Quantitative Proteomic Analysis of Membrane Proteins Involved in

Article pubs.acs.org/jpr

Quantitative Proteomic Analysis of Membrane Proteins Involved in Astroglial Differentiation of Neural Stem Cells by SILAC Labeling Coupled with LC−MS/MS Rui Cao,*,†,§ Ke Chen,†,§ Qin Song,† Yi Zang,‡ Jia Li,‡ Xianchun Wang,† Ping Chen,*,† and Songping Liang*,† †

Key Laboratory of Protein Chemistry and Developmental Biology of Education Committee, College of Life Sciences, Hunan Normal University, Changsha 410081, P.R. China ‡ National Center for Drug Screening, Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 201203, P.R. China S Supporting Information *

ABSTRACT: Membrane proteins play a critical role in the process of neural stem cell selfrenewal and differentiation. Here, we apply the SILAC (stable isotope labeling by amino acids in cell culture) approach to quantitatively compare the membrane proteome of the selfrenewing and the astroglial differentiating cells. High-resolution analysis on a linear ion trapOrbitrap instrument (LTQ-Orbitrap) at sub-ppm mass accuracy resulted in confident identification and quantitation of more than 700 distinct membrane proteins during the astroglial differentiation. Of the 735 quantified proteins, seven cell surface proteins display significantly higher expression levels in the undifferentiated state membrane compared to astroglial differentiating membrane. One cell surface protein transferrin receptor protein 1 may serve as a new candidate for NSCs surface markers. Functional clustering of differentially expressed proteins by Ingenuity Pathway Analysis revealed that most of overexpressed membrane proteins in the astroglial differentiation neural stem cells are involved in cellular growth, nervous system development, and energy metabolic pathway. Taken together, this study increases our understanding of the underlying mechanisms that modulate complex biological processes of neural stem cell proliferation and differentiation. KEYWORDS: neural stem cell, astroglial differentiation, membrane protein, SILAC, tandem mass spectrometry, energy metabolic pathway

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INTRODUCTION Neural stem cells (NSC) are multipotent stem cells found in several selected regions of the CNS such as the hippocampus, cortex, human subependymal zone, and subventricular zone of the brain. These cells are capable of self-renewal and differentiating into neurons, astrocytes, and oligodendrocytes.1 Because of their differentiation potential, neural stem cells are ideal candidates for use in regenerative medicine and treatment of disease.2 Despite extensive research elucidating the processes of self-renewal and lineage-specific differentiation, much remains to be learned about the mechanism underlying the self-renewal and differentiation of NSC before their clinical application.3−5 NSCs can differentiate into various cell types in response to growth factors such as Leukemia inhibitory factor (LIF),6 ciliary neurotrophic factor (CNTF),7 and extracellular chemical factors such as all trans-retinoic acid8 and 5-aminoimidazole4-carboxamide 1-β-D-ribofuranoside (AICAR).9 Upon those growth factors and chemical factors stimulus, membrane proteins play an important biological role during the differentiation processes such as recognize extracellular stimuli and © 2011 American Chemical Society

lead to activation of intracellular signaling. Thus, identification and quantification of the membrane proteins of NSCs during differentiation is an important issue in elucidating the underlying mechanisms. Over past several years, to gain deeper insight into the regulatory networks controlling self-renewal and differentiation of neural stem cell, several comparative proteomic studies have been performed.10−13 However, to our knowledge, little is known about the membrane protein changes of NSCs during differentiation. Foster et al. used a label free method to profile the differential expression of membrane proteins of the human mesenchymal stem cell (MSC) line undergoing osteoblast differentiation.14 More recently, stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative technology was used to compare the membrane proteome of self-renewal and differentiating embryonic stem cells.15 To facilitate the SILAC labeling and further in-depth biological function analysis, here we used a well-characterized Received: July 18, 2011 Published: December 12, 2011 829

