Neurogenesis and major depression: implications from proteomic analyses of hippocampal proteins in a rat depression model

Neuroscience Letters 416 (2007) 252–256

Neurogenesis and major depression: implications from proteomic analyses of hippocampal proteins in a rat depression model Jun Mu a , Peng Xie a,∗ , Ze-Song Yang d , De-Lan Yang b , Fa-Jin Lv c , Tian-You Luo c , Yong Li a a Department of Neurology, The First Affiliated Hospital of Chongqing Medical University, The Key Laboratory of Diagnostic Medicine Designated by the Ministry of Education, Chongqing 400016, China b Department of Psychiatry, The first affiliated Hospital of Chongqing Medical University, Chongqing 400016, China c MRI Center, Chongqing Medical University, Chongqing 400016, China d Department of Hematology, The first affiliated Hospital of Chongqing Medical University, Chongqing 400016, China

Received 25 July 2006; received in revised form 28 December 2006; accepted 20 January 2007

Abstract Major depression is one of the most disabling disorders. Yet, the pathogenesis of this mental disorder is poorly understood. Hippocampus is generally believed to be associated with pathogenesis of depression. In this study, we adopted a proteomic approach to examine possible alterations of protein expression in the hippocampus of a rat depression model. Our results suggest that neurogenesis in hippocampus may play an important role in the pathogenesis of major depression. © 2007 Elsevier Ireland Ltd. All rights reserved. Keywords: Proteomics; Hippocampus; Major depression disorders; MALDI-TOF-MS

Although major depression affects more than 120 million of children and adults each year worldwide [19], no objective evaluation markers are currently available for both diagnosis and prognosis. The pathogenesis of this disease is complicated and involves multiple genetic, psychological, and environmental factors. Although recent studies have identified several candidate genes associated with major depression [13], the roles of the resultant proteins remain largely unknown. Increasing evidences indicate that hippocampus plays an important role in the pathogenesis of major depression disorders [2]. A decrease of hippocampal volume is detected by MRI in both depressive patients and in postmortem studies [15]. In animal model, elevated glucocorticoid levels associated with Major Depression Disorders (MDD) negatively regulate neurogenesis, reduce the levels of neurotrophins and cause excitotoxic damage in the hippocampus [16]. Importantly, chronic antidepressant treatments have been shown to increase adult hippocampal neurogenesis [16], and disrupting antidepressantinduced neurogenesis by x-irradiation of mice hippocampus

∗

Corresponding author. Tel.: +86 23 68485490; fax: +86 23 68485111. E-mail address: Xie peng [email protected] (P. Xie).

0304-3940/$ – see front matter © 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2007.01.067

blocks behavioral responses to treatments of antidepressants [16]. These findings suggest that the behavioral effects of chronic antidepressants may be mediated by the stimulation of neurogenesis in the hippocampus. Interestingly, Khawaja et al. have recently shown that antidepressant venlafaxine or fluoxetine regulates the expression of 33 proteins, including proteins associated with neurogenesis in normal rat hippocampus [11]. We therefore hypothesized that proteins identified by Khawaja et al. might play roles in MDD pathogenesis. To identify protein expression change linked with MDD pathogenesis, in the present study we set up to examine possible alteration of protein expression patterns in the hippocampus of depressed rats without any antidepressant medication. 10 adult male Sprague–Dawley rats from the Chongqing Medical University’s animal center (5–7 weeks old, weighing 250–300 g) were housed with ad lib access to food and water, in groups of five a cage, and maintained on a 12-h light/dark cycle (lights on at 07:00 a.m.), at 22 ◦ C with low humidity. After 1 week of habituation, rats were randomly assigned to one of two groups: chronic stress or no stress. Rats in the chronic stress group were isolated housed, each one in a single cage, subjected to different kinds of stressors each day, known as the chronic unpredictable stress protocol (refer to Willer’s

