In-Source Fragmentation and the Sources of Partially Tryptic Peptides in Shotgun Proteomics

Partially tryptic peptides are often identified in shotgun proteomics using trypsin as the proteolytic enzyme; however, their sources have been controversial. Herein, we investigate the impact of in-source fragmentation on shotgun proteomics profiling of three biological samples: a standard protein mixture, a mouse brain tissue homogenate, and mouse plasma. Because the in-source fragments of peptide ions have the same LC elution time as their parental peptides, partially tryptic peptide ions from in-source fragmentation can be distinguished from other partially tryptic peptides based on their elution time differences from those computationally predicted data. The percentage of partially tryptic peptide identifications resulting from in-source fragmentation in a standard protein digest was observed to be ∼60%. In more complex mouse brain or plasma samples, in-source fragmentation contributed to a lesser degree of 1–3% of all identified peptides due to the limited dynamic range of LC–MS/MS measurements. The other major source of partially tryptic peptides in complex biological samples is presumably proteolytic cleavage by endogenous proteases in the samples. Our work also provides a method to identify such proteolytic-derived partially tryptic peptides due to endogenous proteases in the samples by removing in-source fragmentation artifacts from the identified peptides

In-Source Fragmentation and the Sources of Partially Tryptic Peptides in Shotgun Proteomics

Partially tryptic peptides are often identified in shotgun proteomics using trypsin as the proteolytic enzyme; however, their sources have been controversial. Herein, we investigate the impact of in-source fragmentation on shotgun proteomics profiling of three biological samples: a standard protein mixture, a mouse brain tissue homogenate, and mouse plasma. Because the in-source fragments of peptide ions have the same LC elution time as their parental peptides, partially tryptic peptide ions from in-source fragmentation can be distinguished from other partially tryptic peptides based on their elution time differences from those computationally predicted data. The percentage of partially tryptic peptide identifications resulting from in-source fragmentation in a standard protein digest was observed to be ∼60%. In more complex mouse brain or plasma samples, in-source fragmentation contributed to a lesser degree of 1–3% of all identified peptides due to the limited dynamic range of LC–MS/MS measurements. The other major source of partially tryptic peptides in complex biological samples is presumably proteolytic cleavage by endogenous proteases in the samples. Our work also provides a method to identify such proteolytic-derived partially tryptic peptides due to endogenous proteases in the samples by removing in-source fragmentation artifacts from the identified peptides

MOESM1 of Biodegradation of alkaline lignin by Bacillus ligniniphilus L1

Additional file 1. Up regulation of expressed protein with lignin as carbon source

Resin-Assisted Enrichment of N‑Terminal Peptides for Characterizing Proteolytic Processing

A resin-assisted enrichment method has been developed for specific isolation of protein N-terminal peptides to facilitate LC-MS/MS characterization of proteolytic processing, a major form of posttranslational modifications. In this method, protein thiols are blocked by reduction and alkylation, and protein lysine residues are converted to homoarginines. Protein N-termini are selectively converted to reactive thiol groups, and the thiol-containing N-terminal peptides are then captured by a thiol-affinity resin with high specificity (>97%). The efficiencies of these sequential reactions were demonstrated to be nearly quantitative. The resin-assisted N-terminal peptide enrichment approach was initially applied to a cell lysate of the filamentous fungus <i>Aspergillus niger</i>. Subsequent C-MS/MS analyses resulted in the identification of 1672 unique protein N-termini or proteolytic cleavage sites from 690 unique proteins

Synthesis and Application of an Environmentally Insensitive Cy3-Based Arsenical Fluorescent Probe To Identify Adaptive Microbial Responses Involving Proximal Dithiol Oxidation

Reversible disulfide oxidation between proximal cysteines in proteins represents a common regulatory control mechanism to modulate flux through metabolic pathways in response to changing environmental conditions. To enable <i>in vivo</i> measurements of cellular redox changes linked to disulfide bond formation, we have synthesized a cell-permeable thiol-reactive affinity probe (TRAP) consisting of a monosubstituted cyanine dye derivatized with arsenic (i.e., TRAP_Cy3) to trap and visualize dithiols in cytosolic proteins. Alkylation of reactive thiols prior to displacement of the bound TRAP_Cy3 by ethanedithiol permits facile protein capture and mass spectrometric identification of proximal reduced dithiols to the exclusion of individual cysteines. Applying TRAP_Cy3 to evaluate cellular responses to increases in oxygen and light levels in the photosynthetic microbe <i>Synechococcus</i> sp. PCC7002, we observe large decreases in the abundance of reduced dithiols in cellular proteins, which suggest redox-dependent mechanisms involving the oxidation of proximal disulfides. Under these same growth conditions that result in the oxidation of proximal thiols, there is a reduction in the abundance of post-translational oxidative protein modifications involving methionine sulfoxide and nitrotyrosine. These results suggest that the redox status of proximal cysteines responds to environmental conditions, acting to regulate metabolic flux and minimize the formation of reactive oxygen species to decrease oxidative protein damage

