Enhancing the Power of Liquid Chromatography–Mass Spectrometry-Based Urine Metabolomics in Negative Ion Mode by Optimization of the Additive

Untargeted liquid chromatography–mass spectrometry (LC-MS)-based metabolomics studies are usually carried out in both positive and negative ion modes; however, it is frequently ignored that the optimal conditions in positive ion mode and negative ion mode are often not the same. We carried out a systematic investigation on urine samples to evaluate the additive effects in negative ion mode. It was found that the widely used conditions, 0.1% formic acid (FA) and NH₄Ac at different pH, are far from the optimum for untargeted urine metabolomics studies. Compared to 0.1% FA, the use of 1 mM acetic acid (HAc) resulted in almost three times as many detected peaks (401 vs 148) and around five times the size of the peak area (33.55 × 10⁶ vs 6.47 × 10⁶). The remarkable improvement can be explained by two factors: (i) a significantly enhanced ionization efficiency due to the combination of an appropriate pH at around 4.0–4.5, the reducibility of H⁺, and the high gas-phase basicity of Ac^– and (ii) a reproducible LC separation due to an acceptable buffering capacity. Our study revealed the importance and necessity of additive optimization, which can be of benefit in related metabolomics studies

Enhancing the Power of Liquid Chromatography–Mass Spectrometry-Based Urine Metabolomics in Negative Ion Mode by Optimization of the Additive

Untargeted liquid chromatography–mass spectrometry (LC-MS)-based metabolomics studies are usually carried out in both positive and negative ion modes; however, it is frequently ignored that the optimal conditions in positive ion mode and negative ion mode are often not the same. We carried out a systematic investigation on urine samples to evaluate the additive effects in negative ion mode. It was found that the widely used conditions, 0.1% formic acid (FA) and NH₄Ac at different pH, are far from the optimum for untargeted urine metabolomics studies. Compared to 0.1% FA, the use of 1 mM acetic acid (HAc) resulted in almost three times as many detected peaks (401 vs 148) and around five times the size of the peak area (33.55 × 10⁶ vs 6.47 × 10⁶). The remarkable improvement can be explained by two factors: (i) a significantly enhanced ionization efficiency due to the combination of an appropriate pH at around 4.0–4.5, the reducibility of H⁺, and the high gas-phase basicity of Ac^– and (ii) a reproducible LC separation due to an acceptable buffering capacity. Our study revealed the importance and necessity of additive optimization, which can be of benefit in related metabolomics studies

Enhancing the Power of Liquid Chromatography–Mass Spectrometry-Based Urine Metabolomics in Negative Ion Mode by Optimization of the Additive

Untargeted liquid chromatography–mass spectrometry (LC-MS)-based metabolomics studies are usually carried out in both positive and negative ion modes; however, it is frequently ignored that the optimal conditions in positive ion mode and negative ion mode are often not the same. We carried out a systematic investigation on urine samples to evaluate the additive effects in negative ion mode. It was found that the widely used conditions, 0.1% formic acid (FA) and NH₄Ac at different pH, are far from the optimum for untargeted urine metabolomics studies. Compared to 0.1% FA, the use of 1 mM acetic acid (HAc) resulted in almost three times as many detected peaks (401 vs 148) and around five times the size of the peak area (33.55 × 10⁶ vs 6.47 × 10⁶). The remarkable improvement can be explained by two factors: (i) a significantly enhanced ionization efficiency due to the combination of an appropriate pH at around 4.0–4.5, the reducibility of H⁺, and the high gas-phase basicity of Ac^– and (ii) a reproducible LC separation due to an acceptable buffering capacity. Our study revealed the importance and necessity of additive optimization, which can be of benefit in related metabolomics studies

Enhancing the Power of Liquid Chromatography–Mass Spectrometry-Based Urine Metabolomics in Negative Ion Mode by Optimization of the Additive

Untargeted liquid chromatography–mass spectrometry (LC-MS)-based metabolomics studies are usually carried out in both positive and negative ion modes; however, it is frequently ignored that the optimal conditions in positive ion mode and negative ion mode are often not the same. We carried out a systematic investigation on urine samples to evaluate the additive effects in negative ion mode. It was found that the widely used conditions, 0.1% formic acid (FA) and NH₄Ac at different pH, are far from the optimum for untargeted urine metabolomics studies. Compared to 0.1% FA, the use of 1 mM acetic acid (HAc) resulted in almost three times as many detected peaks (401 vs 148) and around five times the size of the peak area (33.55 × 10⁶ vs 6.47 × 10⁶). The remarkable improvement can be explained by two factors: (i) a significantly enhanced ionization efficiency due to the combination of an appropriate pH at around 4.0–4.5, the reducibility of H⁺, and the high gas-phase basicity of Ac^– and (ii) a reproducible LC separation due to an acceptable buffering capacity. Our study revealed the importance and necessity of additive optimization, which can be of benefit in related metabolomics studies

