One-Step Sampling, Extraction, and Storage Protocol for Peptidomics Using Dihydroxybenzoic Acid

Romanova, Elena V.; Rubakhin, Stanislav S.; Sweedler, Jonathan V.

doi:10.1021/ac7026047

Cited by 33 publications

(40 citation statements)

References 59 publications

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“…Since levels of these intermediates were expected to be low, we utilized the pituitary, a tissue rich in peptides, for our studies; although the neuropeptides in mouse pituitary have been well characterized, these reaction intermediates have not been detected [28], [31], [32]. Characterization of these amidation intermediates is challenging, not only because of their low levels compared to the fully processed peptides, but also due to the restrictions imposed by MS detection limits and dynamic range.…”

Section: Resultsmentioning

confidence: 99%

Probing the Production of Amidated Peptides following Genetic and Dietary Copper Manipulations

et al. 2011

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Amidated neuropeptides play essential roles throughout the nervous and endocrine systems. Mice lacking peptidylglycine α-amidating monooxygenase (PAM), the only enzyme capable of producing amidated peptides, are not viable. In the amidation reaction, the reactant (glycine-extended peptide) is converted into a reaction intermediate (hydroxyglycine-extended peptide) by the copper-dependent peptidylglycine-α-hydroxylating monooxygenase (PHM) domain of PAM. The hydroxyglycine-extended peptide is then converted into amidated product by the peptidyl-α-hydroxyglycine α-amidating lyase (PAL) domain of PAM. PHM and PAL are stitched together in vertebrates, but separated in some invertebrates such as Drosophila and Hydra. In addition to its luminal catalytic domains, PAM includes a cytosolic domain that can enter the nucleus following release from the membrane by γ-secretase. In this work, several glycine- and hydroxyglycine-extended peptides as well as amidated peptides were qualitatively and quantitatively assessed from pituitaries of wild-type mice and mice with a single copy of the Pam gene (PAM+/−) via liquid chromatography-mass spectrometry-based methods. We provide the first evidence for the presence of a peptidyl-α-hydroxyglycine in vivo, indicating that the reaction intermediate becomes free and is not handed directly from PHM to PAL in vertebrates. Wild-type mice fed a copper deficient diet and PAM+/− mice exhibit similar behavioral deficits. While glycine-extended reaction intermediates accumulated in the PAM+/− mice and reflected dietary copper availability, amidated products were far more prevalent under the conditions examined, suggesting that the behavioral deficits observed do not simply reflect a lack of amidated peptides.

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Section: Resultsmentioning

confidence: 99%

Probing the Production of Amidated Peptides following Genetic and Dietary Copper Manipulations

et al. 2011

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“…As developed by Romanova et al [33], the one-step protocol using saDHB solution was easy to perform (Fig. 1B).…”

Section: Resultsmentioning

confidence: 99%

“…Recently, Romanova et al [33] reported that the use of saturated DHB in aqueous solution could provide direct extraction of neuropeptides from neuronal tissues ranging from cellular clusters to intact brain. This one-step protocol is effective and can also be used for a long-term preservation of the neuropeptide extracts.…”

Section: Introductionmentioning

confidence: 99%

Combining tissue extraction and off-line capillary electrophoresis matrix-assisted laser desorption/ionization Fourier transform mass spectrometry for neuropeptide analysis in individual neuronal organs using 2,5-dihydroxybenzoic acid as a multi-functional agent

Wang¹,

Jiang²,

Sturm³

2009

Journal of Chromatography A

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In this study we report an improved protocol that combines simplified sample preparation and microscale separation for mass spectrometric analysis of neuropeptides from individual neuroendocrine organs of crab Cancer borealis. A simple, one-step extraction method with commonly used matrixassisted laser desorption/ionization (MALDI) matrix, 2,5-dihydroxybenzoic acid (DHB), in saturated aqueous solution, is employed for improved extraction of neuropeptides. Furthermore, a novel use of DHB as background electrolyte for capillary electrophoresis (CE) separation in the off-line coupling of CE to MALDI-Fourier transform mass spectrometric (FT-MS) detection is also explored. The new CE electrolyte exhibits full compatibility with MALDI-MS analysis of neuropeptides in that both the peptide extraction process and MALDI detection utilize DHB. In addition, enhanced resolving power and improved sensitivity are also observed for CE-MALDI-MS of peptide mixture analysis. Collectively, the use of DHB has simplified the extraction and reduced the sample loss by elimination of homogenizing, drying, and desalting processes. In the mean time, the concurrent use of DHB as CE separation buffer and subsequent MALDI-MS detection offers improved spectral quality by eliminating the interferences from typical CE electrolyte in MALDI detection.

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“…The requirement for substantial amounts of sample material frequently restricts peptidomics approaches to the analysis of either larger anatomically defined structures (whole brain) or pooled samples [7,8,12–14,18]. An alternative that is suitable for quantitative profiling of minute individual samples is the single-step extraction of signal peptides using 2,5-dihydroxybenzoic acid (DHB), a commonly used matrix for MALDI MS [28]. The method is suitable for fractionation of peptides by capillary electrophoresis using DHB as a background electrolyte [29].…”

Section: Challenges That Drive New Developments In Quantitative Peptimentioning

confidence: 99%

Quantitation of endogenous peptides using mass spectrometry based methods

Romanova

Dowd

Sweedler

2013

Current Opinion in Chemical Biology

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The mass spectrometry-based “omics” sub-discipline that focuses on comprehensive, often exploratory, analyses of endogenous peptides involved in cell-to-cell communication is oftentimes referred to as peptidomics. While the progress in bioanalytical technology development for peptide discovery has been tremendous, perhaps the largest advances have involved robust quantitative mass spectrometric approaches and data mining algorithms. These efforts have accelerated the discovery and validation of biomarkers, functionally important posttranslational modifications, and unexpected molecular interactions, information that aids drug development. In this article we outline the current approaches used in quantitative peptidomics and the technical challenges that stimulate new advances in the field, while also reviewing the newest literature on functional characterizations of endogenous peptides using quantitative mass spectrometry.

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One-Step Sampling, Extraction, and Storage Protocol for Peptidomics Using Dihydroxybenzoic Acid

Cited by 33 publications

References 59 publications

Probing the Production of Amidated Peptides following Genetic and Dietary Copper Manipulations

Probing the Production of Amidated Peptides following Genetic and Dietary Copper Manipulations

Combining tissue extraction and off-line capillary electrophoresis matrix-assisted laser desorption/ionization Fourier transform mass spectrometry for neuropeptide analysis in individual neuronal organs using 2,5-dihydroxybenzoic acid as a multi-functional agent

Quantitation of endogenous peptides using mass spectrometry based methods

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