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Results for “isobaric labeling”

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At least 19 recordsLinked to original sources

PubMed2025

Analytical Considerations for the Development of Plate-Based Proteomics Platforms Using Isobaric Labeling.

Mass spectrometer-based proteomics platforms have great potential to rapidly advance our systematic understanding of complex biological problems, enable drug discovery, decipher drug mechanisms of action, and discover novel biomarkers. As the demand for processing large sets of samples in an automatic manner is constantly increasing, the integration of automation platforms (nanoliter dispensers, liquid handlers, etc.) has become a routinary configuration paired with liquid chromatography-mass spectrometers. The functional integration of all of those instruments into a single unit is what we call a plate-based high-throughput proteomics platform (HT proteomics). The readout of the platform is the quantitative proteome data at the protein or peptide level. In this work, we developed a plate-based HT proteomics standard that we called the HT-sKO. The HT-sKO allows the evaluation of accuracy and the estimation of the relative limit of quantification when the target proteins vary up to 60-fold in abundance. The HT-sKO utilizes nonhuman recombinant proteins that can be spiked into the samples, allowing for sample acquisition and HT proteomics platform evaluation at the same time. We also showed the foundational role of a robust acquisition strategy for developing a stable HT proteomics platform and the value of using a tube-based method as an informant assay on data quality expectations for the platform. Using this new standard, we demonstrated that the intra- and inter-plate variance is around 4-6% for the protein level or around 10% for the peptide-level readout. We also showed that the HT-sKO standard is compatible with whole-proteome, phospho-proteome, and reactive cysteine profiling platforms.

Proteomics↗

PubMed2025

Boosting Quantification of N-Glycans by an Enhanced Isobaric Multiplex Reagents for Carbonyl-Containing Compound (SUGAR) Tagging Strategy.

Glycans are complex molecules composed of various monosaccharides and exhibit diverse, branched polymer structures. Extensive research has been conducted on mass spectrometry (MS)-based qualitative and quantitative glycan analysis due to their critical biological functions. However, traditional data-dependent acquisition (DDA) in MS analysis primarily selects a limited subset of abundant ions during MS1 scans for fragmentation in subsequent MS2 stages. In this study, we introduce an advanced isobaric labeling strategy that incorporates a large amount of content-relevant sample labeled with one isobaric tag channel as an additional boosting channel. This innovation enhances the efficiency of isobaric multiplex reagents for carbonyl-containing compound (SUGAR) tagging in quantitative glycomics. Notably, this approach significantly improves the characterization of low-abundance N-glycans and enables the detection of subtle quantitative differences in N-glycan profiling.

Polysaccharides↗

PubMed2026

High-throughput single-cell proteomics and transcriptomics from same cells with a nanoliter-scale, spin-transfer approach.

Single-cell multiomic platforms provide a comprehensive snapshot of cellular states and cell types by offering critical insights into the spatiotemporal regulation of biomolecular networks at a systems level, thereby defining the basis of multicellularity. Here, we introduce nanoSPINS, an advanced platform that enables high-throughput profiling and integrative analysis of the transcriptome and proteome from the same single cells using RNA sequencing and isobaric labeling LC-MS-based proteomics, respectively. NanoSPINS can efficiently transfer mRNA-containing droplets across two microarrays via a centrifugation-based approach, while proteins are retained on the initial platform. Benchmarking of nanoSPINS on two cell lines demonstrates its ability to generate global proteomic and transcriptomic profiles that align well with previously established methodologies/platforms. The incorporation of isobaric TMTpro labeling into this single-cell multiomics platform significantly enhances the throughput of single-cell proteomic analyses. Through the high-throughput quantification of the proteome and transcriptome, nanoSPINS not only facilitates the identification of molecular features at both mRNA and protein level but also provides larger sample sizes for improved statistical power in clustering and differential abundance. Given the broad applicability of single-cell multiomics in biological research and clinical settings, we believe nanoSPINS represents a powerful platform for the characterization of heterogeneous cell populations.

Single-Cell Analysis↗

PubMed2006

Early events of Bacillus anthracis germination identified by time-course quantitative proteomics.

