Detect more, novel endogenous peptides
Detecting and determining the role of endogenous peptides presents several challenges. Normal peptidase/protease control mechanisms continue after excision and post-mortem proteolytic activity begins immediately, producing protein fragments (peptides) that can mask endogenous peptides present at low levels. Heat stabilization permanently stops protein degradation and enzymatic changes.
Reveal new peptides and determine biological mechanisms
- more effective than snap freezing
Case study: Neuropeptide profiling of the bovine hypothalamus
LC-MS profile: neurosecretory peptides VGF[489–506], CCK[21–44], GnRH, AL11, CLIP, HCNP and BAM-1745 detected in heat-stabilized samples are diminished in snap-frozen samples. Degradation products of stathmin precursor (stathmin [2–20] and stathmin [2–22]) seen only in snap-frozen samples.
Comparison of heat-stabilized and snap-frozen bovine hypothalamus tissue revealed a greater number of neuropeptides in heat-stabilized samples.
Heat stabilization has prevented post-mortem proteolytic degradation from masking neuropeptides during analysis.
Results courtesy of Colgrave et al. Neuropeptide profiling of the bovine hypothalamus: Thermal stabilization is an effective tool in inhibiting post-mortem degradation, Proteomics 2011
Case study: Characterization of neuropeptide functions in nervous and endocrine systems
Analysis of heat-stabilized brain tissue identified 107 potential biologically-active neuropeptides in the tree shrew. Sequence homology analysis showed these peptides to have a higher degree of homology to equivalent human sequences than those in mice or rats, consistent with the close phylogenetic relationship between tree shrews and primates. Results will enable functional characterization of these endogenous peptides in the nervous and endocrine system.
From 25 known neuropeptide-containing precursors, 107 potential biologically-active neuropeptides were identified: 92 identical to sequences found in one or more other species, 24 found in literature as classical neuropeptides.
Results courtesy of Petruzziello et al. Extensive Characterization of Tupaia belangeri Neuropeptidome Using an Integrated Mass Spectrometric Approach J. Proteome Res 2012.
Case study: Role of endogenous peptides and hormones in bovine reproduction
Heat stabilization of bovine hypothalamus, followed by an optimized peptide extraction protocol revealed a greater number of neuropeptide precursors as compared to snap frozen samples. These preliminary findings will be used to elucidate mechanisms and pathways involved in reproduction.
Data courtesy of Dr Michelle Colgrave, CSIRO Livestock Industries, Presentation “Peptidomics of the Bovine Hypothalamus” made at the Annual meeting of Australasian Proteomics Society in Lorne, Australia, 2010
Identify potential biomarkers
Case study: Drug-induced withdrawal behavior
Identification and quantification of endogenous peptides derived from tachykinin and opioid peptide systems in morphine-dependent rats confirmed the presence of several known peptides, pre-stages and their truncated forms. Results suggest that some of the identified peptides are potential biomarker candidates for the study of drug-induced withdrawal behavior.
Identification of endogenous peptides revealed changes in expression of selected precursors (proenkephalin, prodynorphin and preprotachykinin) that were influenced by subsequent treatment, but also appeared to be dependent on the strain of rat. Sample: heat-stabilized striatal brain tissue of morphine-dependent rats from two different strains treated with morphine-naloxone or saline–naloxone during naloxone-precipitated withdrawal.
Results courtesy of Uppsala University (A Quantitative Peptidomic Analysis of Peptides Related to the Endogenous Opioid and Tachykinin Systems in Nucleus Accumbens of Rats Following Naloxone-Precipitated Morphine Withdrawal U. Rossbach et al. J. Proteome Res. 2009 Oct;9(20):4616-26)
Stabilizor Peptide Extraction Kit
MS platform for analysis of endogenous peptides
Visit our User support zone to learn more on how to work with heat-stabilized samples and view guidelines for several downstream analytical methods and the key parameters important to think about for each workflow.