An optimal protocol to analyze the rat spinal cord proteome

Abstract

Since the function of the spinal cord depends on the proteins found there, better defing the normal Spinal Cord Proteome is an important and challenging task. Although brain and cerebrospinal fluid samples from patients with different central nervous system (CNS) disorders have been studied, a thorough examination of specific spinal cord proteins and the changes induced by injury or associated to conditions such as neurodegeneration, spasticity and neuropathies has yet to be performed. In the present study, we aimed to describe total protein content in the spinal cord of healthy rats, employing different proteomics tools. Accordingly, we have developed a fast, easy, and reproducible sequential protocol for protein extraction from rat spinal cords. We employed conventional two dimensional electrophoresis (2DE) in different pH ranges (eg. 4-7, 3-11 NL) combined with identification by mass spectrometry (MALDI-TOF/TOF), as well as first dimension protein separation combined with Liquid Chromatography Mass Spectrometry/Mass Spectrometry (LC-MS/MS) to maximise the benefits of this technology. The value of these techniques is demonstrated here by the identification of several proteins known to be associated with neuroglial structures, neurotransmission, cell survival and nerve growth in the central nervous system. Furthermore this study identified many spinal proteins that have not previously been described in the literature and which may play an important role as either sensitive biomarkers of dysfunction or of recovery after Spinal Cord Injury.

Keywords: Liquid Chromatography Mass Spectrometry/Mass Spectrometry (LC-MS MS); proteome; proteomics; spinal cord; two dimensional electrophoresis (2-DE).

Figure 1.

The proteomic platforms used in this study and a flowchart demonstrating the strategy for the rat spinal cord analysis. Schematic illustration of the proteomics methods used to characterise the rat spinal cord proteome.

Figure 2.

The protocol to extract proteins from the rat spinal cord. A) After surgery the spinal cord tissue was washed in saline buffer to eliminate blood contaminants and tissue was homogenized (Buffer 1) and later a new extraction of proteins was realized using buffer 2. Supernatant A, containing most of the soluble proteins and supernatant B, containing membrane and hydrophobic proteins were analysed separately in 2-DE in order to check the efficiency of the protein extraction protocol. B) Supernatant A and B were mixed and analysed by 2-DE.

Figure 3.

2-DE gel images. 2-DE was performed with IPG strips at different pH ranges: A) pH 4–7 (left) and B) pH 3–11 NL (right). C) 2-DE gel performed with 3–11 NL IPG strip and 9%–16% acrylamide/bisacrylamide.

Figure 4.

Preparative 2-DE Gel (700 μg). Spot Map of the proteins identified. The characterization of the spots identified is shown in Table 1.

Figure 5.

Characterization of the spinal cord proteins identified. A) The functional grouping of all the proteins identified using 2-DE and MALDI-TOF/TOF together with LC-MS/MS are presented. B) Isoelectric point distribution and subcellular localization of the proteins identified. C) Additional classification of the proteins with recognized function in spinal cord.