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

Western blot analysis.

<p>Western blot analysis of proteins extracted from mature spores of wild type (PY79, lane 1), <i>ΔcotGΔcotH</i> (AZ603, lane 2), <i>ΔcotGΔcotH amyE::cotGcotH</i> (AZ608, lane 3), <i>ΔcotGΔcotH amyE::cotG_stopcotH</i> (AZ604, lane 4), <i>cotH::spc</i> (ER220, lane 5) and <i>ΔcotGΔcotH amyE::cotG</i> (AZ607, lane 6 of panel B) strains. For CotA and CotB detection (panel A) the proteins have been extracted by SDS treatment while for CotC and CotU detection (panel B) the NaOH treatment has been used. Proteins (25 µg) were reacted with CotA, CotB and CotC specific rabbit antibodies and then with peroxidase-conjugated secondary antibodies and visualized by the Pierce method. The estimated size of CotB, CotC and CotU is indicated.</p

Production of CotH in a <i>cotG</i> null mutant.

<p>(A) SDS-PAGE fractionation of coat proteins from a wild type strain (PY79) and isogenic strains carrying null mutations in <i>cotG</i> (ER203) or in <i>cotH</i> (ER220). A molecular weight marker is also present and the size of relevant bands indicated. (B) Western blot with anti-CotH antibody of the same three strains analyzed in panel A. The arrow points to the CotH specific band.</p

Germination efficiency and lysozyme-resistance assays.

<p>Spores derived from wild type (PY79, black circles), <i>cotG</i> null (AZ604, white circles), <i>cotH</i> null (ER220, white squares) and <i>cotGcotH</i> null (AZ603, black squares) were tested for germination efficiency (A) and for lysozime resistance (B). Germination was induced by Asn-GFK and measured as percentage of loss of optical density at 580 nm. Similar results were obtained by using L-Ala to induce germination. A <i>cotE</i> null strain (black triangles) known to be sensitive to lysozyme has been used as positive control during the lysozime treatment. Error bars are based on the standard deviation of 4 independent experiments.</p

<i>Bacillus subtilis</i> strains used in this study.

<p><i>Bacillus subtilis</i> strains used in this study.</p

CotG and phosphorylation sites.

<p>Results of a mass spectrometry analysis of peptides derived from trypsine digestion of CotG are reported. Unambiguosly identified sites of phosphorylation are indicated. Tripeptides containing a phosphate moiety are underlined; the random coiled tandem repeats region is in red.</p

Construction of a single <i>cotG</i> mutant.

<p>(A) Thick gray and black arrows indicate the coding parts of <i>cotG</i> and <i>cotH</i>, respectively. Dashed arrow indicates the mRNA produced from the <i>cotG</i> and <i>cotH</i> promoters, as already reported. Site of insertion of the additional base in the <i>cotG</i> coding sequence (wild type sequence) that causes the formation of a premature stop codon (mutant sequence). Western blot analysis with anti-CotH (B) and anti-CotG (C) antibodies of proteins extracted by SDS treatment from wild type and isogenic mutant spores. The mutants genotype relative to the <i>cotG cotH</i> and <i>amyE</i> loci is indicated. Arrows point the CotH and CotG specific bands.</p

Profiling Carbonylated Proteins in Heart and Skeletal Muscle Mitochondria from Trained and Untrained Mice

Understanding the relationship between physical exercise, reactive oxygen species, and skeletal muscle modification is important in order to better identify the benefits or the damages that appropriate or inappropriate exercise can induce. Heart and skeletal muscles have a high density of mitochondria with robust energetic demands, and mitochondria plasticity has an important role in both the cardiovascular system and skeletal muscle responses. The aim of this study was to investigate the influence of regular physical activity on the oxidation profiles of mitochondrial proteins from heart and tibialis anterior muscles. To this end, we used the mouse as animal model. Mice were divided into two groups: untrained and regularly trained. The carbonylated protein pattern was studied by two-dimensional gel electrophoresis followed by Western blot with anti-dinitrophenyl hydrazone antibodies. Mass spectrometry analysis allowed the identification of several different protein oxidation sites, including methionine, cysteine, proline, and leucine residues. A large number of oxidized proteins were found in both untrained and trained animals. Moreover, mitochondria from skeletal muscles and heart showed almost the same carbonylation pattern. Interestingly, exercise training seems to increase the carbonylation level mainly of mitochondrial proteins from skeletal muscle

SDS-PAGE and Fluorescence analysis.

<p>(A) Proteins released after treatment with SDS of spores of the indicated strains were fractionated on a 12,5% polyacrilamide gel. The arrow indicates the 41 kDa band correspoding to CotS (18). The gel was stained with Coomassie brilliant blue. (B) Strains carrying the <i>cotS::gfp</i> fusion were analyzed by phase-contrast (PC) and fluorescence (F) microscopy. The bottom panel reports a merge of the two images. Exposure time was 588 ms in all cases.</p

Calibration curves obtained for the three analytes.

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