dx.doi.org/10.1021/pr200677z | J. Proteome Res. 2012, 11, 829−838

Journal of Proteome Research

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neural stem cell line C17.29,16−18 in our primary proteomic study. C17.2, a neural precursor cell line originally derived from the external germinal layer of neonatal mouse cerebellum, have the ability to self-renew and to differentiate into neurons, astrocytes, and oligodendrocytes, both in vitro and in vivo.9,19−21 In this study, we compared the membrane protein profile of astroglial differentiation NSCs with those of undifferentiated cells by using SILAC-based quantitative proteomics. A total of 1129 proteins were identified, and of them, 728 were membrane proteins. More importantly, our study revealed 15 significantly altered cell surface proteins during differentiation. Seven cell surface proteins displayed 2-fold higher levels in the self-renewal cells, while the remaining eight displayed more abundantly in the differentiating cells. We also systematically studied the differentially expressed membrane proteins in the astroglial differentiation of NSCs. The data indicates that many alterations of proteins were specific to defined protein−protein interaction networks and metabolic pathways in the processes of neural stem cell proliferation and differentiation.

Thermo Fisher Scientific, San Jose, CA, USA), which was equipped with an ESI nanospray source. The digested peptides were injected into an Easy LC system (Proxeon, Odense, Denmark) with a precolumn (2 cm, ID 100 μm, 5 μm, C18). Peptides were eluted from a C18 column (10 cm, ID 75 μm, 3 μm, C18) with a linear gradient of 5−30% solvent B (99.9% acetonitrile with 0.1% formic acid) over 65 min with a constant flow of 200 nL/min. The mass spectrometer was set so that each full MS scan (m/z 350−1800) in profile mode was acquired in the orbitrap with a resolution of 100 000 at m/z 400, followed by 5 MS/MS scans in the ion trap on 5 most intense ions from a MS spectrum with dynamic exclusion setting: a repeat count of 2, a repeat duration of 30 s, and an exclusion duration of 90 s. The data was acquired by Xcalibur software 2.1 (Thermo Fisher Scientific, San Jose, CA, USA). Data Analysis

Mouse immortalized neural stem cell line C17.2 was originally described by Snyder et al.16,17 The C17.2 cells were maintained in 75 cm2 culture flasks in DMEM supplemented with 10% fetal bovine serum (FBS) in a humidified incubator at 37 °C and 5% CO2. For the SILAC experiments, C17.2 cells were cultured in DMEM medium containing light 12C6 or heavy 13C6-labeled Llysine supplemented with dialyzed FBS (Invitrogen, CA, USA) for a minimum of six population doublings. The heavy labeled C17.2 cells were stimulated with 1 mM AICAR (Cell Signaling Technology, MA, USA) at 50−60% confluence. The light labeled C17.2 cells were mock stimulated with water. After a 48 h treatment, the cells were washed with PBS and harvested.

Protein identification and quantification was performed with MaxQuant version 1.0.13.13 according to protocol.22 Raw MS spectra were processed by using MaxQuant 1.0.13.13 software, and the derived peak lists were searched against a combined database from the International Protein Index (IPI) mouse protein database version 3.67 (forward database) and the reversed sequences of all proteins from the same IPI mouse protein database using Mascot (version 2.2.04, Matrix Science, London, UK). Precursor mass tolerance was set at 15 ppm and 0.6 Da for MS/MS fragments. Enzyme specificity was set to trypsin. Carbamidomethylation of cysteine was set as a fixed modification; N-acetyl protein and oxidation of methionine and heavy label lys6 were set as variable modifications. Identification was set to a false discovery rate of 1% at both the protein and peptide levels. Proteins were quantified if the protein has at least one MaxQuant-quantifiable SILAC pair. The final reported protein ratio presents a normalized ratio with variability of heavy versus light SILAC obtained in all replicates where the same protein was identified.

