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 ...

SGS North America Inc.

Oxidative modifications can have significant effects on protein structure in solution. Here, the structures and stabilities of oxidized ubiquitin ions electrosprayed from an aqueous solution (pH 2) are studied by ion mobility spectrometry-mass spectrometry (IMS-MS). IMS-MS has proven to be a valuable technique to assess gas phase and in many cases, solution structures. Herein, in vitro oxidation is performed by Fenton chemistry with Fe(II)/hydrogen peroxide. Most molecules in solution remain unmodified, whereas ∼20% of the population belongs to an M+16 Da oxidized species. Ions of low charge states (+7 and +8) show substantial variance in collision cross section distributions between unmodified and oxidized species. Novel and previously reported gaussian conformers are used to model cross section distributions for +7 and +8 oxidized ubiquitin ions, respectively, in order to correlate variances in observed gas-phase distributions to changes in populations of solution states. Based on gaussian modeling, oxidized ions of charge state +7 have an A-state conformation which is more populated for oxidized relative to unmodified ions. Oxidized ubiquitin ions of charge state +8 have a distribution of conformers arising from native-state ubiquitin and higher intensities of A- and U-state conformers relative to unmodified ions. This work provides evidence that incorporation of a single oxygen atom to ubiquitin leads to destabilization of the native state in an acidic solution (pH ∼2) and to unfolding of gas-phase compact structures.

Effects of Fe(II)/H2O2 oxidation on ubiquitin conformers measured by ion mobility-mass spectrometry.

Copper is an essential micronutrient for plant growth and development, and copper transporter plays a pivotal role for keeping copper homeostasis. However, little is known about copper transporters in wheat. Here, we report a novel copper transporter gene family, TaCT1, in common wheat. Three TaCT1 homoeologous genes were isolated and assigned to group 5 chromosomes. Each of the TaCT1 genes (TaCT1-5A, -5B or -5D) possesses 12 transmembrane domains. TaCT1 genes exhibited higher transcript levels in leaf than in root, culm and spikelet. Excess copper down-regulated the transcript levels of TaCT1 and copper deficiency-induced TaCT1 expression. Subcellular experiments localized the TaCT1 to the Golgi apparatus. Yeast expression experiments and virus-induced gene silencing analysis indicated that the TaCT1 functioned in copper transport. Site-directed mutagenesis demonstrated that three amino acid residues, Met(35), Met(38) and Cys(365), are required for TaCT1 function. Phylogenetic and functional analyses suggested that homologous genes shared high similarity with TaCT1 may exist exclusively in monocot plants. Our work reveals a novel wheat gene family encoding major facilitator superfamily (MFS)-type copper transporters, and provides evidence for their functional involvement in promoting copper uptake and keeping copper homeostasis in common wheat.

Identification and characterization of a novel copper transporter gene family TaCT1 in common wheat.

Proteomics techniques are continuously being developed to further understanding of biology and disease. Many of the pathways that are relevant to disease mechanisms rely on the identification of post-translational modifications (PTMs) such as phosphorylation, acetylation, and glycosylation. Much attention has also been focused on oxidative PTMs which include protein carbonyls, protein nitration, and the incorporation of fatty acids and advanced glycation products to amino acid side chains, amongst others. The introduction of these PTMs in the cell can occur due to the attack of reactive oxygen and nitrogen species (ROS and RNS, respectively) on proteins. ROS and RNS can be present as a result of normal metabolic processes as well as external factors such as UV radiation, disease, and environmental toxins. The imbalance of ROS and RNS with antioxidant cellular defenses leads to a state of oxidative stress, which has been implicated in many diseases. Redox proteomics techniques have been used to characterize oxidative PTMs that result as a part of normal cell signaling processes as well as oxidative stress conditions. This review highlights many of the redox proteomics techniques which are currently available for several oxidative PTMs and brings to the reader's attention the application of redox proteomics for understanding disease pathogenesis in neurodegenerative disorders and others such as cancer, kidney, and heart diseases.

Mass spectrometry and redox proteomics: applications in disease.

