Project PXD002783

PRIDE Assigned Tags:
Biomedical Dataset
Dataset Belongs to:
Human Proteome Project



Protein Profile Changes in the Frontotemporal Lobes in Human Severe Traumatic Brain Injury


Severe traumatic brain injury (sTBI) is a serious public health issue with high morbidity and mortality rates. Previous proteomic studies on sTBI have mainly focused on human cerebrospinal fluid and serum, as well as on brain protein changes in murine models. However, human proteomic data in sTBI brain is still needed. We used proteomics and bioinformatics strategies to investigate variations in protein expression in human brains after sTBI, using samples from the Department of Neurosurgery, Affiliated Hospital of Hebei University (Hebei, P.R. China). Our proteomics data identified 4031 proteins, of which 162 proteins were overexpressed and 5 proteins were downregulated. The biological pathways that showed significant changes in protein expression according to bioinformatics analysis were glial cell differentiation, complement activation, apolipoprotein catalysis in statin pathway, and the blood coagulation cascade. Western blot verification of protein changes in a subset of the available tissue samples showed results that were consistent with the proteomics data. This study is one of the first to investigate the whole proteome of human sTBI brains, and provides a characteristic signature and overall landscape of the sTBI brain proteome.

Sample Processing Protocol

The protocol of the study was approved by the ethics committee of the Institute of Basic Medical Sciences of the Chinese Academy of Medical Sciences, and the samples were collected on the condition that patients and their close relatives provided written informed consent for associated surgery or succumbed to sTBI. The study included all sTBI patients with radiological evidence of a head injury admitted to the Department of Neurosurgery, Affiliated Hospital of Hebei University (Hebei, P.R. China) during the period of one year of the study. Including criteria were: (1) age >18 years with a clear history of TBI; (2) no evidence suggestive of any severe systematic complications or combined injuries; (3) no malignant tumors or other neurological/psychiatric/immunological disorders, alcohol consumption, diabetes, hypertension, infections, previous TBI or neurosurgery; (4) no surgery or trauma experienced 1 month prior to admission; (5) pre-operative GCS ≤ 8; and (6) no apparent intracranial infection during hospitalization. The demographics and clinical common data elements of the patients were collected following the Transforming Research and Clinical Knowledge in TBI (TBI-TRACK) guidelines, including age, gender, site of TBI, GCS at the time of admission, neuroimaging findings (degree of effacement of basal cisterns and mid-line shift), interval time between sTBI and surgical dissection, and post-mortem interval in autopsied cases. All necessary details in this study are shown in Table S1. It should be noted that, according to its customary usage, a GCS score (range: 3-15) of 3 was recognized as the most severe agonal state. The GCS value we adopted was the admission GCS of each patient; as GCS scores do not follow a normal distribution, studies that use mean GCS values and standard statistical analysis can be misleading. The craniotomy samples from TBI brain tissues (n = 12) were collected by the Department of Neurosurgery, Affiliated Hospital of Hebei University (Hebei, P.R. China; details in Table S1). Diagnoses of sTBI were made from patients’ computed-tomography (CT) scans based on standard radiological guidelines. The interval between TBI and neurosurgery and specimen collection was ≤56 hours. A 1-cm3 piece of tissue was collected during surgery at the injured site of each TBI brain and was stored at -80°C for proteomic studies. None of the patients who provided samples for the study had any evidence suggestive of any severe systematic complications or combined injuries, malignant tumors or other neurological/psychiatric/immunological disorders, and had no history of alcohol consumption, diabetes, hypertension, infections, previous TBI or neurosurgery (Table S1). The standard operating procedure of craniotomy was followed in all the surgical dissection of the samples. Control tissues were obtained from eight normal postmortem brains, which were archived in the Chinese Human Brain Bank (Beijing, P.R. China). All donors were healthy, without evidence of any severe systematic complications or combined injuries, malignant tumors or other neurological/psychiatric/immunological disorders, or evidence of alcohol consumption, diabetes, hypertension, infections, previous TBI or neurosurgery. The brain tissues were prepared under modified standard autopsy procedures. A 1-cm3 piece of tissue was collected at the site that was macroscopically similar to the TBI tissues, and was stored at -80°C for proteomic studies. Uniform protocols for the autopsy, tissue preparation, and other processing procedures were applied to all the samples.

