Minimizing Postsampling Degradation of Peptides by a Thermal Benchtop Tissue Stabilization Method

Abstract

Enzymatic degradation is a major concern in peptide analysis. Postmortem metabolism in biological samples entails considerable risk for measurements misrepresentative of true in vivo concentrations. It is therefore vital to find reliable, reproducible, and easy-to-use procedures to inhibit enzymatic activity in fresh tissues before subjecting them to qualitative and quantitative analyses. The aim of this study was to test a benchtop thermal stabilization method to optimize measurement of endogenous opioids in brain tissue. Endogenous opioid peptides are generated from precursor proteins through multiple enzymatic steps that include conversion of one bioactive peptide to another, often with a different function. Ex vivo metabolism may, therefore, lead to erroneous functional interpretations. The efficacy of heat stabilization was systematically evaluated in a number of postmortem handling procedures. Dynorphin B (DYNB), Leu-enkephalin-Arg6 (LARG), and Met-enkephalin-Arg6-Phe7 (MEAP) were measured by radioimmunoassay in rat hypothalamus, striatum (STR), and cingulate cortex (CCX). Also, simplified extraction protocols for stabilized tissue were tested. Stabilization affected all peptide levels to varying degrees compared to those prepared by standard dissection and tissue handling procedures. Stabilization increased DYNB in hypothalamus, but not STR or CCX, whereas LARG generally decreased. MEAP increased in hypothalamus after all stabilization procedures, whereas for STR and CCX, the effect was dependent on the time point for stabilization. The efficacy of stabilization allowed samples to be left for 2 hours in room temperature (20°C) without changes in peptide levels. This study shows that conductive heat transfer is an easy-to-use and efficient procedure for the preservation of the molecular composition in biological samples. Region- and peptide-specific critical steps were identified and stabilization enabled the optimization of tissue handling and opioid peptide analysis. The result is improved diagnostic and research value of the samples with great benefits for basic research and clinical work.

Preserving Neuronal Chemical Messengers: Heat Stabilization Versus Snap Freezing for Improved MALDI Mass Spectrometry Imaging of Brain Tissues

“…Although we did not explicitly perform stability assays with chemical standards, previous studies employing rapid microwave-induced and conductive heat transfer stabilization methods have confirmed the stability of neurotransmitters (dopamine, serotonin, norepinephrine, and acetylcholine) and neuropeptides (substance P, dynorphins, and enkephalins) under conditions analogous to our HS procedure. These results indicate that rapid heating effectively preserves the integrity of these molecules by preventing enzymatic degradation (Goodwin et al 2010;Kanazawa and Jessell 1976;Nylander et al 1997;Segerstrom et al 2016;Wasek et al 2018).…”

“…Heat stabilization is performed by rapid and efficient heat transfer through a lower and upper conductive heat block during vacuum application, ensuring homogenous heating at 95°C in any part of the sample. This approach reduces the degradation processes of proteins and peptides, as well as preserves their post-translational modifications (Segerstrom et al 2016;Skold et al 2007;Svensson et al 2009). This work investigated in detail the postmortem degradation differences among heat-stabilized (HS) and fresh frozen (FF) brain tissues for key neurotransmitters, metabolites, and neuropeptides using MALDI-MSI analysis.…”

Preserving Neuronal Chemical Messengers: Heat Stabilization Versus Snap Freezing for Improved MALDI Mass Spectrometry Imaging of Brain Tissues

“…Although we did not explicitly perform stability assays with chemical standards, previous studies employing rapid microwave-induced and conductive heat transfer stabilization methods have confirmed the stability of neurotransmitters (dopamine, serotonin, norepinephrine, and acetylcholine) and neuropeptides (substance P, dynorphins, and enkephalins) under conditions analogous to our HS procedure. These results indicate that rapid heating effectively preserves the integrity of these molecules by preventing enzymatic degradation (Goodwin et al 2010;Kanazawa and Jessell 1976;Nylander et al 1997;Segerstrom et al 2016;Wasek et al 2018).…”

“…Heat stabilization is performed by rapid and efficient heat transfer through a lower and upper conductive heat block during vacuum application, ensuring homogenous heating at 95°C in any part of the sample. This approach reduces the degradation processes of proteins and peptides, as well as preserves their post-translational modifications (Segerstrom et al 2016;Skold et al 2007;Svensson et al 2009). This work investigated in detail the postmortem degradation differences among heat-stabilized (HS) and fresh frozen (FF) brain tissues for key neurotransmitters, metabolites, and neuropeptides using MALDI-MSI analysis.…”

Improved identification and quantitation of mature endogenous peptides in the rodent hypothalamus using a rapid conductive sample heating system

“…20,23,25–27,30,32,34,37,39,41,45,59–61 To avoid redundancy with previous studies, we did not test and compare the detection of peptides in untreated tissue samples relative to heat-treated ones. Our investigation focuses on comparing the effectiveness of different heat treatment approaches specifically for improved discovery and characterization of the physiologically relevant neuropeptide complement.…”

Episodic Ethanol Exposure in Adolescent Rats Causes Residual Alterations in Endogenous Opioid Peptides

