Additions to the Human Plasma Proteome via a Tandem MARS Depletion iTRAQ-Based Workflow

Robust platforms for determining differentially expressed proteins in biomarker and discovery studies using human plasma are of great interest. While increased depth in proteome coverage is desirable, it is associated with costs of experimental time due to necessary sample fractionation. We evaluated a robust quantitative proteomics workflow for its ability (1) to provide increased depth in plasma proteome coverage and (2) to give statistical insight useful for establishing differentially expressed plasma proteins. The workflow involves dual-stage immunodepletion on a multiple affinity removal system (MARS) column, iTRAQ tagging, offline strong-cation exchange chromatography, and liquid chromatography tandem mass spectrometry (LC-MS/MS). Independent workflow experiments were performed in triplicate on four plasma samples tagged with iTRAQ 4-plex reagents. After stringent criteria were applied to database searched results, 689 proteins with at least two spectral counts (SC) were identified. Depth in proteome coverage was assessed by comparison to the 2010 Human Plasma Proteome Reference Database in which our studies reveal 399 additional proteins which have not been previously reported. Additionally, we report on the technical variation of this quantitative workflow which ranges from ±11 to 30%

Enhanced Sample Multiplexing of Tissues Using Combined Precursor Isotopic Labeling and Isobaric Tagging (cPILOT)

There is an increasing demand to analyze many biological samples for disease understanding and biomarker discovery. Quantitative proteomics strategies that allow simultaneous measurement of multiple samples have become widespread and greatly reduce experimental costs and times. Our laboratory developed a technique called combined precursor isotopic labeling and isobaric tagging (cPILOT), which enhances sample multiplexing of traditional isotopic labeling or isobaric tagging approaches. Global cPILOT can be applied to samples originating from cells, tissues, bodily fluids, or whole organisms and gives information on relative protein abundances across different sample conditions. cPILOT works by 1) using low pH buffer conditions to selectively dimethylate peptide N-termini and 2) using high pH buffer conditions to label primary amines of lysine residues with commercially-available isobaric reagents (see Table of Materials/Reagents). The degree of sample multiplexing available is dependent on the number of precursor labels used and the isobaric tagging reagent. Here, we present a 12-plex analysis using light and heavy dimethylation combined with six-plex isobaric reagents to analyze 12 samples from mouse tissues in a single analysis. Enhanced multiplexing is helpful for reducing experimental time and cost and more importantly, allowing comparison across many sample conditions (biological replicates, disease stage, drug treatments, genotypes, or longitudinal time-points) with less experimental bias and error. In this work, the global cPILOT approach is used to analyze brain, heart, and liver tissues across biological replicates from an Alzheimer's disease mouse model and wild-type controls. Global cPILOT can be applied to study other biological processes and adapted to increase sample multiplexing to greater than 20 samples

Spontaneous DNA damage to the nuclear genome promotes senescence,redox imbalance and aging

Accumulation of senescent cells over time contributes to aging and age-related diseases. However, what drives senescence in vivo is not clear. Here we used a genetic approach to determine if spontaneous nuclear DNA damage is sufficient to initiate senescence in mammals. Ercc1-/Δ mice with reduced expression of ERCC1-XPF endonuclease have impaired capacity to repair the nuclear genome. Ercc1-/Δ mice accumulated spontaneous, oxidative DNA damage more rapidly than wild-type (WT) mice. As a consequence, senescent cells accumulated more rapidly in Ercc1-/Δ mice compared to repair-competent animals. However, the levels of DNA damage and senescent cells in Ercc1-/Δ mice never exceeded that observed in old WT mice. Surprisingly, levels of reactive oxygen species (ROS) were increased in tissues of Ercc1-/Δ mice to an extent identical to naturally-aged WT mice. Increased enzymatic production of ROS and decreased antioxidants contributed to the elevation in oxidative stress in both Ercc1-/Δ and aged WT mice. Chronic treatment of Ercc1-/Δ mice with the mitochondrial-targeted radical scavenger XJB-5–131 attenuated oxidative DNA damage, senescence and age-related pathology. Our findings indicate that nuclear genotoxic stress arises, at least in part, due to mitochondrial-derived ROS, and this spontaneous DNA damage is sufficient to drive increased levels of ROS, cellular senescence, and the consequent age-related physiological decline

A diverse view of science to catalyse change:Valuing diversity leads to scientific excellence, the progress of science and, most importantly, it is simply the right thing to do. we must value diversity not only in words, but also in actions

No abstract available.publishe

A diverse view of science to catalyse change

Valuing diversity leads to scientific excellence, the progress of science and, most importantly, it is simply the right thing to do. We must value diversity not only in words, but also in actions

List of oxidative modifications identified from multiple proteases.

<p>^<i>a</i>Lowercase letters represent the amino acid residues that have oxidative modifications.</p><p>^<i>b</i>Positions of modified residues in the entire ubiquitin sequence are shown and are abbreviated as follows: Oxidation indicates an oxygen addition to the amino acid residue, Dioxidation indicated two oxygens addition to the amino acid residue, carbonylation indicates formaton of carbonyl group with a mass increase of 14 Da, GluSA indicates the carbonylation of arginine to glutamic semialdehyde, AminoAdSA indicates the carbonlyation of lysine to aminoadipic semialdehyde, Oxd'n indicates the carbonlyation of threonine to 2-amino-3-oxo-butanoic acid, Asp indicates the oxidation of histidine to aspartic acid, Deamidation indicates the conversion of -NH₂ to -OH and Decarbonylation indicates the loss of carboxyl group.</p><p>^<i>c</i>MS/MS spectra of each peptide is provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116606#pone.0116606.s001" target="_blank">S1 Fig.</a></p><p>List of oxidative modifications identified from multiple proteases.</p

CID MS/MS spectra of (a) [LIfAGK+H]⁺ with Phe45-Ox as observed by trypsin proteolysis, t_r = 22.16 min, <i>m/z</i> = 664.40; (b) [EGIPPDQQRLIfAGK+2H]²⁺ with Phe45-Ox as observed by Lys-C proteolysis, t_r = 22.71, <i>m/z</i> = 842.95; (c) [TLSDyNIQK+2H]²⁺ with Tyr59-Ox as observed by trypsin proteolysis, t_r = 14.33 min, <i>m/z</i> = 549.28 and (d) [QLEDGRTLSDyNIQK+2H]²⁺ with Tyr59-Ox as observed by Lys-C proteolysis, t_r = 22.14 min, <i>m/z</i> = 898.45.

<p>Note that lowercase letters represent the oxidation of phenylalanine and tyrosine. Ions labeled with asterisks (*) contain modifications.</p

CID MS/MS spectra of (a) [tLSDYNIQK+2H]²⁺ with Thr55-Oxd’n as observed by trypsin proteolysis, t_r = 20.28min, <i>m/z</i> = 540.27 and (b) [MQIFVkTLTGK+2H]²⁺ with Lys6 – AminoAdSA as observed by Lys-C proteolysis, t_r = 26.95, <i>m/z</i> = 632.85.

<p>Note that lowercase letters represent the carbonylation of threonine to 2-amino-3-oxo-butanoic acid and lysine to aminoadipic semialdehyde. Ions labeled with asterisks (*) contain modifications.</p

Precursor ion mass spectra of oxidized ubiquitin.

<p>In the inset is a zoom-in of the +12 charge state that shows unmodified and oxidized ubiquitin species. The observed mass shift between native and oxidized ubiquitin is indicated in the figure.</p