Data Processing Protocol

The TBI tissue (experimental group) and the normal postmortem brain tissue (control group) were homogenized with ice-cold homogenization buffer, consisting of 8 M urea in PBS, pH 8.0, 1× protease inhibitor cocktail, and 1 mM phenylmethanesulfonyl fluoride. Homogenates were centrifuged at 12,000 rpm at 4°C for 15 min to remove cell debris, and the clarified supernatant was transferred to a new 1.5-ml tube. Following estimation of protein concentration with a Nanodrop 2000 (Thermo Scientific), 12.5 μg of protein from each sample within same group was pooled and protein normalization confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on a 10% polyacrylamide gel. TMT labeling At this stage, the extracts were treated with 10 mM dithiothreitol for 1 h at 55°C, and then with 25 mM iodoacetamide for 30 min in the dark at room temperature. Extracts were then digested with 1:100 w/w endopeptidase Lys-C overnight at 37°C. The extracts were diluted with PBS (pH 8.0) to give a final urea concentration of 1.0 M, and were then digested with 1:50 w/w trypsin overnight. The next morning, the extracts were first acidified with 100% formic acid, and then desalted with a reverse-phase column (Oasis HLB; Waters, MC, USA). Extracts were dried with a vacuum concentrator and finally dissolved in 200 mM triethylammonium bicarbonate buffer for labeling with TMT reagents. TMT labeling of peptides was performed according to the manufacturer’s protocol. Briefly, TMT reagent (0.8 mg TMT dissolved in 40 µl 99.9% acetonitrile) was added to the extract and allowed to react for 1 h at room temperature before 5 min termination by 5 µl of 5% hydroxylamine. Different TMT labels were used to label different groups: TMT-131 was used to label the TBI group and TMT-130 was used to label the control group. All of the labeled extracts from the three pools were mixed, desalted, dried as previously described, and dissolved in 100 μl of 0.1% formic acid. High performance liquid chromatography (HPLC) separation Fractionation of the labeled peptides was performed as follows. First, TMT labeled peptides (100 μl in 0.1% formic acid) were transferred to MS tubes for HPLC analysis (UltiMate 3000 UHPLC, Thermo Scientific). An Xbridge BEH300 C18 column was used (1 ml/min, 4.6 × 250 mm, 2.5 µm, Waters). Column temperature was maintained at 45°C with a flow rate of 1.0 ml/min, and detection was performed by UV absorbance at 214 nm. Fractions were collected every 1.5 min in 50 tubes before dissolving in 20 µl 0.1% formic acid for further liquid chromatography (LC)-MS/MS analysis. Peptide analysis by LC-MS/MS LC-MS/MS analysis was conducted on a Q Exactive mass spectrometer (Thermo Scientific). The labeled digestion fractions were separated using a 120 min gradient elution at a flow rate of 0.30 l/min. The UltiMate 3000 RSLCnano System (Thermo Scientific) was interfaced with the Thermo Q Exactive Benchtop mass spectrometer (Thermo Scientific). The analytical column was a silica capillary column (75 µm ID, 150 mm length; Upchurch, Oak Harbor, WA) packed with C18 resin (300 Å, 5 µm; Varian, Lexington, MA). To set the Q Exactive mass spectrometer in data-dependent acquisition mode, we utilized Xcalibur 2.1.2 software for manipulation. A single full-scan mass spectrum in Orbitrap (400-1, 800 m/z, 60,000 resolution) was followed by 10 data-dependent MS/MS scans at 27% normalized collision energy (higher-energy C-trap dissociation). Proteome Discoverer 1.3 software (Thermo Scientific) was used for the human FASTA database from UniProt (released 12/01/2014), on which the MS/MS spectra of each LC-MS/MS run was analyzed. The criteria for searching was set according to the software recommendations with the following modifications: full tryptic specificity with no more than two missed cleavages permitted; set carbamidomethylation (C, +57.021 Da) and MT plex (lysine, K and any N-terminal) as static modifications; and oxidation (methionine, M) as a dynamic modification. The fragment ion mass tolerance was corrected to 20 mmu (all MS2 spectra) and the precursor ion mass tolerances were set at 20 ppm (all MS in an Orbitrap mass analyzer). Based on the reporter ion intensities per peptide, protein levels were quantified relative to the manufacturer’s instructions. Quantitative precision was presented with protein ratio variability. The ratio relationships of the extracts from each group were predetermined as follows: TMT-131 for the sTBI group and TMT-130 for the control group. The upregulation and downregulation thresholds were set at 1.5 and 0.67, respectively.


Wei Ge, National Key Laboratory of Medical Molecular Biology & Department of Immunology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences
Wei Ge, National Key Laboratory of Medical Molecular Biology and Department of Immunology; Institute of Basic Medical Sciences; Chinese Academy of Medical Sciences ( lab head )

Submission Date


Publication Date





Q Exactive


Not available

Experiment Type

Shotgun proteomics


    Xu B, Tian R, Wang X, Zhan S, Wang R, Guo Y, Ge W. Protein profile changes in the frontotemporal lobes in human severe traumatic brain injury. Brain Res. 2016 Apr 8. pii: S0006-8993(16)30199-8 PubMed: 27067185