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标题: Differential Expression Profiling of Membrane Proteins by Quantitative Proteomic [打印本页]

作者: 江边孤钓    时间: 2009-3-5 10:48     标题: Differential Expression Profiling of Membrane Proteins by Quantitative Proteomic

a UBC Center for Proteomics, Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia, Canada;
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b Center for Experimental BioInformatics,
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5 O& Y9 d+ q8 l6 B6 C' M0 w  R* Kc Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense M, Denmark;
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d Department of Endocrinology, University Hospital of Odense, Odense C, Denmark
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Key Words. Proteome ? Osteoblast ? Differentiation ? Stem cells ? Mesenchymal stem cells ? Membrane proteins
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Correspondence: Moustapha Kassem, M.D., Ph.D., D.Sci., Department of Endocrinology and Metabolism, University Hospital of Odense, DK-5000 Odense C, Denmark. Telephone: 45-6541-1606; Fax: 45-6591-9653; e-mail: moustapha.kassem@ouh.fyns-amt.dk+ \0 i% _/ @. w# d6 y
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ABSTRACT
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Human mesenchymal stem cells (hMSCs) are multipotent stem cells found in the bone marrow stroma and possibly the stroma of many other organs . These cells are able to differentiate into multiple mesoderm-type cells, including osteoblasts (OBs), adipocytes, and chondrocytes. Due to their versatile growth and differentiation potential, hMSCs are ideal candidates for use in regenerative medicine and cell transplantation protocols . Traditionally, hMSCs have been isolated from bone marrow using physicochemical properties (adhesions to plastic or other extracellular matrix), and only a small number of surface markers are currently available for their isolation . Also, study of their differentiation potential (e.g., into OB) has relied on monitoring changes in a small number of proteins . Thus, identification of marker proteins that can be used to identify and distinguish hMSCs from other cell types, as well as to monitor their differentiation progress, is needed to make use of the full therapeutic potential of hMSCs. In addition, comprehensive profiles of MSC membrane proteins can provide novel biological insights into the proliferation and differentiation of these cells, including the potential for identifying therapeutic targets.
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( w2 n, b" P9 I9 I% CFew studies have tried to identify new molecular markers of hMSCs because most investigations have used hybridoma technology to obtain monoclonal antibodies against subsets of MSCs . Although informative, this method is too focused to provide a global understanding of changes in the membrane phenotype of MSCs during differentiation. Mass spectrometry (MS)–based proteomics tools can be applied qualitatively to gain a more holistic view of biological systems, as seen in recent studies of keratinocytes  and the osteoclast secretome . However, quantitative proteomics methods allow dynamic changes in cell differentiation stages to be followed on the same broad scale, potentially revealing much more insight into the system of interest. Of particular relevance to this study, Conrads et al.  have used isotope-coded affinity tags to examine the effects of inorganic phosphate on the murine OB proteome. The choice of MS instrument and database search parameters can also have a large impact on the reliability of protein identifications , leading to unacceptably high levels of false-positive identifications if criteria are not stringent enough.
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Application of proteomics to human stem cell biology has been hampered by the lack of physiologically relevant cell models that can be expanded to generate the levels of material required for such studies. We have recently developed a cell model for hMSCs by overexpressing the human telomerase reverse transcription (hTERT) gene in normal hMSCs and thus created a cell line termed hMSC-TERT that maintains the phenotypic characteristics and hormonal responsiveness of normal hMSCs despite extensive cell proliferation . To gain some understanding of the process of OB differentiation of hMSCs, we have enriched membrane proteins from hMSC-TERT before and after induction of OB lineage commitment in short-term culture in vitro. Using quantitative, MS-based proteomics, we have identified 463 proteins with very high confidence and have measured their changes in expression induced by OB differentiation. Several marker proteins of stem cells and OB were identified, along with several new candidate proteins with putative roles in stem cell proliferation and differentiation. For those gene products changing most dramatically, we have also measured the concomitant changes in their mRNA expression levels by reverse transcription (RT) real-time polymerase chain reaction (PCR) and confirmed the protein level changes for three of them by cytochemistry and confocal immunofluorescence microscopy.
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, `1 H; @9 j0 G, x; YMATERIALS AND METHODS2 P' T+ X; k/ K6 B( `4 ^