Membrane Preparation

Protein Pathway Analysis

Two different cell stages generated in triplicate were pooled and selected for proteomic analysis. Harvested cells were homogenized in homogenization buffer (pH 7.4) containing 50 mM Tris-HCl, 0.1 M NaCl, 1 mM EDTA, and protease inhibitor cocktail (Sigma-Aldrich China Inc., Shanghai, China) by Dounce homogenizer (30 strokes) and centrifuged at 2000g for 10 min (Eppendorf 5804 R, Hamburg, Germany) at 4 °C to pellet unbroken cells and nuclei. The resulting supernatant was centrifuged at 100 000g for 1 h. The membrane pellet was collected and washed with 0.1 M sodium carbonate and then was solubilized in lysis buffer (50 mM Tris-HCl pH 8.0, 1 mM EDTA, 4% SDS, 1 mM DTT, and protease inhibitor cocktail). The protein quantification was performed using a RC DC protein assay kit (Bio-Rad, CA, USA).

GO annotation of the identified proteins was obtained using DAVID (version 6.7); see http://david.abcc.ncifcrf.gov/ summary.jsp.23 Differentially expressed proteins were analyzed using Ingenuity Pathway Analysis (IPA, Ingenuity Systems; see www.ingenuity.com). The over-represented biological processes, molecular functions, and canonical pathways were generated based on information contained in the Ingenuity Pathways Knowledge Base.

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MATERIALS AND METHODS

NSCs Culture and Differentiation

Western Blotting

Biological replicates were used for Western blotting analysis of astroglial differentiation. Western blotting analysis was performed as described previously.24,25 In brief, lysates from whole cell extracts or membrane pellets containing 50 μg of proteins were subjected to gel electrophoresis. Proteins were then transferred to PVDF membranes (Millipore, CA, USA). The blots were blocked in 4% BSA in TBST solution for 30 min at room temperature and then incubated at 4 °C overnight with the primary antibody. GFAP (1:4000), β -III tubulin (1:1000), and nestin (1:1000) were purchased from Abcam (Cambridge, MA, USA). Annexin A2 (1:5000) was purchased from BD Pharmingen (San Diego, California, USA). Transferrin receptor 1 (1:1000) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-β-actin (1:2000) was obtained from Protein Tech (Wuhan, Hubei, China). After incubation with secondary antibodies (1:2000, Millipore, CA,

SDS-PAGE and In-Gel Digestion

The equal amounts of light and heavy lysates were mixed. One hundred micrograms of the mixed sample was separated by 10% SDS-PAGE, and visualized by Colloidal Coomassie staining. The gel lane was excised and cut into 40 gel slices. The gel slices were subjected to in-gel digestion with trypsin. The tryptic peptides were extracted and lyophilized by SpeedVac (Thermo Savant, NY, USA). LC−MS/MS Analysis

The peptide mixtures were analyzed by a linear ion trapOrbitrap hybrid mass spectrometer (LTQ-Orbitrap XL, 830



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USA) at room temperature for 1 h, the blot was visualized by Chemi Doc XRS imaging system (Bio Rad, CA, USA). Immunocytochemistry

C17.2-NSCs were seeded at 5 × 104 cells/mL on 12-mm polyL-lysine coated glass coverslips for immunofluorescence analysis. The cells were fixed in 4% para-formaldehyde and processed for immunofluorescence as described previously.9 The following antibodies were used to detect antigen: β-III tubulin (Abcam, 1:200), nestin (Abcam, 1:200), glial fibrillary acidic protein (GFAP) (Abcam, 1:500), and Alexa dyeconjugated secondary antibodies (Millipore, 1:200). Hoechst 33342 (5 μg/mL, Molecular Probes/Invitrogen, CA, USA) staining was used to label nuclei. Images were captured on an Olympus fluorescent microscope (IX51) equipped with a cool snap camera spot system (Roper Scientific Inc., USA), with 20× and 40× objectives. Images were combined for figures using Adobe Photoshop CS2. Statistics

Figure 1. Induction of astroglial differentiation from C17.2 NSCs by AICAR. A, photomicrographs of C17.2-NSC self-renewal (left) and astroglial differentiation cells (right). B, immunofluorescence analysis of C17.2-NSC undifferentiating (self-renewal) and astroglial differentiating cells, green stained for nestin (neural stem cell marker) or GFAP (astrocyte cell differentiation marker) and red-stained for β-III tubulin (neuron-specific marker). Nuclei was counterstained with Hoechst 33342. Scale bar is 50 μm. C, Western blotting analysis of the differentiation of C17.2 NSCs.