J. Mu et al. / Neuroscience Letters 416 (2007) 252–256

method) [18]: food deprivation (24 h), water deprivation (24 h), tail clamping 1 min, 45 ◦ C atmosphere 5 min, inversion of the light/dark cycle (7:00 p.m. lights on), horizontal vibration 30 min (160 rpm), ice water swimming 5 min (4 ◦ C). On the whole, one of these seven stressors was randomly applied daily for total 3 weeks before the behavioral testing. All rats were tested for sucrose consumption and given 1% (w/v) sucrose solution in water for 24 h in place of their regular drinking water in their home cages, following 24 h period of water deprivation on the 1st, 8th, 15th, and the 22nd day, respectively. The bottles were weighed prior to being given to the rats and at the conclusion of the test (7:00 a.m. the next morning). A large plywood box (80 cm × 80 cm × 40 cm) painted brown with a black grid (16 cm × 16 cm) on the floor was used for exploration testing. The rat was placed into a corner of the box and allowed to explore freely for 3 min. The box was thoroughly cleaned between subjects. All test sessions were videotaped in a soundproof room. The number of rears (animal on hind limbs) and crossing (grid boxes entered) were recorded. The protein extraction was performed essentially as reported [20,7]. The rats were decapitated under deep anesthesia and hippocampi were dissected rapidly from the whole brain. Samples from each group were pooled and stored at −85 ◦ C. Hippocampal tissue was powderised in liquid nitrogen and suspended in 2 ml of acetone solution containing 0.2% (w/v) DTT and 10% (w/v) TCA. After being homogenized, the suspension was left at −20 ◦ C to stay overnight, before it was centrifuged at 35,000 × g for 30 min, at 4 ◦ C. The supernatant was decanted. The pellet was again suspended in 2 ml of pre-cooled acetone solution containing 0.2% (w/v) DTT, left at −20 ◦ C for 1 h and centrifuged at 35,000 × g for 30 min, at 4 ◦ C. The supernatant was decanted and pellet dried in the fuming cupboard. Each sample was dissolved in 2 ml of 2-D lysis buffer (7 M urea, 2 M thiourea, 4% CHAPS, 40 mM Tris, 65 mM DTT, 0.3 mg/mlEDTA, 35 ug/mlPMSF, 0.7 ug/mlPepstatin, 0.5 ug/mlleupeptin, 0.5, v/v CA) and centrifuged at 40,000 × g for 60 min at 15 ◦ C. The protein content of the supernatant was determined by the Coomassie blue method [1]. First dimensional isoelectric focusing (IEF) was carried out on 18-cm immobilized pH gradient (IPG) strips (pH 3–10, Amersham Pharmacia Biotech) using an IPGphor unit (Amersham Pharmacia Biotech). Each strip was rehydrated with sample lysate in a final volume of 350 ␮l of IEF solution containing 7 M urea, 2 M thiourea, 4% CHAPS, 40 mM DTT, 0.5% IPG buffer, and 0.002% (w/v) bromophenol blue (Amersham Pharmacia Biotech). IEF was then carried out using the following conditions: focusing started at (1) 30 V, 360 Vh; kept constant at (2) 200 V, 200 Vh (3) 500 V, 500 Vh; (4) 1000 V, 1000 Vh; the voltage was gradually ascended to 8000 V, with a total focusing 45000 Vh. Strips were then subjected to a two-step equilibration, in 10 ml balanced solution I (50 mM Tris–HCl, pH 8.8; 2% SDS; 30% glycerol; 6 M urea; 100 mg DTT) and 10 ml balanced solution II (50 mM Tris–HCl, pH 8.8; 6 M urea; 30% glycerol; 2% SDS; bromophenol blue; 250 mg iodoacetamide). And the strips