Quantitative Profiling of Protein S‑Glutathionylation Reveals Redox-Dependent Regulation of Macrophage Function during Nanoparticle-Induced Oxidative Stress

Engineered nanoparticles (ENPs) are increasingly utilized for commercial and medical applications; thus, understanding their potential adverse effects is an important societal issue. Herein, we investigated protein S-glutathionylation (SSG) as an underlying regulatory mechanism by which ENPs may alter macrophage innate immune functions, using a quantitative redox proteomics approach for site-specific measurement of SSG modifications. Three high-volume production ENPs (SiO₂, Fe₃O₄, and CoO) were selected as representatives which induce low, moderate, and high propensity, respectively, to stimulate cellular reactive oxygen species (ROS) and disrupt macrophage function. The SSG modifications identified highlighted a broad set of redox sensitive proteins and specific Cys residues which correlated well with the overall level of cellular redox stress and impairment of macrophage phagocytic function (CoO > Fe₃O₄ ≫ SiO₂). Moreover, our data revealed pathway-specific differences in susceptibility to SSG between ENPs which induce moderate <i>versus</i> high levels of ROS. Pathways regulating protein translation and protein stability indicative of ER stress responses and proteins involved in phagocytosis were among the most sensitive to SSG in response to ENPs that induce subcytoxic levels of redox stress. At higher levels of redox stress, the pattern of SSG modifications displayed reduced specificity and a broader set pathways involving classical stress responses and mitochondrial energetics (<i>e.g.,</i> glycolysis) associated with apoptotic mechanisms. An important role for SSG in regulation of macrophage innate immune function was also confirmed by RNA silencing of glutaredoxin, a major enzyme which reverses SSG modifications. Our results provide unique insights into the protein signatures and pathways that serve as ROS sensors and may facilitate cellular adaption to ENPs, <i>versus</i> intracellular targets of ENP-induced oxidative stress that are linked to irreversible cell outcomes

Antibody-Independent, Deep-Dive Targeted Quantification of Proteins at 10 pg/mL Levels in Non-Depleted Human Serum/Plasma (ASMS 2016)

<p>Targeted proteomics approaches, such as selected reaction monitoring (SRM), have emerged as powerful tools for sensitive, quantitative protein analysis in systems biology and biomedical research. Chromatography or affinity based sample pre-fractionation/enrichment is typically performed before SRM analysis when higher sensitivity is needed, especially in the analysis of complex biological samples. However, even with these existing techniques targeted quantitative analysis of extremely low abundance (e.g., <50 pg/mL) proteins in complex biological samples, such as serum or plasma, remains challenging. To address this need, we developed an antibody-independent, two-dimensional (2D) offline liquid chromatography (LC)-based Deep-Dive (DD)-SRM approach for quantification of proteins at low pg/ml levels in human serum/plasma without the need for major protein depletion. </p

Overview of the A) subcellular location, B) molecular size distribution and C) molecular and cellular functions of the 135 proteins that showed significant variation between irradiated and sham skin tissues.

<p>Others: subcellular location known, but not being included in the main categories; Unknown: subcellular location unknown. Protein may have more than one function.</p

Representative proteins that showed A) consistent decrease and B) consistent increase with increasing radiation dose.

<p>The fold change of protein abundance at each dose was represented by the log2 ratio with respect to the value of sham group. * represents statistically significant (P<0.05, Dunnett adjusted 2 sided t-test). Detailed statistical analysis values, such as fold change and p values are listed in Supporting Information <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092332#pone.0092332.s001" target="_blank">Table S1</a>.</p

Selected list of statistically significant proteins secreted into the medium by a full thickness skin tissue model at different doses (3, 10 and 200 cGy) of X-rays when compared with sham controls (0 cGy).

<p>Fold changes between treatment and sham groups were represented as group differences, only proteins with changes >1.5 fold (0.58 in log2 scale) for up- or <2.0 fold for down-regulation (−1 in log2 scale) were shown (full list of statistically significant proteins is shown in Supporting Information <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092332#pone.0092332.s001" target="_blank">Table S1</a>). Dunnet adjusted T-test was used to assess the statistical significance of the change. P values <0.05 were deemed statistically significant. Dunnet trend: 0, no significant change; 1, upregulation with statistical significance; −1, downregulation with statistical significance. Trend: general trend of the average abundance of proteins with increasing radiation dose; down, downregulation with increasing dose; up, upregulation with increasing dose; ∧, 10 cGy group had the highest abundance; V, 10 cGy group had the lowest abundance. Protein subcellular locations and biological functions were derived from Uniprot.</p