All quantitative data were presented as mean ± standard deviation (SD). For Western blotting, comparisons between two groups were performed by Student’s t test. Statistical significance was defined as *p < 0.05 and **p < 0.01.

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RESULTS

Astroglial Differentiation of SILAC-Labeled Neural Stem Cells

combined raw data search by Mascot 2.2 against IPI mouse database v3.67 yield 1129 distinct proteins including 728 membrane proteins with 99% confidence (P < 0.01, Table S1 in the Supporting Information) and 735 proteins were quantified with quantitative ratios. For quantitative analysis, previously studies using SILAC and LC−MS/MS have applied cutoff ranging from 1.3- to 2.0-fold.26 On the basis of our Western blotting results, we used a 1.5-fold cutoff, which identifies the neural stem cell maker protein nestin (membrane-associated protein, showed a nearly 1.5-fold down regulation). With 1.5fold cutoff value, 105 membrane proteins were found upregulated, and 100 proteins were found down-regulated upon astroglial differentiation of NSCs for further considerations (Table S2, Supporting Information).

SILAC labeling of primary NSCs is a difficult task due to the passage limits. Here, we used the immortalized C17.2 neural stem cell line as a model for SILAC labeling and further quantitative proteomic analysis. C17.2 NSCs were subjected to a well-established astrocytes differentiation method with AICAR as previously reported.9,18 First, we compared the morphology of NSCs with or without AICAR treatment because morphology is one of the criteria indicating the differentiation of C17.2 NSCs. As shown in Figure 1, after 5 passages (about 10 doubling of C17.2 cells), in the presence of 1 mM AICAR for 48 h, dramatic changes in morphology of C17.2 NSCs occur when compared to the absence of AICAR treatment cells. The self-renewal state and astrocytes differentiating state were further demonstrated by an expression analysis of several self-renewal and differentiation markers by Western blotting and immunochemistry staining (Figure 1). The Western blotting result shows with AICAR treatment for 2 days, nestin (a marker for NSCs and neural progenitors) and β-III tubulin (marker for early neuronal cell) expression are decreased, and while astrocytes make glial fibrillary acidic protein (GFAP), expression is increased. Immunochemistry staining analysis of the AICAR untreated C17.2-NSCs shows nearly all C17.2 cells express nestin. At 48 h of treatment with AICAR, we found that around 80% of C17.2 cells displayed morphological changes and expressed GFAP (Figure 1b). These data indicate that the in vitro model of astroglial differentiation of NSCs was efficiently established and agree well with the result from our previously study.9

Cell Surface Proteins of Undifferentiated and Differentiated NSCs

To identify undifferentiated and differentiated NSC cell surface makers from a pool of membrane proteins, we focused on a plasma membrane protein expression level that has significant changes between the self-renewing and differentiating neural stem cells (>2.0 fold). A total of 15 high-priority putative candidates were identified by a mass spectrometer from at least two of the three repeats, of which eight were up regulated, and seven proteins were down regulated in the process of astroglial differentiation compared with controls (Table 1). An interesting finding was that the fibronectin 1(Fn1) shows upregulated about 4-fold upon astroglial differentiation. We also found integrin β-1 (Itgb1), one receptor for fibronectin, shows up-regulated about 1.5-fold correspondly (Table S2, Supporting Information). Another protein that we are interested in, transferrin receptor 1 (TfR1, Figure 2), shows a decreased expression level on the astroglial differentiation, which could be used as a novel cell surface marker of NSCs.