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were pendulated on the shaker for 15 min before proceeding to SDS-PAGE. Separation in the second dimension was carried out on 10% SDS polyacrylamide gels (methylene-acrylamide; acrylamide; 1.5 M Tris–HCl; 0.4% SDS, pH 8.8; TEMED; 10% APS; 180 mm × 200 mm × 1.0 mm), using an Ettan DALT II 2 D electrophoresis unit (Amersham Pharmacia Biotech), at a constant voltage of 5 W/gel at 25 ◦ C for 30 min, and then 25 W/gel, until the bromophenol blue was seen near the bottom, with a maximum of 180 W. After protein fixation for 1 h in 50% methanol and 10% acetic acid, the gels were stained with colloidal Coomassie blue R250 (Sigma, USA) for 30 min. Excess of dye was washed out from the gels with distilled water. Silver staining of the gels was carried out using a silver stain kit (Genomic Solutions). All steps were carried out at room temperature, gently agitating the trays on a rotary shaker at low speed. The gel was analysed using the Image Master 2 D Elite software (version 5.0, Amersham Bioscience) and determined as a percentage volume of total protein in the area of interest. Stained gels were scanned with Image Scanner and Labscan software (Amersham Pharmacia Biotech). Protein spots were outlined (first automatically and then manually) and quantified using the ImageMaster v.5.0 software (Amersham Pharmacia Biotech). The software calculated the relative spot volume of the proteins compared to the total protein in the gel. The ImageMaster software was used to crossmatch (“synchronize”) and identify protein spots between control gels and depressive gels that were different from control in integrated intensity by at least a factor of 1.5. This population of protein spots was then reanalyzed to determine the subset of spots that crossmatched within the depressive gels subdivided as either up- or downregulated. These Coomassie brilliant blue-stained protein spots were excised automatically with the ProPic system for in-gel trypsin digestion. After Zip-tip clean-up, mass analyses of the individual samples were carried out using a matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) (Bruker). Peptide mixture was applied with an equal volume of matrix. For each sample, spectra were acquired in the delayed extraction and reflector mode and an average of 150 spectra that passed the accepted criterion of peak intensity was automatically selected and saved. Measured peptide masses were excluded if their masses corresponded to trypsin autodigestion products. Peptide matching and protein searches were carried out automatically in SWISS-PROT and NCBI nonredundant databases. Peptide masses were compared to theoretical values for all available proteins from all species. Monoisotopic masses were used, and a mass tolerance of 0.1 Da was allowed. Unmatched peptides or miscleavage sites were not considered. All mass searches were carried out using a mass window between 1 and 100 kDa and included rat sequences. Search parameters allowed for Nterminal formylation and oxidation of methionine. Criteria for positive identification of proteins were set as follows: (1) the MS match consisted of a minimum of four peptides; (2) the matched peptides covered at least 15% of the whole protein sequence; (3) 50 ppm or better mass accuracy; (4) the molecular weight and pI

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J. Mu et al. / Neuroscience Letters 416 (2007) 252–256

Table 1 Sucrose consumption in chronic stress rats (ml, mean ± S.D.) Group Stress Non-stress

n

Day 1

Day 8

Day 15

Day 22

5 5

75.64 ± 2.41 74.57 ± 5.43

73.24 ± 3.56 75.21 ± 2.15

68.33 ± 76.32 ± 4.17

2.89a

65.65 ± 3.86a 76.51 ± 1.45

n, number of rats in each group. a P < 0.01.

of identified proteins matched estimated values obtained from image analysis; and (5) the protein exhibited a significant difference in the number of matched peptides to the next potential hit. The measures of stressed and non-stressed control rats were compared with an independent t-test, using SPSS 11.0 software, in the open field test, on preference of sucrose solution (sucrose consumed). To determine whether the experimental animals show the major depression symptom anhedonia, we measured the sucrose consumption levels in both stressed and non-stressed rats. As shown in Table 1, compared with the non-stressed group the sucrose consumption of rats under chronic unpredictable stress was significantly decreased at 15 days after treatment. The decrease of sucrose consumption was more obvious at 22 days after stress treatment, suggesting a time-dependent reduction of anhedonia in stressed animals. We performed open field test to examine the motivation level of the experimental rats. Compared with the non-stress group, animals subjected to chronic unpredictable stress for 21 days showed a significant decrease in crossings and rears of open field activity (Table 2). These data implicate that stressed animals have reduced interest in moving. The hippocampal extracts from stressed or stress-free rats were pooled and subjected to 2-D gel (2-DE) analysis. The number of spots found in the gels from the stressed group was 1594, 1624, 1609, respectively, with an average of 1609.00 ± 15.0. For stress-free group, the number was 1571, 1628, 1648, respectively, with an average of 1615.67 ± 29.78. The matching rate between the groups was 72%. The figures shown below are representative 2-DE maps obtained from each group (Fig. 1). There were 27 spots showing differential protein expression, ranging from pH 4.0 to 9.7, 25–70 kDa. The protein expression was upregulated in four of the 27 spots (spot 15–18). The values were determined by the image analyzing. Table 3 shows a list of the identified protein spots. The circles delineate the regions containing spots that showed differential expression between the stressed group (A) and the non-stress group (B). The spots were excised, trypsinized, and analyzed by MALDI-TOF-MS. The proteins

were identified subsequently by their tryptic peptide mass fingerprints. To examine possible alterations of protein expression in the depressive hippocampus, in the present study we adopted a rat depression model. In agreement with the recent studies by Magdalena and colleagues, chronic mild stress (CMS) significantly decreased sucrose consumption in the rat hippocampal [9]. Mammalian hippocampus plays critical roles in memory, learning, neural plasticity and emotion. Substantial evidences have demonstrated that new neurons are generated in the den-