Chromosome segmental introgression lines (ILs) are an effective way to utilize germplasm resources in crops. To improve agronomic traits of wheat cultivar (Triticum aestivum) Shi 4185, four sets of ILs were developed. The donors were Chinese endemic subspecies accessions Yunnan wheat (T. aestivum ssp. yunnanense) YN3, Tibetan semi-wild wheat (T. aestivum ssp. tibetanum) XZ-ZM19450, and Xinjiang wheat (T. aestivum ssp. petropavlovskyi) XJ5, and synthetic wheat HC-XM1620 derived from a cross between T. durum acc. D67.2/P66.270 with Aegilops tauschii acc. 218. Totals of 356, 366, 445 and 457 simple sequence repeat (SSR) markers were polymorphic between Shi 4185 and YN3, XZ-ZM19450, XJ5 and HC-XM1620, respectively. In total, 991 ILs were identified, including 300 derived from YN3, covering 95% of the genome of Shi 4185, 218 from XZ-ZM19450 (79%), 279 from XJ5 (97%), and 194 from HC-ZX1620 (84%). The sizes and locations of each introgression were determined from a consensus SSR linkage map. Using the ILs, 11 putative quantitative trait loci (QTLs) were identified for plant height (PH), spike length (SL) and grain number per spike (GNS). Comparative analyses of 24 elite ILs with the parents revealed that the four donor parents could be important resources to improve wheat SL and GNS. Our work offers a case for utilizing endemic landraces for QTL mapping and improvement of wheat cultivars using introgression lines.

Development, identification and utilization of introgression lines using Chinese endemic and synthetic wheat as donors.

Cysteine-selective proteomics approaches simplify complex protein mixtures and improve the chance of detecting low abundant proteins. It is possible that cysteinyl-peptide/protein enrichment methods could be coupled to isotopic labeling and isobaric tagging methods for quantitative proteomics analyses in as few as two or up to 10 samples, respectively. Here we present two novel cysteine-selective proteomics approaches: cysteine-selective dimethyl labeling (cysDML) and cysteine-selective combined precursor isotopic labeling and isobaric tagging (cPILOT). CysDML is a duplex precursor quantification technique that couples cysteinyl-peptide enrichment with on-resin stable-isotope dimethyl labeling. Cysteine-selective cPILOT is a novel 12-plex workflow based on cysteinyl-peptide enrichment, on-resin stable-isotope dimethyl labeling, and iodoTMT tagging on cysteine residues. To demonstrate the broad applicability of the approaches, we applied cysDML and cPILOT methods to liver tissues from an Alzheimer's disease (AD) mouse model and wild-type (WT) controls. From the cysDML experiments, an average of 850 proteins were identified and 594 were quantified, whereas from the cPILOT experiment, 330 and 151 proteins were identified and quantified, respectively. Overall, 2259 unique total proteins were detected from both cysDML and cPILOT experiments. There is tremendous overlap in the proteins identified and quantified between both experiments, and many proteins have AD/WT fold-change values that are within ~20% error. A total of 65 statistically significant proteins are differentially expressed in the liver proteome of AD mice relative to WT. The performance of cysDML and cPILOT are demonstrated and advantages and limitations of using multiple duplex experiments versus a single 12-plex experiment are highlighted.

Sample multiplexing with cysteine-selective approaches: cysDML and cPILOT.

A quantitative proteomics strategy called combined precursor isotopic labeling and isobaric tagging (cPILOT) was designed to discover alterations in the amyloid precursor protein/presenilin-1 (APP/PS-1) mouse liver proteome. The multiplexing strategy allows simultaneous quantitation of 12 samples in a single experiment.

Global cPILOT analysis of the APP/PS-1 mouse liver proteome.

Proteins present in cellular environments with high levels of reactive oxygen and nitrogen species and/or low levels of antioxidants are highly susceptible to oxidative post-translational modification (PTM). Irreversible oxidative PTMs can generate a complex distribution of modified protein molecules, recently termed as proteoforms. Using ubiquitin as a model system, we mapped oxidative modification sites using trypsin, Lys-C, and Glu-C peptides. Several M+16 Da proteoforms were detected as well as proteoforms that include other previously unidentified oxidative modifications. This work highlights the use of multiple protease digestions to give insights to the complexity of oxidative modifications possible in bottom-up analyses.

Multiple proteases to localize oxidation sites.