Identification and Quantification of Membrane Proteins of Undifferentiated and Differentiated NSCs

Quantitative analysis of membrane protein changes between the self-renewal and astrocytes differentiating cells was performed with three replicates of C17.2 cells using SILAC to compare quantitatively the light labeled self-renewal cells with the heavy labeled astrocyte differentiating cells. The 831



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Table 1. Differentially Expressed Cell Surface Proteins during Astroglial Differentiation of NSC Identified through SILACBased Proteomicsa accession number

protein name

TMb

ratio diff/undiff (SD)c

function

Q60928 Q3TCU5 P11276 P48036 Q9D2N4 P05202 Q8BH64 Q62470 P07356 P10107 P46935 P97855 Q3V3R4 Q62351 P39061

CD224 tapasin fibronectin 1 annexin A5 dystrobrevin alpha aspartate aminotransferase EH domain-containing protein 2 integrin alpha-3 annexin A2 annexin A1 E3 ubiquitin-protein ligase NEDD4 GAP SH3 domain-binding protein 1 integrin alpha-1 transferrin receptor protein 1 collagen alpha-1(XVIII) chain

1 1 0 0 0 0 0 1 0 0 0 0 1 1 0

17.01(N/A) 4.75(0.27) 4.57(0.42) 3.67(0.68) 2.72(0.16) 2.65(0.07) 2.38(N/A) 2.11(0.07) 0.47(0.03) 0.46(0.04) 0.39(N/A) 0.38(0.02) 0.37(0.06) 0.19(0.09) 0.17(0.06)

glutathione metabolism antigen processing cell adhesion and signal transduction blood coagulation calcium ion binding organic acid metabolic process actin cytoskeleton organization neuron migration cellular component organization cell proliferation proteolysis ATP binding signal transduction signal transduction cell morphogenesis and cell proliferation

a

Predicted subcellular localization was obtained using Gene Ontology. bNumber of predicted transmembrane domains. cQuantification data shown as mean ± SD.

Figure 2. LC−MS/MS analysis. A, examples of SILAC peptide pairs for the corresponding transferrin receptor protein 1 from self-renewal and astroglial differentiating NSCs. B, corresponding tandem mass spectra of SILAC peptides. The sequences of two peptides were identified as DESLAYYIENQFHEFK and TAAEVAGQLIIK, which form transferrin receptor protein 1.

Bioinformatics Analysis of the Differentially Expressed Membrane Proteins

in mitochondria (Figure 3a). IPA was used to assign identified proteins into different functional groups based on the Ingenuity Pathways Analysis literature database. All the differentially expressed proteins were uploaded to the IPA server. For molecular and cellular functions, the data indicated that many

The DAVID Bioinformatics Resource 6.7 was used for annotation of the cellular component. We found that the majority of the membrane proteins we identified were resident 832



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Figure 3. continued

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Figure 3. Biological function analysis. A, the distribution of annotation for a cellular component for differentially expressed proteins; the cellular location was determined by GO annotations from DAVID database. B, molecular and cellular function analysis of differential expression of membrane proteins under differentiation of C17.2 cells. RNA post-transcriptional modification, protein synthesis, cellular growth and proliferation, cell-to-cell signaling/interaction, and molecular transport are the top listed functions, as determined by the p value. C, physiological system development and function analysis of differential expression of membrane proteins under differentiation of C17.2 cells. The tissue development and nervous system development are top listed biological process; −log (p-value) > 1.5 is significant. D, canonical pathways associated to the mitochondrial metabolisms that showed significant changes in expression undergoes astroglial differentiation.