Table 2 Open field activity in chronic stressed rats (score, mean ± S.D.) Group Stress Non-stress

n

Crossing

5 5

21.27 ± 35.70 ± 7.87

n, number of rats in each group. a P < 0.01.

Rears 5.89a

7.58 ± 2.65a 11.56 ± 2.45 Fig. 1. Silver-stained 2-DE maps of proteins from rat hippocampus.

J. Mu et al. / Neuroscience Letters 416 (2007) 252–256

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Table 3 Identification of rat hippocampal proteins with altered expression levels after chronic unpredictable stress Spot number

Protein

Neurogenic involvement S1 gi|55855 S2 gi|78000203 S5 gi|266566 S19

gi|6754632

S4 S7 S8

gi|6680986 gi|60678254 gi|13591997

S9 S15 S10

gi|38541404 gi|19705578 gi|34856405

S21 S18 S16 S22

gi|16758446 gi|206681 gi|18266700 gi|62638424

Signal transduction mediation S17 gi|13928824

Description

pI

Calreticulin precursor Tropomyosin 1, alpha isoform i Dual specificity mitogen-activated protein kinase kinase 1 Mitogen activated protein kinase 1 oxidative metabolism Cytochrome c oxidase, subunit Va Creatine kinase Methylmalonate semialdehyde dehydrogenase gene Aconitase 2, mitochondrial Vacuolar H + ATPase B2 Predicted: similar to Coiled-coil-helix-coiled-coil-helix domain containing 6 Isocitrate dehydrogenase 3 (NAD+) alpha Rieske Fe-S protein precursor Transcription heterogeneous nuclear ribonucleoprotein H1 Predicted: similar to RIKEN cDNA 2900041A09

4.05 4.73 6.4

Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein

tate gyrus of human hippocampus [5] and that the production and survival of new hippocampal neurons can be regulated by hormones and experience [6]. Interestingly, recent study shows that decreased neurogenesis in the hippocampus may play an important role in precipitating episodes of depression [4]. To further understand the involvement of neurogenesis in the pathogenesis of depression, Khawaja, et al. has examined the proteomic profiling of hippocampal cytosolic extracts from adult rats treated with antidepressant venlafaxine or fluoxetine. They found that 33 protein spots were modulated by both drug compared to controls. The functions of these proteins were found to be associated with: (1) neurogenesis, including insulin like growth factor 1 (IGF-1) and glia maturation factor 9 (GMF-beta); (2) outgrowth/maintenance of neuronal processes, including hippocampal cholinergic neurostimulating peptide (HCNP) and PCTAIRE-3; (3) neural regeneration/axonal guidance collapsin response mediator protein (CRMP-2); (4) neuronal vesicular cell trafficking and synaptic plasticity, including Ras-related protein 4a (Rab4a), Ras-related protein 1b (Rab1b), heat shock protein 10 (HSP10); (5) neurosteroidogenic protein such as hydroxysteroid sulfotransferase A; and (6) anti-apoptotic signaling, including dimethylargininase-1 lN,N-dimethylarginine dimethylaminohydrolase-1 (DDAH-1), pyruvate dehydrogenase-E1 (PDH-E1) and antioxidant protein2 (AOP-2) pathway-mediated regulatory events [11]. Johnston and colleagues examined protein expression in human frontal cortex of 89 postmortem individuals with schizophrenia, bipolar disorder, major depressive disorder, and non-psychiatric controls. They identified eight protein species using the 2DE analysis. Of the eight proteins, six showed

M (kDa)

Peptides matched (%)

Value in control

Value in depression

62 34 755

12/26(46) 8/16(50) 9/24(38)

78086 168293 53724

64607 104703 39901

6.92

41

6/22(27)

77436

49866

5.23 8.07 8.09

16 49 66

6/17(35) 10/28(36) 8/11(73)