Germination of Bacillus anthracis spores involves rehydration of the spore interior and rapid degradation of several of the protective layers, including the spore coat. Here, we examine the temporal changes that occur during B. anthracis spore germination using an isobaric tagging system. Over the course of 17 min from the onset of germination, the levels of at least 19 spore proteins significantly decrease. Included are acid-soluble proteins, several known and predicted coat proteins, and proteins of unknown function. Over half of these proteins are small (less than 100 amino acids) and would have been undetectable by conventional gel-based analysis. We also identified 20 proteins, whose levels modestly increased at the later time points when metabolism has likely resumed. Taken together, our data show that isobaric labeling of complex mixtures is particularly effective for temporal studies. Furthermore, we describe a rigorous statistical approach to define relevant changes that takes into account the nature of data obtained from multidimensional protein identification technology coupled with the use of isobaric tags. This study provides an expanded list of the proteins that may be involved in germination of the B. anthracis spore and their relative levels during germination.

Amino Acid Sequence↗

PubMed2006

Phosphopeptide quantitation using amine-reactive isobaric tagging reagents and tandem mass spectrometry: application to proteins isolated by gel electrophoresis.

Polyacrylamide gel electrophoresis is widely used for protein separation and it is frequently the final step in protein purification in biochemistry and proteomics. Using a commercially available amine-reactive isobaric tagging reagent (iTRAQ) and mass spectrometry we obtained reproducible, quantitative data from peptides derived by tryptic in-gel digestion of proteins and phosphoproteins. The protocol combines optimized reaction conditions, miniaturized peptide handling techniques and tandem mass spectrometry to quantify low- to sub-picomole amounts of (phospho)proteins that were isolated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Immobilized metal affinity chromatography (FeIII-IMAC) was efficient for removal of excess reagents and for enrichment of derivatized phosphopeptides prior to matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis. Phosphopeptide abundance was determined by liquid chromatography/tandem mass (LC/MS/MS) using either MALDI time-of-flight/time-of-flight (TOF/TOF) MS/MS or electrospray ionization quadrupole time-of-flight (ESI-QTOF) MS/MS instruments. Chemically labeled isobaric phosphopeptides, differing only by the position of the phosphate group, were distinguished and characterized by LC/MS/MS based on their LC elution profile and distinct MS/MS spectra. We expect this quantitative mass spectrometry method to be suitable for systematic, comparative analysis of molecular variants of proteins isolated by gel electrophoresis.

Amines↗

PubMed2005

Analysis of cell membrane aminophospholipids as isotope-tagged derivatives.

Glycerophosphoethanolamine (GPEtn) and glycerophosphoserine (GPSer) lipids were reacted with a multiplexed set of differentially isotopically enriched N-methylpiperazine acetic acid N-hydroxysuccinimide ester reagents, which place isobaric mass labels at a primary amino group. The resulting derivatized aminophospholipids were isobaric and chromatographically indistinguishable but yielded positive reporter ions (m/z 114 or 117) after collisional activation that could be used to identify and quantify individual members of the multiplex set. The chromatographic and mass spectrometric response of N-methylpiperazine amide-tagged aminophospholipids was probed using glycerophosphoethanolamine and glycerophosphoserine lipid standards. The [M+H]+ of each tagged aminophospholipid shifted 144 Da, and during collision-induced dissociation the major fragmentation ion was either m/z 114 or 117. This mode of detecting aminophospholipids was useful for an unbiased analysis of plasmalogen GPEtn lipids. Molecular species information on the esterified fatty acyl substituents was obtained by collisional activation of the [M-H]- ions. The isotope-tagged reagents were used to assess changes in the distribution of GPEtn lipids after exposure of liposomes made from phospholipids extracted from RAW 264.7 cells to Cu2+/H2O2 to illustrate the ability of these reagents to aid in the mass spectrometric identification of aminophospholipid changes that occur during biological stimuli.

Animals↗

PubMed2005

Temporal quantitation of mutant Kit tyrosine kinase signaling attenuated by a novel thiophene kinase inhibitor OSI-930.