/ q1 X4 d3 k$ PThe primary aim of this study was to identify and quantify changes in plasma membrane proteins, as well as those of the biosynthetic and endosomal pathways in hMSCs during OB differentiation. We first enriched integral membrane proteins and membrane-associated proteins by eliminating the highly abundant nuclear and mitochondrial proteins before collecting the remaining membranes by an ultra-high-speed centrifugation step. Resuspending the membranes in Na2CO3 at pH 11 reduced the potential masking effects of highly abundant cytosolic proteins bound loosely to the membrane pellet. Pairs of membrane samples prepared in this way (three control and three OB-differentiated cells) were digested to peptides and analyzed by information-dependent acquisition on two different classes of hybrid mass spectrometers: QSTAR Pulsar quadrupole TOF instruments or a LTQ-FT linear ion-trap/Fourier transform ion cyclotron resonance instrument . In each case, the instrument software selected peptide ions from the survey scan (Fig. 1A) to be fragmented (Fig. 1B). The total ion chromatogram in each pair of samples (Fig. 1C) looked superficially similar but was far too complex to serve as a basis for quantitative comparison. Therefore, using an adaptation of MSQuant , we first correlated the retention times (see Materials and Methods) of high-confidence peptides (IonsScore >40) identified in both members of each pair of hMSC and OB-differentiated cell membranes (Fig. 1D) before extracting the ion chromatograms for each monoisotopic ion for all identified peptides (Fig. 1E). By making use of the retention time correlation and high-mass accuracy to locate unsequenced peptide ions in the opposing sample, this strategy only required a peptide to be identified in one of the two samples. The effect of OB differentiation was determined by dividing the results obtained in OB-differentiated samples by that in control samples (OB/control ratios) for each protein based on the average for all peptides observed from that protein across all replicates. The OB/control ratios measured for these proteins ranged from 6-fold downregulation to 27-fold upregulation (Fig. 1F)." {: n4 a- x0 T, r+ v  o
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Figure 1. Liquid chromatography/tandem mass spectrometry. (A): Survey scans of peptides eluted from reversed-phase columns were acquired as described in Materials and Methods and used to select peptide ions for fragmentation. Fragment spectra (B) of the 539.79-m/z ion from (A) was found to match the indicated peptide from versican core protein. (C): Representative total ion chromatographs from hMSCs and OB-differentiated samples. (D): Plot of elution times for peptides sequenced in both samples of a pair. The calculated correlation coefficient is greater than 0.99. (E): Representative ion chromatograms for peptide LLASDAGLYR of versican core protein measured in hMSCs (solid line, near baseline) and OB-differentiated (dashed line) samples. (F): Plot of ratios for all proteins quantified here, ordered from largest positive change with OB differentiation toward largest negative change. Abbreviations: hMSC, human mesenchymal stem cell; OB, osteoblast.
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Compilation of the data from all three analyses resulted in the identification of 463 proteins seen in at least two of the three analyses and with at least two unique peptides. This set of proteins represents an extremely high confidence profile of membrane proteins in hMSCs because, to be considered identified, proteins had to be observed in at least two different samples and had to have at least two sequenced peptides detected with high mass accuracy. This means that it is extremely unlikely that there is even one false-positive identification among the 463 (see Materials and Methods). A complete list of all proteins and peptides identified in this study is presented in the online supplementary material.
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- Y  c+ v8 m4 a3 h  O( ^Classification of the subcellular localizations of 463 proteins based on annotations in the UniProt Knowledgebase(http://www.ebi.uniprot.org/index.shtml) and the Gene Ontology Consortium revealed several classes of enriched proteins (Fig. 2A). Sixty-six percent are integral membrane proteins, proteins with known or predicted membrane anchors or proteins known to interact with other membrane proteins. These included all known markers of MSCs, including ALP, 5'-nucleotidase (CD73), Thy-1 glycoprotein (CD90), neprilysin (CD10), myeloid plasma membrane glycoprotein (CD13), endoglin (CD105), activated leukocyte-cell adhesion molecule (CD166), HOP26 (CD63), integrin ?1 (CD29), integrin 5 (CD49e), integrin 4 (CD49d), phagocytic glycoprotein I (CD44), fibronectin, collagen type VI, and epidermal growth factor receptor . Other CD antigens detected were CD98, CD59, CD51, CD107b, CD107a, CD 91, CD99, CD71, CD47, and CD108. Also, several members of the integrins, integrin alpha 11, integrin beta-5, integrin alpha-2 (CD49b), integrin alpha-6 (CD49f), integrin alpha-V (CD51), and integrin alpha-3 (CD49c), were detected (see supplemental material).
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Figure 2. Distribution of identified proteins. (A): Subcellular distribution of all identified proteins according to their annotation in the Swiss-Prot Knowledgebase. (B): Number of proteins with different numbers of transmembrane domains, predicted as described in Materials and Methods.
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Membrane proteins, particularly of the plasmalemma, are important in defining the unique characteristics of stem cells. To better understand the nature of the MSC membrane proteome, we took amino acid sequences of all proteins identified here and predicted their membrane topology using publicly available bioinformatics tools. Interestingly, 133 of the 463 proteins had at least one -helical transmembrane domain (TMD) predicted in their primary sequence, 11 more than were classified as integral membrane based on their UniProt annotations. Most proteins contained either one or two TMDs, but many proteins with several TMDs were also detected (Fig. 2B and supplemental online material). Likewise, the success of the membrane enrichment is also reflected by the observation that only 11% of the identified proteins originated from nuclei or mitochondria.
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Induction of OB differentiation of hMSC-TERT cells in our experiments was confirmed by the presence of increased expression of four OB-specific genes: ALP, collagen type I (COL1), osteopontin and osteocalcin, and bone sialoprotein 2 (BSP2) (Fig. 3A). Our working hypothesis in this study was that markers of OB differentiation should show an OB/hMSC ratio greater than or less than 1. As expected, ALP, detected here with 9% sequence coverage, displayed the highest OB/hMSC ratio (>27, Fig. 1C and supplemental online material) of all the proteins quantified in this study. The expression levels of several other proteins also increased or decreased with OB differentiation. To determine a significance threshold for these changes, we first considered the measurement errors in this analysis. The average relative SD of OB/hMSC ratios measured here was 47%, so we considered any protein whose expression level changed by at least this, or twofold in either direction, to be of potential interest. Based on these criteria, we identified 83 proteins that increased at least twofold and 21 proteins that decreased at least twofold (Table 1 and supplemental online material).
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Figure 3. Marker proteins. (A): Fold changes in mRNA levels of Runx2 (CBFA-1), alkaline phosphatase (ALP), collagen type I (Col I), osteopontin (OPN), osteocalcin (OC), and bone sialoprotein 2 (BSP2) between human mesenchymal stem cells (hMSCs) (black bars) and osteoblast (OB)-differentiated cells (open bars). (B): ALP enzyme activity in hMSCs and OB-differentiated cells, detected as described in Materials and Methods. (C): Collagen type I 1 (Col I) and transferrin receptor (TfR) levels on hMSCs and OB-differentiated cells. (D): Versican and TfR levels on hMSCs and OB-differentiated cells. Scale bar = 50 μm.
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3 F! N6 j5 [: x& Y1 TTable 1. Proteins changing expression during differentiation) g7 \7 h; Q* y1 k" l. |