proteins are involved in top five biological processes (p < 0.001): RNA post-transcriptional modification, protein synthesis, cellular growth and proliferation, cell-to-cell signaling/ interaction, and molecular transport (Figure 3b). For physiological system development and function, the tissue development and nervous system development function were indicated by our data (Figure 3c). With regard to canonical pathways, we found that some membrane proteins involved in the citrate cycle were up-regulated in the process of astroglial differentiation. Protein expression for important enzymes of the citrate cycle, such as succinyl-CoA ligase subunit beta (SUCLA2), succinate dehydrogenase iron−sulfur subunit (SDHB), alpha-ketoglutaratedehydrogenase (Ogdh), phosphoenolpyruvate carboxykinase (PCK2), succinyl-CoA ligase subunit alpha (SUCLG1), aconitate hydratase (ACO2), fumarate hydratase (FH), isocitrate dehydrogenase (NADP), and isocitrate dehydrogenase 3 (NAD+) beta (IDH3B), were all significantly up-regulated (Table 2 and Supporting Information Table S2). Correspondingly, we found that pyruvate metabolism, butanoate metabolism, oxidative phosphorylation, glycolysis/gluconeogenesis, and fatty acid metabolism were all coordinated up-regulated (Figure 3d). Apparently, those metabolism pathways undergo expression changes in a highly correlated pattern, indicating that the activation of oxidation is a metabolic signature of neural stem cell differentiation. Pathway analysis was also used to analyze different cellular functional networks to determine which were altered in differentiating NSCs. In our study, several networks were grouped by IPA, such as there are 30 membrane proteins involved in gene expression, RNA post-transcriptional modification, cellular assembly and organization were grouped as top 1 network, which had the highest score 41 (Figure 4a). Additionally, 20 proteins, such as Fn1, Itga3, etc., could be

Table 2. Differentially Expressed Mitochondrial Membrane Proteins or Membrane-Bound Proteins of the Citrate Cycle Undergo Astroglial Differentiation of Neural Stem Cells IPI number

protein name

IPI00126635

isocitrate dehydrogenase 3 (NAD+) beta aconitate hydratase alpha-ketoglutarate dehydrogenase succinate dehydrogenase succinyl-CoA ligase [ADPforming] subunit beta succinyl-CoA ligase [GDPforming] subunit alpha isocitrate dehydrogenase [NADP] fumarate hydratase pyruvate dehydrogenase E1 component subunit beta dihydrolipoyl dehydrogenase phosphoenolpyruvate carboxykinase [GTP]

IPI00116074 IPI00845652 IPI00338536 IPI00261627 IPI00406442 IPI00875110 IPI00129928 IPI00132042 IPI00874456 IPI00223060

gene name

ratio diff/ undiff

SD

Idh3b

2.4763

n/a

Aco2 Ogdh

2.7973 2.1513

n/a 0.037

Sdhb Sucla2

1.643 2.1492

n/a 0.04

Suclg1

2.015

0.037

Idh2

1.8205

0.061

Fh Pdhb

1.8756 1.7123

0.068 0.038

Dld

1.7836

0.012

Pck2

1.9574

n/a

grouped into specific disease associations, such as hematological disease, cancer, respiratory disease (Figure 4b). Validation of the Expression of Selected Cell Surface Proteins

For validation purposes, the expression of one constitutive proteins (β-actin), two down-regulated proteins (annexin a2, transferrin receptor 1), two up-regulated proteins (fibronectin 1 and integrin β-1) quantified in the present quantitative study were validated by Western blotting (Figure 5). Similar to the quantitative proteomics results, compared with the self-renewal 834



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Figure 4. Molecular pathway analysis of regulated membrane protein changes in NSCs as they undergo astroglial differentiation. Proteins and their expression levels of change were imported into the Ingenuity Pathways Analysis, and interacting pathways were constructed. A, network 1 group by IPA shows it was primarily involved in gene expression RNA post-transcriptional modification, cellular assembly, and organization. Red represents an increase in protein expression, whereas green represents a decrease in expression level; the color intensity represents the degree of abundance change. A solid line indicates a direct interaction, and a dashed line indicates an indirect interaction. B, network 3 primarily involved in hematological disease, cancer, and respiratory disease.