OSI-930, a potent thiophene inhibitor of the Kit, KDR, and platelet-derived growth factor receptor tyrosine kinases, was used to selectively inhibit tyrosine phosphorylation downstream of juxtamembrane mutant Kit in the mast cell leukemia line HMC-1. Inhibition of Kit kinase activity resulted in a rapid dephosphorylation of Kit and inhibition of the downstream signaling pathways. Attenuation of Ras-Raf-Erk (phospho-Erk, phospho-p38), phosphatidyl inositol-3' kinase (phospho-p85, phospho-Akt, phospho-S6), and signal transducers and activators of transcription signaling pathways (phospho-STAT3/5/6) were measured by affinity liquid chromatography tandem mass spectrometry, by immunoblot, and by tissue microarrays of fixed cell pellets. To more globally define additional components of Kit signaling temporally altered by kinase inhibition, a novel multiplex quantitative isobaric peptide labeling approach was used. This approach allowed clustering of proteins by temporal expression patterns. Kit kinase, which dephosphorylates rapidly upon kinase inhibition, was shown to regulate both Shp-1 and BDP-1 tyrosine phosphatases and the phosphatase-interacting protein PSTPIP2. Interactions with SH2 domain adapters [growth factor receptor binding protein 2 (Grb2), Cbl, Slp-76] and SH3 domain adapters (HS1, cortactin, CD2BP3) were attenuated by inhibition of Kit kinase activity. Functional crosstalk between Kit and the non-receptor tyrosine kinases Fes/Fps, Fer, Btk, and Syk was observed. Inhibition of Kit modulated phosphorylation-dependent interactions with pathways controlling focal adhesion (paxillin, leupaxin, p130CAS, FAK1, the Src family kinase Lyn, Wasp, Fhl-3, G25K, Ack-1, Nap1, SH3P12/ponsin) and septin-actin complexes (NEDD5, cdc11, actin). The combined use of isobaric protein quantitation and expression clustering, immunoblot, and tissue microarray strategies allowed temporal measurement signaling pathways modulated by mutant Kit inhibition in a model of mast cell leukemia.

Antineoplastic Agents↗

PubMed2024

Proteome-Scale Tissue Mapping Using Mass Spectrometry Based on Label-Free and Multiplexed Workflows.

Multiplexed bimolecular profiling of tissue microenvironment, or spatial omics, can provide deep insight into cellular compositions and interactions in healthy and diseased tissues. Proteome-scale tissue mapping, which aims to unbiasedly visualize all the proteins in a whole tissue section or region of interest, has attracted significant interest because it holds great potential to directly reveal diagnostic biomarkers and therapeutic targets. While many approaches are available, however, proteome mapping still exhibits significant technical challenges in both protein coverage and analytical throughput. Since many of these existing challenges are associated with mass spectrometry-based protein identification and quantification, we performed a detailed benchmarking study of three protein quantification methods for spatial proteome mapping, including label-free, TMT-MS2, and TMT-MS3. Our study indicates label-free method provided the deepest coverages of ∼3500 proteins at a spatial resolution of 50 μm and the highest quantification dynamic range, while TMT-MS2 method holds great benefit in mapping throughput at >125 pixels per day. The evaluation also indicates both label-free and TMT-MS2 provides robust protein quantifications in identifying differentially abundant proteins and spatially covariable clusters. In the study of pancreatic islet microenvironment, we demonstrated deep proteome mapping not only enables the identification of protein markers specific to different cell types, but more importantly, it also reveals unknown or hidden protein patterns by spatial coexpression analysis.

Proteome↗

PubMed2006

Qualitative and quantitative analyses of protein phosphorylation in naive and stimulated mouse synaptosomal preparations.

Activity-dependent protein phosphorylation is a highly dynamic yet tightly regulated process essential for cellular signaling. Although recognized as critical for neuronal functions, the extent and stoichiometry of phosphorylation in brain cells remain undetermined. In this study, we resolved activity-dependent changes in phosphorylation stoichiometry at specific sites in distinct subcellular compartments of brain cells. Following highly sensitive phosphopeptide enrichment using immobilized metal affinity chromatography and mass spectrometry, we isolated and identified 974 unique phosphorylation sites on 499 proteins, many of which are novel. To further explore the significance of specific phosphorylation sites, we used isobaric peptide labels and determined the absolute quantity of both phosphorylated and non-phosphorylated peptides of candidate phosphoproteins and estimated phosphorylation stoichiometry. The analyses of phosphorylation dynamics using differentially stimulated synaptic terminal preparations revealed activity-dependent changes in phosphorylation stoichiometry of target proteins. Using this method, we were able to differentiate between distinct isoforms of Ca2+/calmodulin-dependent protein kinase (CaMKII) and identify a novel activity-regulated phosphorylation site on the glutamate receptor subunit GluR1. Together these data illustrate that mass spectrometry-based methods can be used to determine activity-dependent changes in phosphorylation stoichiometry on candidate phosphopeptides following large scale phosphoproteome analysis of brain tissue.

