Daniele Canzani, PhD

"Human cells make use of hundreds of unique ubiquitin E3 ligases to ensure proteome fidelity and control cellular functions by promoting protein degradation. These processes require exquisite selectivity, but the individual roles of most E3s remain poorly characterized in part due to the challenges associated with identifying, quantifying, and validating substrates for each E3. We report an integrative mass spectrometry (MS) strategy for characterizing protein fragments that interact with… Show more

"Human cells make use of hundreds of unique ubiquitin E3 ligases to ensure proteome fidelity and control cellular functions by promoting protein degradation. These processes require exquisite selectivity, but the individual roles of most E3s remain poorly characterized in part due to the challenges associated with identifying, quantifying, and validating substrates for each E3. We report an integrative mass spectrometry (MS) strategy for characterizing protein fragments that interact with KLHDC2, a human E3 that recognizes the extreme C-terminus of substrates. Using a combination of native MS, native top-down MS, MS of destabilized samples, and liquid chromatography MS, we identified and quantified a near complete fraction of the KLHDC2-binding peptidome in E. coli cells. This degronome includes peptides that originate from a variety of proteins. Although all identified protein fragments are terminated by diglycine or glycylalanine, the preceding amino acids are diverse. These results significantly expand our understanding of the sequences that can be recognized by KLHDC2, which provides insight into the potential substrates of this E3 in humans. We anticipate that this integrative MS strategy could be leveraged more broadly to characterize the degronomes of other E3 ligase substrate receptors, including those that adhere to the more common N-end rule for substrate recognition. Therefore, this work advances “degronomics,” i.e., identifying, quantifying, and validating functional E3:peptide interactions in order to determine the individual roles of each E3." Show less

Florida citrus depends on a breakthrough in the fight against citrus greening disease. Of the antibiotics used to treat this disease, penicillin G has been one of the most effective. Because orange fruit grown in the state of Florida are mainly used to produce orange juice, we have validated an ultra-HPLC tandem MS method to screen for penicillin G and its metabolites (penillic and penilloic acids) at the chemical residue level after treatment. In this method, three spike levels (0.25, 1, and… Show more

Florida citrus depends on a breakthrough in the fight against citrus greening disease. Of the antibiotics used to treat this disease, penicillin G has been one of the most effective. Because orange fruit grown in the state of Florida are mainly used to produce orange juice, we have validated an ultra-HPLC tandem MS method to screen for penicillin G and its metabolites (penillic and penilloic acids) at the chemical residue level after treatment. In this method, three spike levels (0.25, 1, and 20 ng/g) were tested in triplicate. Absolute recoveries for penillic and penilloic acids were 60–75% depending on the matrix used, whereas corrected recoveries of penicillin G using an isotopically labeled internal standard were ~100%. Two product ion transitions per analyte were required for identification, which contributes to a high degree of selectivity. Show less

In recent years, citrus greening disease, also known as huanglongbing
(HLB), has devastated citrus crops throughout the world. Penicillin
G has been used to treat HLB-infected trees with promising
results. However, the metabolites produced from the degradation
of penicillin G are known to cause potentially life-threatening
allergic reactions. Therefore, the concentration and presence of
the metabolites must be carefully monitored. We have developed
and revised an analytical… Show more

In recent years, citrus greening disease, also known as huanglongbing
(HLB), has devastated citrus crops throughout the world. Penicillin
G has been used to treat HLB-infected trees with promising
results. However, the metabolites produced from the degradation
of penicillin G are known to cause potentially life-threatening
allergic reactions. Therefore, the concentration and presence of
the metabolites must be carefully monitored. We have developed
and revised an analytical method based on ultrahigh-pressure
liquid chromatography (UHPLC) in combination with tandem mass
spectrometry (MS/MS) to identify and quantitate penicillin G and its
major metabolites, penillic acid and penilloic acid, in citrus fruit and
juice. Here, we discuss the chromatographic conditions and revisions
that improved the precision and accuracy of our measurements. Show less

Penicillin G has been applied to citrus trees as a potential treatment in the fight against Huanglongbing (HLB). Here, we have developed and validated a method to identify and quantitate penicillin G and two of its metabolites, penillic acid and penilloic acid, in citrus fruit using ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). This method improves upon a previous method by incorporating isotopically labeled internal standards, namely, penillic acid-D 5… Show more

Penicillin G has been applied to citrus trees as a potential treatment in the fight against Huanglongbing (HLB). Here, we have developed and validated a method to identify and quantitate penicillin G and two of its metabolites, penillic acid and penilloic acid, in citrus fruit using ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). This method improves upon a previous method by incorporating isotopically labeled internal standards, namely, penillic acid-D 5, and penilloic acid-D 5. These standards greatly enhanced the accuracy and precision of our measurements by compensating for recovery losses, degradation, and matrix effects. When 2g of citrus fruit sample is extracted, the limits of detection (LOD) were determined to be 0.1ng/g for penicillin G and penilloic acid, and 0.25ng/g for penillic acid. At fortification levels of 0.1, 0.25, 1, and 10ng/g, absolute recoveries for penillic and penilloic acids were generally between 50-70%. Recoveries corrected with the isotopically labeled standards were approximately 90-110%. This method will be useful for the identification and quantitation of drug residues and their degradation products using isotopically labeled standards and UHPLC-MS/MS. Show less

In this work, we investigate the stability of penicillin G in various conditions including acidic, alkaline, natural acidic matrix, and after treatment of citrus trees that are infected with the citrus greening disease. The identification, confirmation, and quantitation of penicillin G and its various metabolites were evaluated using two UHPLC-MS/MS systems with variable capabilities (i.e. Thermo Q Exactive Orbitrap and Sciex 6500 Qtrap). Our data show that under acidic and alkaline conditions,… Show more

In this work, we investigate the stability of penicillin G in various conditions including acidic, alkaline, natural acidic matrix, and after treatment of citrus trees that are infected with the citrus greening disease. The identification, confirmation, and quantitation of penicillin G and its various metabolites were evaluated using two UHPLC-MS/MS systems with variable capabilities (i.e. Thermo Q Exactive Orbitrap and Sciex 6500 Qtrap). Our data show that under acidic and alkaline conditions, penicillin G at 100 ng/mL degrades fast, with a determined half-life time of approximately 2 hours. Penillic acid, penicilloic acid, and penilloic acid are found to be the most abundant metabolites of penicillin G. These major metabolites, along with isopenillic acid, are found when penicillin G is used for treatment of the citrus greening infected trees. The findings of this study will provide insight regarding penicillin G residues for food safety purposes, in agricultural and biological applications. Show less