7 l9 l/ R2 F' A, j4 `* Y/ v. FWe next sought to verify some of the proteins that showed the largest changes during early-stage OB differentiation. By staining cells for ALP activity, we observed significantly increased levels of the enzyme in OB-differentiated cells compared with control cultures (Fig. 3B). Furthermore, we used dual-color immunofluorescence to double-label CD71/versican and CD71/COLI in nonpermeabilized hMSC-TERT and OB-differentiated cells. We selected CD71 (transferrin receptor) as a control in these experiments because a portion of it is found on the plasma membrane and, in our quantitative analysis described above, it did not change upon induction of OB differentiation (1.0 ± 0.5). Using CD71 staining intensities as a baseline, COLI and versican were both more abundant in the OB-differentiated cells compared with control cells (Figs. 3C, 3D).
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' n, n# I  e( V' lAlthough the quantitative analysis of hMSC and OB membranes revealed several proteins whose expression levels changed dramatically, our dataset also contains a great deal of additional information. We asked if there are any functional classes of proteins identified in this study that are coregulated during OB differentiation. Mapping GO terms (see Materials and Methods) to the proteins identified here revealed several functional classes of proteins displaying very close coregulation (Table 2). In particular, all nine proteins annotated in the cell-matrix adhesion and integrin receptor signaling pathway GO categories displayed remarkably consistent changes in their expression levels, increasing an average of 2.0 ± 0.5–fold with OB differentiation. All five heteronuclear ribonuclear proteins (hnRNPs) increased even more dramatically with OB differentiation (6.9 ± 4.0–fold).% N* G/ T: D# T6 q6 A8 W
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Table 2. Coregulated functional families
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& C2 N2 e# ~8 i( F, ~$ o' g" V+ cAlthough proteins are the primary effectors of biological function, large-scale alterations in the levels of gene expression are often diagnostic of the changing roles of cells or tissues. We used quantitative real-time PCR to ask if the changes in levels of protein expression were mirrored by changes in gene expression. The mRNA expression levels for the genes corresponding to the 41 proteins whose expression levels change more than threefold were measured, but only the level of ALP mRNA changed significantly with OB differentiation (supplemental online table).1 m" U5 d# U" [7 n, F
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DISCUSSION+ g3 a. G7 Z" f