Cysteine is widely involved in redox signaling pathways through a number of reversible and irreversible modifications. Reversible modifications (e.g., S-glutathionylation, S-nitrosylation, disulfide bonds, and sulfenic acid) are used to protect proteins from oxidative attack and maintain cellular homeostasis, while irreversible oxidations (e.g., sulfinic acid and sulfonic acid) serve as hallmarks of oxidative stress. Proteomic analysis of cysteine-enriched peptides coupled with reduction of oxidized thiols can be used to measure the oxidation states of cysteine, which is helpful for elucidating the role that oxidative stress plays in biology and disease. As an extension of our previously reported cysDML method, we have developed oxidized cysteine-selective dimethylation (OxcysDML), to investigate the site-specific total oxidation of cysteine residues in biologically relevant samples. OxcysDML employs (1) blocking of free thiols by a cysteine-reactive reagent, (2) enrichment of peptides containing reversibly oxidized cysteine by a solid phase resin, and (3) isotopic labeling of peptide amino groups to quantify cysteine modifications arising from different biological conditions. On-resin enrichment and labeling minimizes sample handing time and improves efficiency in comparison with other redox proteomic methods. OxcysDML is also inexpensive and flexible, as it can accommodate the exploration of various cysteine modifications. Here, we applied the method to liver tissues from a late-stage Alzheimer's disease (AD) mouse model and wild-type (WT) controls. Because we have previously characterized this proteome using the cysDML approach, we are able here to probe deeper into the redox status of cysteine in AD. OxcysDML identified 1129 cysteine sites (from 527 proteins), among which 828 cysteine sites underwent oxidative modifications. Nineteen oxidized cysteine sites had significant alteration levels in AD and represent proteins involved in metabolic processes. Overall, we have demonstrated OxcysDML as a simple, rapid, robust, and inexpensive redox proteomic approach that is useful for gaining deeper insight into the proteome of AD.

A simple isotopic labeling method to study cysteine oxidation in Alzheimer's disease: oxidized cysteine-selective dimethylation (OxcysDML).

Reversible cysteine modifications play important physiological roles such as modulating enzymatic catalysis, maintaining redox homeostasis and conducting cellular signaling. These roles can be critical in the context of disease. Oxidative modifications such as S-nitrosylation (SNO) are signatures of neurodestruction in conditions of oxidative stress however are also indicators of neuroprotection and normal signaling in cellular environments with low concentrations of reactive oxygen and nitrogen species. SNO is a dynamic and low abundance modification and requires sensitive and selective analytical methods for its detection in biological tissues. Here we present an enhanced multiplexing strategy to study SNO in complex mixtures arising from tissues. This method, termed oxidized cysteine-selective cPILOT (OxcyscPILOT), allows simultaneous analysis of SNO-modified peptides in 12 samples. OxcyscPILOT has three primary steps: (1) blocking of free thiols by a cysteine-reactive reagent, (2) enrichment of peptides containing SNO on a solid phase resin, and (3) isotopic labeling and isobaric tagging of enriched peptides on the solid phase resin. This approach offers the advantage of allowing total protein abundance levels to be measured simultaneously with endogenous SNO levels and measurement of SNO levels across four biological replicates in a single analysis. Furthermore, the relative amount of SNO on a specific cysteine site can also be determined. A well-known model of Alzheimer's disease, the APP/PS-1 transgenic mouse model, was selected for demonstration of the method as several SNO-modified proteins have previously been reported in brain and synaptosomes from AD subjects. OxcyscPILOT analysis resulted in identification of 138 SNO-modified cysteines in brain homogenates that correspond to 135 proteins. Many of these SNO-modified proteins were only present in wild-type or AD mice, whereas 93 proteins had SNO signals in both WT and AD. Pathway analysis links SNO-modified proteins to various biological pathways especially metabolism and signal transduction, consistent with previous reports in the literature. The OxcyscPILOT strategy provides enhanced multiplexing capability to current redox proteomics methods to study oxidative modifications of cysteine.

High-throughput endogenous measurement of S-nitrosylation in Alzheimer's disease using oxidized cysteine-selective cPILOT.

Cysteine is a highly reactive amino acid and is subject to a variety of reversible post-translational modifications (PTMs), including nitrosylation, glutathionylation, palmitoylation, as well as formation of sulfenic acid and disulfides. These modifications are not only involved in normal biological activities, such as enzymatic catalysis, redox signaling, and cellular homeostasis, but can also be the result of oxidative damage. Especially in aging and neurodegenerative diseases, oxidative stress leads to aberrant cysteine oxidations that affect protein structure and function leading to neurodegeneration as well as other detrimental effects. Methods that can identify cysteine modifications by type, including the site of modification, as well as the relative stoichiometry of the modification can be very helpful for understanding the role of the thiol proteome and redox homeostasis in the context of disease. Cysteine reversible modifications however, are challenging to investigate as they are low abundant, diverse, and labile especially under endogenous conditions. Thanks to the development of redox proteomic approaches, large-scale quantification of cysteine reversible modifications is possible. These approaches cover a range of strategies to enrich, identify, and quantify cysteine reversible modifications from biological samples. This review will focus on nongel-based redox proteomics workflows that give quantitative information about cysteine PTMs and highlight how these strategies have been useful for investigating the redox thiol proteome in aging and neurodegenerative diseases.

Liqing Gu

SGS North America Inc.

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