Figure 5. A, verification of the expression of selected proteins by Western blotting. The total proteins were separated by 10% SDS-PAGE and transferred to a PVDF membrane. Itgb1, Tfrc, Anxa2, and Fn1 were detected with respective antibodies. Beta actin was used as an internal control. B, bands were analyzed by densitometry using Quality-One software (Bio-Rad laboratories, Richmond, CA). The X axis shows the relative intensity. All data were from at least three independent experiments and shown as mean ± SD.

this study, we applied SILAC technology to compare the membrane proteome of self-renewal and astrocytes differentiating NSCs in order to identify a neural stem cell surface maker as well as increase our understanding of the underlying signaling events of NSC differentiation.

cells, we observed a decrease in protein level of the annexin a2, transferrin receptor protein 1, and a marked increase in protein level of fibronectin 1 and integrin β-1. Without any surprise, the Western blotting results are constant with the SILAC quantitative result, demonstrating that the ratio observed from mass spectrometry is reliable.

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Identification and Quantification of Membrane Proteins

We have used the well-characterized immortalized neural stem cell line C17.2, which can be directly induced into astroglial differentiation by AICAR via activating the JAK-STAT3 pathway as CNTF and LIF did. For monitoring membrane changes during a differentiation, a global quantitative method,

DISCUSSION

Differentiation of NSCs and subsequent outgrowth of neurite and/or astroglial phenotypic changes, all are processes that are tightly regulated by membrane protein signaling pathway. In 835



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into several networks with high possibility. Through IPA analysis, we found 28 membrane proteins involved in gene expression, cellular assembly, and organization that were grouped as the top network (Figure 4a). Interestingly, network 3 is relevant to some specific disease associations, such as cancer and respiratory disease (Figure 4b). Multiple proteins, including Fn1 protein, and ITGA3 protein in the middle of a network detected by IPA have been reported to be directly related to cell adhesion and cell motility.36,37 Expansion of a quantitative membrane proteomic analysis, for example, measuring changes in protein phosphorylation or glycosylation, may further the investigation of possible links between astroglial differentiation and hematological disease or some kinds of cancer. The changed proteins, group as cancer and respiratory disease, also could be caused by the limitation of the current IPA database or many biological events share the same proteins of some network. On the basis of our SLIAC quantitative data, we found that astroglial differentiation leads to and/or accompanies extensive changes in mitochondrial protein expression. These changes are functionally correlated and revealed that almost every important aspect of energy metabolism is activated during neural stem cell differentiation, such as citrate cycle (TCA), pyruvate metabolism, oxidative phosphorylation, glycolysis/ gluconeogenesis, and fatty acid metabolism. This is consistent with previous reports that the intracellular oxidation state regulates the balance between embryonic stem cell self-renewal and differentiation.24,38 On the basis of our results, we suspect that the activation of oxidation metabolism is important to NSC differentiation. Apparently, the activation of oxidation metabolism is linked to or caused by the additive AICAR. AICAR is a well-known activator of AMP-activated protein kinase (AMPK),39 and its effects on lipid and glucose metabolism have been studied.40,41 Our findings may also help to elucidate the molecular mechanism of AICAR on cell differentiation. In summary, we have applied SILAC to quantitatively measure the regulation of more than 700 membrane or membrane associated proteins during the astroglial differentiation of a mouse's neural stem cells that are induced by AICAR. In addition to familiar proteins, one cell surface protein transferrin receptor protein 1 was preliminarily validated in this study and is a promising maker for neural stem cells. Beyond the identified surface markers, we also elucidated alterations in other membranes such as the mitochondria membrane at protein expression level. Our membrane proteome data reveals that energy metabolic pathways are activated during the astroglial differentiation process. This will help to gain insight into potential pathways and regulatory networks involved in both the maintenance of NSC multipotent state and the processes of specific cell lineage differentiation.