Amino Acid Sequence↗

PubMed2006

Warm ischemia-induced alterations in oxidative and inflammatory proteins in hepatic Kupffer cells in rats.

The aim of the study was to investigate the impact of ischemia/reperfusion injury on the proteome of Kupffer cells. Lean Zucker rats (n = 6 each group) were randomized to 75 min of warm ischemia or sham operation. After reperfusion for 8 h, Kupffer cells were isolated by enzymatic perfusion and density gradient centrifugation. Proteins were tryptically digested into peptides and differentially labeled with iTRAQ (isobaric tags for relative and absolute quantitation) reagent. After fractionation by cation exchange chromatography, peptides were identified by mass spectrometry (ESI-LC-MS/MS). Spectra were interrogated against the Swiss-Prot database and quantified using ProteinProspector. The results for heat shock protein 70 and myeloperoxidase were validated by ELISA. Quantitative information for more than 1559 proteins was obtained. In the ischemia group proteins involved in inflammation were significantly up-regulated. The ratio for calgranulin B in the ischemia/sham group was 1.81 +/- 0.97 (p < 0.0001), for complement C3 the ratio was 1.81 +/- 0.49 (p < 0.0001), and for myeloperoxidase the ratio was 1.30 +/- 0.32. Myeloperoxidase was only recently documented in Kupffer cells. The antioxidative proteins Cu,Zn-superoxide dismutase (1.34 +/- 0.19; p < 0.001) and catalase (1.23 +/- 0.43; p < 0.001) were also elevated. In conclusion, ischemia/reperfusion injury induces alterations in the Kupffer cell proteome. Isotope ratio mass spectrometry is a powerful tool to investigate these reactions. The ability to simultaneously monitor several pathways involved in reperfusion stress may result in important mechanistic insight and possibly new treatment options.

Animals↗

PubMed2005

Assessing the effects of diurnal variation on the composition of human parotid saliva: quantitative analysis of native peptides using iTRAQ reagents.

Changes in salivary composition correlate with disease susceptibility, disease state, or both. However, use of saliva for diagnostic purposes is complicated by the gland-specific effects of circadian rhythm or diurnal variation. We recently characterized a suite of peptides in the < or =10-kDa fraction of human parotid saliva that included many novel species. In this study, we used novel iTRAQ labeling chemistry to investigate possible diurnal effects on peptide generation. We collected samples produced by gustatory stimulation as the ductal secretions at four time points under conditions that minimized proteolysis, pooled them according to collection time, and isolated the LMW fractions. Samples collected at each collection time were derivatized with a different isobaric iTRAQ reagent. The labeled samples were combined, separated by reversed-phase HPLC, co-spotted with matrix on MALDI targets, and analyzed by MALDI TOF/TOF mass spectrometry. With this approach, we achieved relative quantification of the parotid peptides at four time points. In several cases, abundance during the day changed dramatically. iTRAQ tagging improved the efficiency of MS/MS fragmentation, which in turn allowed the identification of several novel peptides. Our results demonstrated both the utility of this method and the importance of diurnal effects on the composition of the human parotid saliva peptidome.

Amino Acid Sequence↗

PubMed2007

A novel quantitative proteomics strategy to study phosphorylation-dependent peptide-protein interactions.

Phosphorylation-dependent protein-protein interactions provide the mechanism for a large number of intracellular signal transduction pathways. One of the goals of signal transduction research is to understand more precisely the nature of these phosphorylation-dependent interactions. Here, we report a novel strategy based on quantitative proteomics that allows for the rapid analysis of peptide-protein interactions with more than one phosphorylation site involved. The phosphorylation of two tyrosine residues, Y342 and Y346, within the linker B region of the protein-tyrosine kinase Syk is important for optimal signaling from the B cell receptor for antigen. We employed four amino-specific, isobaric reagents to differentially label proteins interacting in vitro with four Syk peptides containing none, one, or two phosphates on tyrosine residues Y342 and Y346, respectively. In total, 76 proteins were identified and quantified, 11 of which were dependent on the phosphorylation of individual tyrosine residues. One of the proteins, peroxiredoxin 1, preferably bound to phosphorylated Y346, which was further verified by Western blotting results. Thus, we demonstrate that the use of 4-fold multiplexing allows for relative protein measurements simultaneously for the identification of interacting proteins dependent on the phosphorylation of specific residues.

