Each measurement represents the average of three technical replicates.</p

Calibration curves obtained for the three analytes.

<p>Calibration curves obtained for the three analytes.</p

Quantitative determination of free D-Asp, L-Asp and N-methyl-D-aspartate in mouse brain tissues by chiral separation and Multiple Reaction Monitoring tandem mass spectrometry

<div><p>Several studies have suggested that free d-Asp has a crucial role in N-methyl d-Asp receptor-mediated neurotransmission playing very important functions in physiological and pathological processes. This paper describes the development of an analytical procedure for the direct and simultaneous determination of free d-Asp, l-Asp and N-methyl d-Asp in specimens of different mouse brain tissues using chiral LC-MS/MS in Multiple Reaction Monitoring scan mode. After comparing three procedures and different buffers and extraction solvents, a simple preparation procedure was selected the analytes of extraction. The method was validated by analyzing l-Asp, d-Asp and N-methyl d-Asp recovery at different spiked concentrations (50, 100 and 200 pg/μl) yielding satisfactory recoveries (75–110%), and good repeatability. Limits of detection (LOD) resulted to be 0.52 pg/μl for d-Asp, 0.46 pg/μl for l-Asp and 0.54 pg/μl for NMDA, respectively. Limits of quantification (LOQ) were 1.57 pg/μl for d-Asp, 1.41 pg/μl for l-Asp and 1.64 pg/μl for NMDA, respectively. Different concentration levels were used for constructing the calibration curves which showed good linearity. The validated method was then successfully applied to the simultaneous detection of d-Asp, l-Asp and NMDA in mouse brain tissues. The concurrent, sensitive, fast, and reproducible measurement of these metabolites in brain tissues will be useful to correlate the amount of free d-Asp with relevant neurological processes, making the LC-MS/MS MRM method well suited, not only for research work but also for clinical analyses.</p></div

TIC and MRM chromatograms recorded for the three analytes in a sample from prefrontal cortex of wild type mouse.

<p>The specific MRM transitions for each analyte are indicated.</p

Total ion current (TIC) chromatogram for the analysis of 100 pg/μl standard mixture solution of l-Asp, d-Asp and NMDA.

<p>Peaks corresponding to each analyte are labelled.</p

Quantitative analysis of d-Asp (left panel) and d-Asp versus l-Asp ratio (right panel) in samples from mouse prefrontal cortex using the developed LC-MS/MS MRM procedure.

<p><i>DDO</i>^+/+, wild type mouse; <i>DDO</i>^-/-, d-aspartate oxidase (DDO) knockout mouse.</p

LCMSMS analyses in MRM mode of test samples from mouse brain tissues to optimise the sample treatment procedure.

<p>Some experimental conditions for sample extraction and precipitation are reported in the figure. Peaks corresponding to each analyte are labelled.</p

Validation parameters of the analytical method developed for the quantitative determination of l-Asp, d-Asp and NMDA.

<p>Each measurement represents the average of three technical replicates.</p