In vivo aging of rat skeletal muscle sarcoplasmic reticulum Ca-ATPase. Chemical analysis and quantitative simulation by exposure to low levels of peroxyl radicals

AbstractSarcoplasmic reticulum (SR) Ca-ATPase of young adult (5 months) and aged (28 months) Fischer 344 male rat skeletal muscle was analyzed for posttranslational modifications as a result of biological aging and their potential functional consequences. The significant differences in the amino acid composition were a 6.8% lower content of sulfhydryl groups and a ca. 4% lower content of Arg residues of the Ca-ATPase from old as compared to young rats. Based on a total of 24 Cys residues the difference in protein thiols corresponds to a loss of 1.5 mol Cys/mol Ca-ATPase as a result of in vivo aging. The loss of Cys residues was not accompanied by a loss of enzyme activity though the `aged' Ca-ATPase was more sensitive to heat inactivation, aggregation, and tryptic digestion. A comparison of the total sulfhydryl content of all SR proteins present revealed a 13% lower amount for SR vesicles isolated from aged rats. Compared to the alterations of Cys and Arg, there was only a slight and probably physiologically insignificant increase of protein carbonyls with aging, i.e. from 0.32 to 0.46 mol carbonyl groups per mol of Ca-ATPase. When SR vesicles from young rats were exposed to AAPH-derived peroxyl radicals, there was a loss of ca. 1.38×10−4 M total SR sulfhydryl groups per 4 mg SR protein/ml (corresponding to ca. 25%) and a loss of 9.6×10−5 M Ca-ATPase sulfhydryl groups (corresponding to ca. 31%) per 1.6×10−5 M initiating peroxyl radicals, indicating that the stoichiometry of sulfhydryl oxidation was ≥6 oxidized thiols per initiating AAPH-derived peroxyl radical. Besides Cys, the exposure to AAPH-derived radicals caused a slight loss of Ca-ATPase Arg, Met, and Ser residues. Most importantly, the SR Ca-ATPase exposed to this low concentration of peroxyl radicals displayed physical and functional properties quantitatively comparable to those of SR Ca-ATPase isolated from aged rats, i.e. no immediate loss of activity, increased susceptibility to heat inactivation, aggregation, and tryptic digestion. Moreover, a comparison of kinetically early tryptic fragments by HPLC-electrospray MS and N-terminal sequencing revealed that similar peptide fragments were produced from `aged' and AAPH-oxidized Ca-ATPase which were not (or kinetically significantly later) generated from the `young' Ca-ATPase, suggesting some conformational changes of the Ca-ATPase as a result of aging and AAPH-exposure. All except one of these peptides originated from locations remote from the nucleotide-binding and calcium-binding sites. The latter results suggest that aging and AAPH-exposure may target similar Cys residues, mainly at locations remote from the nucleotide-binding and calcium-binding sites, rationalizing the fact that Cys oxidation did not immediately cause inactivation of the Ca-ATPase. Our results provide a quantitative estimate of a net concentration of reactive oxygen species, here peroxyl radicals, which induces physical and chemical alterations of the SR Ca-ATPase quantitatively comparable to those induced by in vivo aging

First Community-Wide, Comparative Cross-Linking Mass Spectrometry Study

The number of publications in the field of chemical cross-linking combined with mass spectrometry (XL-MS) to derive constraints for protein three-dimensional structure modeling and to probe protein-protein interactions has increased during the last years. As the technique is now becoming routine for in vitro and in vivo applications in proteomics and structural biology there is a pressing need to define protocols as well as data analysis and reporting formats. Such consensus formats should become accepted in the field and be shown to lead to reproducible results. This first, community-based harmonization study on XL-MS is based on the results of 32 groups participating worldwide. The aim of this paper is to summarize the status quo of XL-MS and to compare and evaluate existing cross-linking strategies. Our study therefore builds the framework for establishing best practice guidelines to conduct cross-linking experiments, perform data analysis, and define reporting formats with the ultimate goal of assisting scientists to generate accurate and reproducible XL-MS results

Top-Down Hydrogen–Deuterium Exchange Analysis of Protein Structures Using Ultraviolet Photodissociation

Top-down hydrogen–deuterium exchange (HDX) analysis using electron capture or transfer dissociation Fourier transform mass spectrometry (FTMS) is a powerful method for the analysis of secondary structure of proteins in solution. The resolution of the method is a function of the degree of fragmentation of backbone bonds in the proteins. While fragmentation is usually extensive near the N- and C-termini, electron capture (ECD) or electron transfer dissociation (ETD) fragmentation methods sometimes lack good coverage of certain regions of the protein, most often in the middle of the sequence. Ultraviolet photodissociation (UVPD) is a recently developed fast-fragmentation technique, which provides extensive backbone fragmentation that can be complementary in sequence coverage to the aforementioned electron-based fragmentation techniques. Here, we explore the application of electrospray ionization (ESI)-UVPD FTMS on an Orbitrap Fusion Lumos Tribrid mass spectrometer to top-down HDX analysis of proteins. We have incorporated UVPD-specific fragment-ion types and fragment-ion mixtures into our isotopic envelope fitting software (HDX Match) for the top-down HDX analysis. We have shown that UVPD data is complementary to ETD, thus improving the overall resolution when used as a combined approach