Insulin Increases Phosphorylation of Mitochondrial Proteins in Human Skeletal Muscle <i>in Vivo</i>

There is increasing evidence that multiple proteins involved in key regulatory processes in mitochondria are phosphorylated in mammalian tissues. Insulin regulates glucose metabolism by phosphorylation-dependent signaling and has been shown to stimulate ATP synthesis in human skeletal muscle. Here, we investigated the effect of insulin on the phosphorylation of mitochondrial proteins in human skeletal muscle <i>in vivo.</i> Using a combination of TiO₂ phosphopeptide-enrichment, HILIC fractionation, and LC–MS/MS, we compared the phosphoproteomes of isolated mitochondria from skeletal muscle samples obtained from healthy individuals before and after 4 h of insulin infusion. In total, we identified 207 phosphorylation sites in 95 mitochondrial proteins. Of these phosphorylation sites, 45% were identified in both basal and insulin-stimulated samples. Insulin caused a 2-fold increase in the number of different mitochondrial phosphopeptides (87 ± 7 vs 40 ± 7, <i>p</i> = 0.015) and phosphoproteins (46 ± 2 vs 26 ± 3, <i>p</i> = 0.005) identified in each mitochondrial preparation. Almost half of the mitochondrial phosphorylation sites (<i>n</i> = 94) were exclusively identified in the insulin-stimulated state and included the majority of novel sites. Phosphorylation sites detected more often or exclusively in insulin-stimulated samples include multiple sites in mitochondrial proteins involved in oxidative phosphorylation, tricarboxylic acid cycle, and fatty acid metabolism, as well as several components of the newly defined mitochondrial inner membrane organizing system (MINOS). In conclusion, the present study demonstrates that insulin increases the phosphorylation of several mitochondrial proteins in human skeletal muscle <i>in vivo</i> and provides a first step in the understanding of how insulin potentially regulates mitochondrial processes by phosphorylation-dependent mechanisms

Optimized IMAC−IMAC Protocol for Phosphopeptide Recovery from Complex Biological Samples

Immobilized metal ion affinity chromatography (IMAC) is widely used for phosphopeptide enrichment. However, the robustness, efficiency, and specificity of this technique in large-scale phosphoproteomics studies are still disputed. In this study, we first compared three widely used IMAC materials under three different conditions. Fe(III)−nitrilotriacetic acid (NTA) IMAC resin was chosen due to its superior performance in all tests. We further investigated the solution ionization efficiency change of the phosphoryl group and carboxylic group in different acetonitrile−water solutions and observed that the ionization efficiencies of the phosphoryl group and carboxylic group changed differently when the acetonitrile concentration was increased. A magnified difference was achieved in high acetonitrile content solutions. On the basis of this concept, an optimized phosphopeptide enrichment protocol was established using Fe(III)−NTA IMAC resin and it proved to be highly selective in the phosphopeptide enrichment of a highly diluted standard sample (1:1000) prior to MALDI MS analysis. We also observed that a higher iron purity led to an increased IMAC enrichment efficiency. The optimized method was then adapted to phosphoproteome analyses of cell lysates of high protein complexity. From either 20 μg of mouse sample or 50 μg of Drosophila melanogaster sample, more than 1000 phosphorylation sites were identified in each study using IMAC−IMAC and LC−MS/MS. We demonstrate efficient separation of multiply phosphorylated peptides from singly phosphorylated peptides with successive IMAC enrichments. The rational improvements to the IMAC protocol described in this study provide more insights into the factors that affect IMAC performance for phosphopeptide recovery. The improved IMAC−IMAC method should allow more detailed characterization of phosphoproteins in functional phosphoproteomics research projects

Optimized IMAC−IMAC Protocol for Phosphopeptide Recovery from Complex Biological Samples

Immobilized metal ion affinity chromatography (IMAC) is widely used for phosphopeptide enrichment. However, the robustness, efficiency, and specificity of this technique in large-scale phosphoproteomics studies are still disputed. In this study, we first compared three widely used IMAC materials under three different conditions. Fe(III)−nitrilotriacetic acid (NTA) IMAC resin was chosen due to its superior performance in all tests. We further investigated the solution ionization efficiency change of the phosphoryl group and carboxylic group in different acetonitrile−water solutions and observed that the ionization efficiencies of the phosphoryl group and carboxylic group changed differently when the acetonitrile concentration was increased. A magnified difference was achieved in high acetonitrile content solutions. On the basis of this concept, an optimized phosphopeptide enrichment protocol was established using Fe(III)−NTA IMAC resin and it proved to be highly selective in the phosphopeptide enrichment of a highly diluted standard sample (1:1000) prior to MALDI MS analysis. We also observed that a higher iron purity led to an increased IMAC enrichment efficiency. The optimized method was then adapted to phosphoproteome analyses of cell lysates of high protein complexity. From either 20 μg of mouse sample or 50 μg of Drosophila melanogaster sample, more than 1000 phosphorylation sites were identified in each study using IMAC−IMAC and LC−MS/MS. We demonstrate efficient separation of multiply phosphorylated peptides from singly phosphorylated peptides with successive IMAC enrichments. The rational improvements to the IMAC protocol described in this study provide more insights into the factors that affect IMAC performance for phosphopeptide recovery. The improved IMAC−IMAC method should allow more detailed characterization of phosphoproteins in functional phosphoproteomics research projects