SILAC was used in combination with a high accuracy mass spectrometry. SILAC labeled C17.2 cells displayed a remarkable morphological change during cultivation in the presence of AICAR. The Western blot and ICC results have demonstrated that the in vitro model of astroglial differentiation of NSCs was successful (Figure 1). Our data shows that we have identified 1129 proteins and relatively quantified 735 proteins of differentiating NSCs using only lys0 and lys6-labeling. Among them, 205 proteins revealed to be 1.5-fold up- or down-regulated. This is the first relatively quantified membrane proteomics data set published to date relevant to NSCs differentiation. In this study, we focused on several cell surface proteins that show significant expression changes, indication they could be a new cell surface marker for the status of NSC cells. An interesting finding was that, e.g., transferrin receptor 1 shows a decreased expression level on the astrocyte-linage differentiation, which could be used as a novel cell surface marker of NSCs. Transferrin receptor 1, a major mediator of iron uptake in mammalian cells, was demonstrated to be a crucial downstream target of c-myc.27 The abundance of c-myc protein can directly control the stem cell self-renewal and differentiation.28 Another interesting find was that integrin β-1 and fibronectin 1, were both up regulated on astrocyte-linage differentiation in our study. Integrin β-1 is an adhesion receptor that links the extracellular matrix to the cytoskeleton and plays an important role in many biological processes such as neural stem cell maintenance22 and radial glial scaffold.29 FN1 is a multifunctional extracellular matrix (ECM) glycoprotein that binds to cell surface receptor protein integrins.30 It was reported that the interactions between fibronectin 1 and integrin α5/β1 are crucial for osteoblast differentiation in rat calvarial osteoblasts.31 Here, our data suggests that fibronectin 1 and its receptor integrin β-1 may play an important role during astroglial differentiation of NSCs. We also found a 2-fold lower expression level of annexin A2 (Anxa2) in the membrane fraction of a self-renewal neural stem cell than a differentiation neural stem cell by SILAC proteomic analysis. Anxa2 is a member of the calcium-dependent phospholipid-binding protein family and plays a role in the regulation of cellular growth and in the signal transduction pathways.32 Another member of the annexin family, annexin A5, was found up-regulated on the astroglial differentiation of NSCs. Annexin A5, a small Ca2+ dependent phospholipid-binding protein, was originally reported as an anticoagulant protein by competing for phosphatidylserine binding sites with prothrombin and also a protein kinase C inhibitory protein.33 Later, it was reported that annexin A5 are involved in an antiproliferative mechanism in endometrial cancer cells.34 It was also reported that annexin A5 inhibits the apoptotic body formation.35 The increased level of annexin A5 may play a role in antiproliferation effects during the differentiation of NSCs. The higher level of Itgb1 and Fn1 and lower level of the Tfrc and Anxa2 in self-renewal vs astroglial differentiation were also confirmed by Western blotting, which showed a good correlation between quantitative proteomic data and true protein expression level.

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ASSOCIATED CONTENT

S Supporting Information *

Supplementary Table 1, all proteins identified in the membrane proteome analysis. Supplementary Table 2, proteins quantified in the membrane proteome analysis. This material is available free of charge via the Internet at http://pubs.acs.org.

Molecular Function and Network Pathway Analysis

IPA was used to analyze the quantification data, investigate potential changes in molecular functions and group proteins 836



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AUTHOR INFORMATION

Corresponding Author

*Tel: 86-731-8886-1304. Fax: 86-731-8886-1304. E-mail: [email protected] (P.C.); [email protected] (S.L.); [email protected] (R.C.). Author Contributions §

These authors contribued equally to this work.

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ACKNOWLEDGMENTS We are grateful to Dr. David B. Hill (The University of North Carolina at Chapel Hill) for the critical reading of the manuscript. This work was supported by grants from the National Natural Science Foundation of China 31000375 (to R.C.) and 81070353 (to P.C.).

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Quantitative Proteomic Analysis of Membrane Proteins Involved in

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