“…The tissue samples were stabilized by heat denaturation (95°C) with a bench-top Stabilizor T1 (Denator AB, Uppsala, Sweden) according to the manufacturer's manual. The stabilization process involves a combination of conductive heat transfer and pressure under vacuum to prevent enzymatic degradation (e.g., of peptides) during freeze-thawing ( 22 ). Whole brains were placed in a Maintainor Tissue card (Denator AB, Uppsala, Sweden) and stabilized in the “frozen structure preserve mode” and thereafter in the “fresh structure preserve mode” to ensure an adequate treatment.…”

Preserving Neuronal Chemical Messengers: Heat Stabilization Versus Snap Freezing for Improved MALDI Mass Spectrometry Imaging of Brain Tissues

“…Although we did not explicitly perform stability assays with chemical standards, previous studies employing rapid microwave-induced and conductive heat transfer stabilization methods have confirmed the stability of neurotransmitters (dopamine, serotonin, norepinephrine, and acetylcholine) and neuropeptides (substance P, dynorphins, and enkephalins) under conditions analogous to our HS procedure. These results indicate that rapid heating effectively preserves the integrity of these molecules by preventing enzymatic degradation (Goodwin et al 2010;Kanazawa and Jessell 1976;Nylander et al 1997;Segerstrom et al 2016;Wasek et al 2018).…”

“…Heat stabilization is performed by rapid and efficient heat transfer through a lower and upper conductive heat block during vacuum application, ensuring homogenous heating at 95°C in any part of the sample. This approach reduces the degradation processes of proteins and peptides, as well as preserves their post-translational modifications (Segerstrom et al 2016;Skold et al 2007;Svensson et al 2009). This work investigated in detail the postmortem degradation differences among heat-stabilized (HS) and fresh frozen (FF) brain tissues for key neurotransmitters, metabolites, and neuropeptides using MALDI-MSI analysis.…”

Improved identification and quantitation of mature endogenous peptides in the rodent hypothalamus using a rapid conductive sample heating system

“…20,23,25–27,30,32,34,37,39,41,45,59–61 To avoid redundancy with previous studies, we did not test and compare the detection of peptides in untreated tissue samples relative to heat-treated ones. Our investigation focuses on comparing the effectiveness of different heat treatment approaches specifically for improved discovery and characterization of the physiologically relevant neuropeptide complement.…”

Episodic Ethanol Exposure in Adolescent Rats Causes Residual Alterations in Endogenous Opioid Peptides

“…The tissue samples were stabilized by heat denaturation (95°C) with a bench-top Stabilizor T1 (Denator AB, Uppsala, Sweden) according to the manufacturer's manual. The stabilization process involves a combination of conductive heat transfer and pressure under vacuum to prevent enzymatic degradation (e.g., of peptides) during freeze-thawing ( 22 ). Whole brains were placed in a Maintainor Tissue card (Denator AB, Uppsala, Sweden) and stabilized in the “frozen structure preserve mode” and thereafter in the “fresh structure preserve mode” to ensure an adequate treatment.…”

Preserving Neuronal Chemical Messengers: Heat Stabilization Versus Snap Freezing for Improved MALDI Mass Spectrometry Imaging of Brain Tissues

“…Although we did not explicitly perform stability assays with chemical standards, previous studies employing rapid microwave-induced and conductive heat transfer stabilization methods have confirmed the stability of neurotransmitters (dopamine, serotonin, norepinephrine, and acetylcholine) and neuropeptides (substance P, dynorphins, and enkephalins) under conditions analogous to our HS procedure. These results indicate that rapid heating effectively preserves the integrity of these molecules by preventing enzymatic degradation (Goodwin et al 2010;Kanazawa and Jessell 1976;Nylander et al 1997;Segerstrom et al 2016;Wasek et al 2018).…”

“…Heat stabilization is performed by rapid and efficient heat transfer through a lower and upper conductive heat block during vacuum application, ensuring homogenous heating at 95°C in any part of the sample. This approach reduces the degradation processes of proteins and peptides, as well as preserves their post-translational modifications (Segerstrom et al 2016;Skold et al 2007;Svensson et al 2009). This work investigated in detail the postmortem degradation differences among heat-stabilized (HS) and fresh frozen (FF) brain tissues for key neurotransmitters, metabolites, and neuropeptides using MALDI-MSI analysis.…”

Improved identification and quantitation of mature endogenous peptides in the rodent hypothalamus using a rapid conductive sample heating system

“…20,23,25–27,30,32,34,37,39,41,45,59–61 To avoid redundancy with previous studies, we did not test and compare the detection of peptides in untreated tissue samples relative to heat-treated ones. Our investigation focuses on comparing the effectiveness of different heat treatment approaches specifically for improved discovery and characterization of the physiologically relevant neuropeptide complement.…”

Episodic Ethanol Exposure in Adolescent Rats Causes Residual Alterations in Endogenous Opioid Peptides

“…The tissue samples were stabilized by heat denaturation (95°C) with a bench-top Stabilizor T1 (Denator AB, Uppsala, Sweden) according to the manufacturer's manual. The stabilization process involves a combination of conductive heat transfer and pressure under vacuum to prevent enzymatic degradation (e.g., of peptides) during freeze-thawing ( 22 ). Whole brains were placed in a Maintainor Tissue card (Denator AB, Uppsala, Sweden) and stabilized in the “frozen structure preserve mode” and thereafter in the “fresh structure preserve mode” to ensure an adequate treatment.…”