Algorithms↗

PubMed2005

Quantitative proteomic analysis using isobaric protein tags enables rapid comparison of changes in transcript and protein levels in transformed cells.

Isobaric tags for relative and absolute quantitation, an approach to concurrent, relative quantification of proteins present in four cell preparations, have recently been described. To validate this approach using complex mammalian cell samples that show subtle differences in protein levels, a model stem cell-like cell line (FDCP-mix) in the presence or absence of the leukemogenic oncogene TEL/PDGFRbeta has been studied. Cell lysates were proteolytically digested, and peptides within each sample were labeled with one of four isobaric, isotope-coded tags via their N-terminal and/or lysine side chains. The four labeled samples are mixed and peptides separated by two-dimensional liquid chromatography online to a mass spectrometer (LC-MS). Upon peptide fragmentation, each tag releases a distinct mass reporter ion; the ratio of the four reporters therefore gives relative abundances of the given peptide. Relative quantification of proteins is derived using summed data from a number of peptides. TEL/PDGFRbeta leukemic oncogene-mediated changes in protein levels were compared with those seen in microarray analysis of control and transfected FDCP-mix cells. Changes at the protein level in most cases reflected those seen at the transcriptome level. Nonetheless, novel differences in protein expression were found that indicate potential mechanisms for effects of this oncogene.

Amino Acid Sequence↗

PubMed1993

Collision-induced dissociation of carboxylate anions from derivatized 5-lipoxygenase metabolites of arachidonic acid.

The low-energy collision-induced dissociations (CID) of carboxylate anions derived from pentafluorobenzyl ester, trimethylsilyl ether derivatives of four arachidonic acid metabolites of the 5-lipoxygenase pathway have been determined. These molecules include leukotriene B4 (LTB4), a potent chemotactic factor for the human neutrophil; 20-carboxy-LTB4, an inactive metabolite; 5-hydroxyeicosatetraenoic acid (5-HETE), a useful marker of 5-lipoxygenase activity within cells; and 5-hydroxyeicosanoic acid (5-HEA), which has been previously used for the quantitation of leukotriene E4. The carboxylate anion of 5-HEA (m/z 399) was found to decompose by the loss of trimethylsilanol as well as the loss of 146 u corresponding to the loss of trimethylsilanol followed by acrolein, a process specific for 5-hydroxy-containing saturated fatty acids. The loss of trimethylsilanol by a remote site mechanism is the major transition observed for the 5-HETE carboxylate anion (m/z 391). The ion formed (m/z 301) further decomposes by the loss of CO2 (m/z 257). The loss of trimethylsilanol is also seen at m/z 389 after collisional activation of the carboxylate anion of LTB4 (m/z 479) by a complex charge-driven mechanism, not the remote site fragmentation mechanism as expected. The loss of an olefinic proton possibly from carbon-7 is involved as well as an oxygen atom derived from the carboxylic acid moiety. The loss of two trimethylsilanol neutral molecules gives rise to ions seen at m/z 299. Isotopic labeling studies revealed that two isobaric ions are present at m/z 299. Both of these ions involve the loss of trimethylsilanol from the carbon-12 position according to remote site mechanisms, but only one has lost the olefinic proton at carbon-7 and, therefore, likely originates from the further decomposition of the ion (m/z 389) described above. An additional ion seen at m/z 317 is attributed to the loss of trimethylsilyl ether (TMS-O-TMS) following a charge-driven mechanism involving the oxygen atom at carbon-12. The 20-carboxy-LTB4 carboxylate anion (m/z 689) decomposes primarily through the loss of one and two trimethylsilanol moieties, but the base peak (m/z 491) is due to the loss of pentafluorobenzyl alcohol. This ion, likely a ketene, further gives rise to three ions by the sequential loss of one and two trimethylsilanols and TMS-O-TMS. All collision-induced decompositions of the carboxylate anions of these eicosanoids are characterized by losses of small neutral molecules from the derivatizing groups (TMS and pentafluorobenzyl) and little fragmentation of the carbon backbone.

Arachidonate 5-Lipoxygenase↗

PubMed2006

Relative quantification in proteomics: new approaches for biochemistry.