Optimized IMAC−IMAC Protocol for Phosphopeptide Recovery from Complex Biological Samples

Immobilized metal ion affinity chromatography (IMAC) is widely used for phosphopeptide enrichment. However, the robustness, efficiency, and specificity of this technique in large-scale phosphoproteomics studies are still disputed. In this study, we first compared three widely used IMAC materials under three different conditions. Fe(III)−nitrilotriacetic acid (NTA) IMAC resin was chosen due to its superior performance in all tests. We further investigated the solution ionization efficiency change of the phosphoryl group and carboxylic group in different acetonitrile−water solutions and observed that the ionization efficiencies of the phosphoryl group and carboxylic group changed differently when the acetonitrile concentration was increased. A magnified difference was achieved in high acetonitrile content solutions. On the basis of this concept, an optimized phosphopeptide enrichment protocol was established using Fe(III)−NTA IMAC resin and it proved to be highly selective in the phosphopeptide enrichment of a highly diluted standard sample (1:1000) prior to MALDI MS analysis. We also observed that a higher iron purity led to an increased IMAC enrichment efficiency. The optimized method was then adapted to phosphoproteome analyses of cell lysates of high protein complexity. From either 20 μg of mouse sample or 50 μg of Drosophila melanogaster sample, more than 1000 phosphorylation sites were identified in each study using IMAC−IMAC and LC−MS/MS. We demonstrate efficient separation of multiply phosphorylated peptides from singly phosphorylated peptides with successive IMAC enrichments. The rational improvements to the IMAC protocol described in this study provide more insights into the factors that affect IMAC performance for phosphopeptide recovery. The improved IMAC−IMAC method should allow more detailed characterization of phosphoproteins in functional phosphoproteomics research projects

Optimized IMAC−IMAC Protocol for Phosphopeptide Recovery from Complex Biological Samples

Immobilized metal ion affinity chromatography (IMAC) is widely used for phosphopeptide enrichment. However, the robustness, efficiency, and specificity of this technique in large-scale phosphoproteomics studies are still disputed. In this study, we first compared three widely used IMAC materials under three different conditions. Fe(III)−nitrilotriacetic acid (NTA) IMAC resin was chosen due to its superior performance in all tests. We further investigated the solution ionization efficiency change of the phosphoryl group and carboxylic group in different acetonitrile−water solutions and observed that the ionization efficiencies of the phosphoryl group and carboxylic group changed differently when the acetonitrile concentration was increased. A magnified difference was achieved in high acetonitrile content solutions. On the basis of this concept, an optimized phosphopeptide enrichment protocol was established using Fe(III)−NTA IMAC resin and it proved to be highly selective in the phosphopeptide enrichment of a highly diluted standard sample (1:1000) prior to MALDI MS analysis. We also observed that a higher iron purity led to an increased IMAC enrichment efficiency. The optimized method was then adapted to phosphoproteome analyses of cell lysates of high protein complexity. From either 20 μg of mouse sample or 50 μg of Drosophila melanogaster sample, more than 1000 phosphorylation sites were identified in each study using IMAC−IMAC and LC−MS/MS. We demonstrate efficient separation of multiply phosphorylated peptides from singly phosphorylated peptides with successive IMAC enrichments. The rational improvements to the IMAC protocol described in this study provide more insights into the factors that affect IMAC performance for phosphopeptide recovery. The improved IMAC−IMAC method should allow more detailed characterization of phosphoproteins in functional phosphoproteomics research projects

Comparative Proteomic Analysis of Histone Post-translational Modifications upon Ischemia/Reperfusion-Induced Retinal Injury

We present a detailed quantitative map of single and coexisting histone post-translational modifications (PTMs) in rat retinas affected by ischemia and reperfusion (I/R) injury. Retinal I/R injury contributes to serious ocular diseases, which can lead to vision loss and blindness. We applied linear ion trap–orbitrap hybrid tandem mass spectrometry (MS/MS) to quantify 131 single histone marks and 143 combinations of multiple histone marks in noninjured and injured retinas. We observed 34 histone PTMs that exhibited significantly (<i>p</i> < 0.05) different abundance between healthy and I/R injured eyes, of which we confirmed three H4 histone marks by Western blotting. H4K20me2 was up to 4-fold change up-regulated after the injury and is associated with the response to DNA damage as demonstrated by an increase in the phosphorylation of p53 and Chk1. This study demonstrates that quantitative MS provides a sensitive and accurate way to dissect the changes in the histone code after retinal injury. Specifically, DNA damage associated histone PTMs may contribute to neurovascular degeneration during the process of ischemia/reperfusion injury