A gel-based proteomic comparison of human cerebrospinal fluid between inflicted and non-inflicted pediatric traumatic brain injury

Abstract

Traumatic brain injury (TBI) is the most common cause of traumatic death in infancy, and inflicted TBI (iTBI) is the predominant cause. Like other central nervous system pathologies, TBI changes the composition of cerebrospinal fluid (CSF), which may represent a unique clinical window on brain pathophysiology. Proteomic analysis, including two-dimensional (2-D) difference in gel electrophoresis (DIGE) combined with mass spectrometry (MS), was used to compare the CSF protein profile of two pooled samples from pediatric iTBI (n = 13) and non-inflicted TBI (nTBI; n = 13) patients with severe injury. CSF proteins from iTBI and nTBI were fluorescently labeled in triplicate using different fluorescent Cy dyes and separated by 2-D gel electrophoresis. Approximately 250 protein spots were found in CSF, with 90% between-gel reproducibility of the 2-D gel. Following in-gel digestion, the tryptic peptides were analyzed by MS for protein identification. The acute phase reactant, haptoglobin (HP) isoforms, showed an approximate fourfold increase in nTBI versus iTBI. In contrast, the levels of prostaglandin D(2) synthase (PGDS) and cystatin C (CC) were 12-fold and sevenfold higher in iTBI versus nTBI, respectively. The changes of HP, PGDS, and CC were confirmed by Western blot. These initial results with conventional gel-based proteomics show new protein changes that may ultimately help to understand pathophysiological differences between iTBI and nTBI.

FIG. 1

A representative composite difference in gel electrophoresis (DIGE) of inflicted traumatic brain injury (iTBI) and noninflicted traumatic brain injury (nTBI). Cerebrospinal fluid (CSF) samples from iTBI and nTBI were labeled with Cy5 (red) and Cy3 (green), respectively. Yellow spots mean that the protein levels were not different in the two samples. Red spots mean that the protein levels were higher in iTBI, and the green spots mean that the protein levels were higher in nTBI. HP, haptoglobin; PGDS, prostaglandin D₂ synthase; CC, cystatin C. All the proteins were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS).

FIG. 2

Tandem mass spectrometry identification of haptoglobin (HP; A), prostaglandin D₂ synthase (PGDS; B), and cystatin C (CC; C). Analysis of the tryptic peptides with electrospray quadrupole-time of flight (Q-TOF), coupled with nanoflow capillary high-pressure liquid chromatography (HPLC). The tandem mass spectrometry fragmentation spectra (obtained after trypsin digestion) of VGYVSGWGR for HP, GPGEDFR for PGDS, and GGPMDASVEEEGVR for CC are shown. There were 24, 6, and 13 matched peptides from the liquid chromatography-tandem mass spectrometry (LC-MS/MS) for HP, PGDS, and CC, with total scores of 671, 208, and 332, respectively. The resultant MS/MS data were searched using MASCOT.

FIG. 3

Immunoblotting analyses for haptoglobin (HP; A), prostaglandin D₂ synthase (PGDS; B), and cystatin C (CC; C) in pooled inflicted traumatic brain injury (iTBI) and non-inflicted traumatic brain injury (nTBI) cerebrospinal fluid (CSF), and CC (D) in the 26 individual CSF from 13 iTBI and 13 nTBI patients. In parts A-C, both sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) image and quantification of the bands using BioImage were shown; in part D, only the quantification analysis was shown.