23895 449272 45641

4119992 416427 34439

7.87 5.88 8.51

92 64 40

11/21(52) 10/21(48) 8/17(47)

311685 96912 31487

249442 112139 15744

5.65 8.31 6.16 9.72

45 28 61 29

6/15(40) 6/9(67) 6/20(30) 6/9(67)

122208 7111 57901 245378

103052 17364 74079 136362

4.56

34

6/11(55)

74883

92770

decreased levels compared with the non-psychiatric controls for one or more diseases. Four of these are forms of glial fibrillary acidic protein (GFAP), one is dihydropyrimidinase-related protein 2, and the sixth is ubiquinone cytochrome c reductase core protein 1. Two spots, the carbonic anhydrase 1 and fructose biphosphate aldolase C, show increase in one or more diseases compared to controls [10]. No data is available from hippocampus of the postmortem tissues. Most of the proteins identified in these postmortem tissues are involved in the process of energy metabolism. These results are similar to our findings in the present study, in which 6 out of 8 oxidative metabolism-involved proteins decreased in rat hippocampus. These data suggest that altered oxidation metabolism may be involved in the frontal cortex of in major depression. However, impaired energy metabolism might be common phenomenon in psychiatric disorders. In the present study, we used a proteomic approach to examine hippocampal protein expression in a depressed rat model. We identified 27 proteins that might play roles in the pathogenesis of MDD. The identified proteins fall into four broadly defined functional categories: (i) neurogenesis, (ii) oxidative metabolism, (iii) transcription and (iv) signal transduction. One of these proteins is calreticulin precursor, a developmentally regulated protein, primarily participates in protein synthesis or maintenance [17]. This protein also involved in tumor development and progression [8]. The other example of the identified proteins is mitogen-activated protein kinase kinase (MAPK). MAPK plays a pivotal role in the development of the nervous system by mediating both neurogenesis and neuronal differentiation. Evidence indicates that MAPK is involved in axonal

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transport and the organization of neurofilaments (NFs) in axonal neuritis [3]. Abnormally sustained activation of MAPK is shown to cause neurodegeneration. In familial amyloidotic polyneuropathy, MAPK was down-regulated [12]. Tropomyosin 1 is another protein identified in the present study. The expression of tropomyosin 1 is found to down-regulated in aging as well as in neurodegenerative disorders [14]. The rat depression model under the UCMS procedure is used to simulate the major manifestations of human major depression. Although our study suggests that alterations in hippocampal neurogenesis might play a role in mediating the pathogenesis of depression, we do not exclude the other possibilities. For example, increased glucocorticoids in major depression might exert direct effects on cerebral cortex, hippocampus and other subcortical areas, such as amygdala. Serotonin neurotransmission might be also altered in brain stem, subcortical sites and cortex. All of these changes, acting in concert, may give rise to the complex syndrome of depression. Acknowledgements This work is supported by a grant from National Nature Science Foundation of China (No.30570657). We thank professor Liu shao-jun in the Military Medical University for his guidance and Yang Shu-guang for his assistance on 2-D image analysis. We are grateful to Mr. Derek Porter for editing the manuscript. References [1] M.M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem. 72 (1976) 248–254. [2] S. Campbell, G. Macqueen, The role of the hippocampus in the pathophysiology of major depression, J. Psychiatry Neurosci. 29 (6) (2004) 417–426. [3] W.K. Chan, A. Dickerson, D. Ortiz, A.F. Pimenta, C.M. Moran, J. Motil, S.J. Snyder, K. Malik, H.C. Pant, T.B. Shea, Mitogen-activated protein kinase regulates neurofilament axonal transport, J. Cell. Sci. 15(117, Pt 20) (2004) 4629–4642. [4] R.S. Duman, J. Malberg, S. Nakagawa, C. D’Sa, Neuronal plasticity and survival in mood disorders, Biol. Psychiatry 48 (8) (2001) 732–739. [5] P.S. Eriksson, E. Perfilieva, T. Bjork-Eriksson, A.M. Alborn, C. Nordborg, D.A. Peterson, F.H. Gage, Neurogenesis in the adult human hippocampus, Nat. Med. 4 (11) (1998) 1313–1317. [6] E. Gould, P. Tanapat, T. Rydel, N. Hastings, Regulation of hippocampal neurogenesis in adulthood, Biol. Psychiatry 48 (8) (2000) 713–714.

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