Proteomic Quantification and Site-Mapping of <i>S</i>‑Nitrosylated Proteins Using Isobaric iodoTMT Reagents

<i>S</i>-Nitrosylation is a redox-based protein post-translational modification in response to nitric oxide signaling and is involved in a wide range of biological processes. Detection and quantification of protein <i>S</i>-nitrosylation have been challenging tasks due to instability and low abundance of the modification. Many studies have used mass spectrometry (MS)-based methods with different thiol-reactive reagents to label and identify proteins with <i>S</i>-nitrosylated cysteine (SNO-Cys). In this study, we developed a novel iodoTMT switch assay (ISA) using an isobaric set of thiol-reactive iodoTMTsixplex reagents to specifically detect and quantify protein <i>S</i>-nitrosylation. Irreversible labeling of SNO-Cys with the iodoTMTsixplex reagents enables immune-affinity detection of <i>S</i>-nitrosylated proteins, enrichment of iodoTMT-labeled peptides by anti-TMT resin, and importantly, unambiguous modification site-mapping and multiplex quantification by liquid chromatography–tandem MS. Additionally, we significantly improved anti-TMT peptide enrichment efficiency by competitive elution. Using ISA, we identified a set of SNO-Cys sites responding to lipopolysaccharide (LPS) stimulation in murine BV-2 microglial cells and revealed effects of <i>S</i>-allyl cysteine from garlic on LPS-induced protein <i>S</i>-nitrosylation in antioxidative signaling and mitochondrial metabolic pathways. ISA proved to be an effective proteomic approach for quantitative analysis of <i>S</i>-nitrosylation in complex samples and will facilitate the elucidation of molecular mechanisms of nitrosative stress in disease

Proteomic Quantification and Site-Mapping of <i>S</i>‑Nitrosylated Proteins Using Isobaric iodoTMT Reagents

<i>S</i>-Nitrosylation is a redox-based protein post-translational modification in response to nitric oxide signaling and is involved in a wide range of biological processes. Detection and quantification of protein <i>S</i>-nitrosylation have been challenging tasks due to instability and low abundance of the modification. Many studies have used mass spectrometry (MS)-based methods with different thiol-reactive reagents to label and identify proteins with <i>S</i>-nitrosylated cysteine (SNO-Cys). In this study, we developed a novel iodoTMT switch assay (ISA) using an isobaric set of thiol-reactive iodoTMTsixplex reagents to specifically detect and quantify protein <i>S</i>-nitrosylation. Irreversible labeling of SNO-Cys with the iodoTMTsixplex reagents enables immune-affinity detection of <i>S</i>-nitrosylated proteins, enrichment of iodoTMT-labeled peptides by anti-TMT resin, and importantly, unambiguous modification site-mapping and multiplex quantification by liquid chromatography–tandem MS. Additionally, we significantly improved anti-TMT peptide enrichment efficiency by competitive elution. Using ISA, we identified a set of SNO-Cys sites responding to lipopolysaccharide (LPS) stimulation in murine BV-2 microglial cells and revealed effects of <i>S</i>-allyl cysteine from garlic on LPS-induced protein <i>S</i>-nitrosylation in antioxidative signaling and mitochondrial metabolic pathways. ISA proved to be an effective proteomic approach for quantitative analysis of <i>S</i>-nitrosylation in complex samples and will facilitate the elucidation of molecular mechanisms of nitrosative stress in disease

Proteomic Quantification and Site-Mapping of <i>S</i>‑Nitrosylated Proteins Using Isobaric iodoTMT Reagents

<i>S</i>-Nitrosylation is a redox-based protein post-translational modification in response to nitric oxide signaling and is involved in a wide range of biological processes. Detection and quantification of protein <i>S</i>-nitrosylation have been challenging tasks due to instability and low abundance of the modification. Many studies have used mass spectrometry (MS)-based methods with different thiol-reactive reagents to label and identify proteins with <i>S</i>-nitrosylated cysteine (SNO-Cys). In this study, we developed a novel iodoTMT switch assay (ISA) using an isobaric set of thiol-reactive iodoTMTsixplex reagents to specifically detect and quantify protein <i>S</i>-nitrosylation. Irreversible labeling of SNO-Cys with the iodoTMTsixplex reagents enables immune-affinity detection of <i>S</i>-nitrosylated proteins, enrichment of iodoTMT-labeled peptides by anti-TMT resin, and importantly, unambiguous modification site-mapping and multiplex quantification by liquid chromatography–tandem MS. Additionally, we significantly improved anti-TMT peptide enrichment efficiency by competitive elution. Using ISA, we identified a set of SNO-Cys sites responding to lipopolysaccharide (LPS) stimulation in murine BV-2 microglial cells and revealed effects of <i>S</i>-allyl cysteine from garlic on LPS-induced protein <i>S</i>-nitrosylation in antioxidative signaling and mitochondrial metabolic pathways. ISA proved to be an effective proteomic approach for quantitative analysis of <i>S</i>-nitrosylation in complex samples and will facilitate the elucidation of molecular mechanisms of nitrosative stress in disease