|Home | About | Journals | Submit | Contact Us | Français|
Due to the complexity of the mammalian central nervous system neuropeptidomic studies in mammals often yield very complicated mass spectra that make data analysis difficult. Careful sample preparation and extraction protocols must be employed in order to minimize spectral complexity and enable extraction of useful information on neuropeptides from a given sample. Controlling post-mortem protease activity is essential to simplifying mass spectra and to identifying low-abundance neuropeptides in tissue samples. Post-mortem microwave-irradiation coupled with cryostat dissection has proven to be effective in arresting protease activity to allow detection of endogenous neuropeptides instead of protein degradation products.
Neuropeptides are small (5 to 100 amino acid residues) endogenous biomolecules that have the ability to act as neurotransmitters, neuromodulators, or neurohomones in the nervous system. These biomolecules play a role in many physiological functions including feeding, sleeping, learning, pain, anxiety, circadian rhythms, and memory (1,2). The biogenesis of neuropeptides occurs in the cell body of neurons. Here, pre-propeptides are synthesized in the rough endoplasmic reticulum (RER), secreted from the RER after the signal sequence is removed, and packaged into vesicles by the Golgi apparatus. Within these vesicles, propeptides are further processed and undergo post-translational modifications (i.e., glycosylation, C-terminal amidation, acetylation, phosphorylation, and disulfide bond formation) generating bioactive peptides (3).
The important biological role neuropeptides play has made these targets of many investigations. Many neuropeptides have been studied employing traditional techniques such as immunohistochemistry, radioimmunoassay, and Edman degradation. Although these techniques are valuable, mass spectrometry (MS) based techniques do not require a priori knowledge of peptide identity and allow for rapid elucidation of molecular species in a complex mixture. Mass spectrometry offers rapid and sensitive detection of ionizable species, but spectra can be complicated and low abundance target species (i.e., neuropeptides) can be masked due to salts, lipids, and surfactants (4). Thus, appropriate sample preparation methods that allow preferential identification of neuropeptides with minimal interference are often key to successful MS-based studies.
Important considerations must be taken during sample collection and preparation in order to obtain useful information in any neuropeptidomic study. The inhibition of active post-mortem proteases is one of the most important considerations a researcher must take into account. Once the animal is sacrificed and the tissue of interest harvested, proteases rapidly degrade larger proteins into smaller fragments that fall into the mass range of neuropeptides. These abundant protein fragments may suppress neuropeptide signal and make mass spectra interpretation very difficult. To minimize this spectra clouding from protein degradation, focused microwave-irradiation animal sacrifice (5-9), post-sacrifice microwave-irradiation of tissue (10), and cryostat dissection followed by a boiling extraction buffer (11) methods have all been used. Each of these techniques can effectively minimize the post-mortem protein degradation, but these techniques also possess their own drawbacks. Focused microwave-irradiation animal sacrifice, although an effective means to stop protease degradation, requires an expensive targeted-microwave emitting instrument and can introduce unnecessary stress on the animal that may affect neuropeptide expression. The use of a household microwave for post-mortem tissue fixation allows for the animal to be sacrificed by conventional methods, but can require a number of animals to develop a consistent protocol. Cryostat dissection followed by a boiling extraction buffer inhibits protease activity, but has a longer time gap between sacrifice and protease inhibition which allows some extracellular processing to occur.
Presented in this chapter is a neuropeptidomic procedure that utilizes conventional microwave to inhibit proteases post-mortem, cryostat dissection to isolate specific tissue, acidified methanol to extract neuropeptides from tissue, and LC-QTOF-MS(/MS) to analyze the tissue extract. Figure 1 depicts a flowchart of the MS-based neuropeptide analysis procedure.
A Waters nanoAcquity UPLC system was used to deliver the specified volume of sample to a trap column (Waters Symmetry® C18, 180 μm × 20mm; Milford, MA) via a 5% flow of mobile phase B at a rate of 10 μL/min for 1.50 min. The flow rate was then switched to 200 nL/min, and the peptides were flushed onto the analytical column (Microtech C18, 75 μm x 15 cm; Vista, CA) and eluted via a mobile phase B: 5 – 95% linear gradient over 60 minutes into a nESI-QTOF mass spectrometer (see Note 15).
Mass spectra (MS) were collected for all eluted peptides and tandem mass spectra (MS2) were collected in data-dependent acquisition mode where MS2 survey scans were made when an eluted peptide had an ion count of 15 or greater.
Bioactive neuropeptides are oftentimes found to undergo extensive proteolytic cleavages or post-translational modifications, making it difficult to identify what protein precursor a neuropeptide originates from. Recently a binary logistic regression model (12) trained on mammalian prohormone cleavages has been developed that helps determine novel bioactive peptides from an organism’s genetic sequence information. Once optimized, this bioinformatics tool will minimize the time and effort required to analyze MS data and determine novel bioactive peptides from genetic sequence information. Currently database searching and de novo sequencing are the methods of choice for determining peptide identity.
The SwePep (13) database (http://www.swepep.org/) was developed to increase the throughput of identifying endogenous peptides in complex tissue samples analyzed by MS. This is a good place to begin when trying to identify neuropeptides in tissue extracts. Online databases like Mascot (Matrix Science; http://www.matrixscience.com/) and SEQUEST (Thermo Corp.; http://www.thermo.com/) are also powerful tools for identifying neuropeptides (14,15). In this case, Mascot was used to analyze QTOF data.
This work was supported in part by National Science Foundation CAREER Award (CHE-0449991), National Institutes of Health through grant 1R01DK071801, and an Alfred P. Sloan Research Fellowship (L.L.). R.M.S. acknowledges the National Institutes of Health Clinical Neuroengineering Training Program Grant 5T90DK070079. J.A.D. acknowledges an American Foundation for Pharmaceutical Education (AFPE) predoctoral fellowship.
Robert M. Sturm, Department of Chemistry, University of Wisconsin-Madison, 777 Highland Avenue, Madison, WI 53705-2222, USA.
James A. Dowell, School of Pharmacy, University of Wisconsin-Madison, 777 Highland Avenue, Madison, WI 53705-2222, USA.
Lingjun Li, School of Pharmacy & Department of Chemistry, University of Wisconsin-Madison, 777 Highland Avenue, Madison, WI 53705-2222, USA.