Recent developments in mass spectrometry and protein arrays provide opportunities to derive systematically proteomic information from small samples of cellular material. Relative quantification among samples can be achieved with either gel-based or gel-free approaches. Furthermore, the adaptation of specific techniques facilitates absolute quantification. Here, relative quantification in two-dimensional gel electrophoresis is contrasted with that in non-gel-based approaches such as isobaric tagging of peptides, pre-labelling of living cells with isotopomeric forms of essential amino acids and protein array platforms. In addition, novel flow-cytometry-based approaches are considered. These technologies can all be used to determine accurately the levels of proteins or biomarkers in a wide range of samples.

Biochemistry↗

PubMed2007

Protein labeling by iTRAQ: a new tool for quantitative mass spectrometry in proteome research.

A novel, MS-based approach for the relative quantification of proteins, relying on the derivatization of primary amino groups in intact proteins using isobaric tag for relative and absolute quantitation (iTRAQ) is presented. Due to the isobaric mass design of the iTRAQ reagents, differentially labeled proteins do not differ in mass; accordingly, their corresponding proteolytic peptides appear as single peaks in MS scans. Because quantitative information is provided by isotope-encoded reporter ions that can only be observed in MS/MS spectra, we analyzed the fragmentation behavior of ESI and MALDI ions of peptides generated from iTRAQ-labeled proteins using a TOF/TOF and/or a QTOF instrument. We observed efficient liberation of reporter ions for singly protonated peptides at low-energy collision conditions. In contrast, increased collision energies were required to liberate the iTRAQ label from lysine side chains of doubly charged peptides and, thus, to observe reporter ions suitable for relative quantification of proteins with high accuracy. We then developed a quantitative strategy that comprises labeling of intact proteins by iTRAQ followed by gel electrophoresis and peptide MS/MS analyses. As proof of principle, mixtures of five different proteins in various concentration ratios were quantified, demonstrating the general applicability of the approach presented here to quantitative MS-based proteomics.

Chromatography, Liquid↗

PubMed2004

A SIFT ion-molecule study of some reactions in Titan's atmosphere. reactions of N(+), N(2)(+), and HCN(+) with CH(4), C(2)H(2), and C(2)H(4).

The results of a study of the ion-molecule reactions of N(+), N(2)(+), and HCN(+) with methane, acetylene, and ethylene are reported. These studies were performed using the FA-SIFT at the University of Canterbury. The reactions studied here are important to understanding the ion chemistry in Titan's atmosphere. N(+) and N(2)(+) are the primary ions formed by photo-ionization and electron impact in Titan's ionosphere and drive Titan's ion chemistry. It is therefore very important to know how these ions react with the principal trace neutral species in Titan's atmosphere: Methane, acetylene, and ethylene. While these reactions have been studied before the product channels have been difficult to define as several potential isobaric products make a definitive answer difficult. Mass overlap causes difficulties in making unambiguous species assignments in these systems. Two discriminators have been used in this study to resolve the mass overlap problem. They are deuterium labeling and also the differences in reactivities of each isobar with various neutral reactants. Several differences have been found from the products in previous work. The HCN(+) ion is important in both Titan's atmosphere and in the laboratory.

Atmosphere↗

PubMed1995

Membrane protein alterations in rodent erythrocytes and synaptosomes due to aging and hyperoxia.

We have applied the technique of electron paramagnetic resonance (EPR) protein-specific spin labeling to the study of membrane protein alterations occurring during age and exposure to isobaric hyperoxia. Cortical synaptosomes and erythrocyte membranes (ghosts) were isolated from young rodents (Fisher 344 rats or mongolian gerbils, 3-4 months of age) and aged rodents (age 22-27 months for rats, greater than 15 months for gerbils). Membrane proteins were spin labeled with the thiol-specific spin label MAL-6 (2,2,6,6,-tetramehtyl-4-maleimido-piperdin-1-oxyl). The relevant EPR spectral parameter of MAL-6 labeled membranes, the W/S ratio, decreased significantly with age of animal in both synaptosomes and ghosts (P < 0.001). As a paradigm for accelerated oxidative stress, young and aged gerbils were exposed to an atmosphere of 90-100% O2 for 0-48 h. In both young and aged gerbils, the W/S ratio decreased significantly with hyperoxic stress (P < 0.003). The W/S ratio of synaptosomes isolated from aged gerbils decreased continually from 0-48 h hyperoxia, whereas the W/S ratio of synaptosomes from young animals demonstrated a pronounced rebound effect from 24-48 h. The results are discussed with reference to membrane protein oxidation in aging.

Aging↗