/ y8 E* r7 ^# u+ d/ |+ WL.J.F. and P.A.Z. contributed equally to this study. We thank Drs. Jorge Burns, Irina Kratchmarova-Blagoev, and Basem Abdallah for advice on the hMSC-TERT cell cultures, Shao-En Ong for programmatic assistance, Christian Ravnsborg Ingrell for help with protein sequence analysis, members of all of our groups for fruitful discussions, and the Max Planck Institute for Biochemistry, Martinsried, Germany, for the generous loan of the LTQ-FT. The study was supported by grants from Danish Medical Research Council, Danish Center for Stem Cell Research, the Novo Nordisk Foundation, and the Karen Elise Jensen’s Foundation. The Center for Experimental Bioinformatics is supported the Danish National Research Foundation.* x2 ?  u8 Z% [1 }

; |8 e, x+ C2 ZDISCLOSURES
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The authors indicate no potential conflicts of interest.. u/ D* [. @; e! b2 y  ?! ~+ P7 T
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谢谢干细胞之家提供资料
作者: biobio    时间: 2016-4-7 20:51

水至清则无鱼,人至贱则无敌!  
作者: biopxl    时间: 2016-4-13 19:18

真是有你的!  
作者: 20130827    时间: 2016-4-24 19:52

干细胞研究人员的天堂
作者: 桦子    时间: 2016-5-12 13:35

努力,努力,再努力!!!!!!!!!!!  
作者: 糊涂小蜗牛    时间: 2016-5-26 17:18

帮你项项吧  
作者: 海小鱼    时间: 2016-6-27 20:27

谢谢分享了!   
作者: 初夏洒脱    时间: 2016-6-28 19:27

细胞治疗行业  
作者: tuting    时间: 2016-7-2 09:52

支持一下  
作者: ladybird    时间: 2016-7-9 19:10

孜孜不倦, 吾等楷模 …………  
作者: 石头111    时间: 2016-7-29 22:10

哎 怎么说那~~  
作者: 考拉    时间: 2016-8-9 04:35

对不起,我走错地方了,呵呵  
作者: 陈晴    时间: 2016-8-12 03:26

顶顶更健康,越顶吃的越香。  
作者: 剑啸寒    时间: 2016-9-21 11:35

先看看怎么样!  
作者: 求索迷茫    时间: 2016-9-22 08:18

哈哈,看的人少,回一下  
作者: 杏花    时间: 2016-10-25 20:54

观看中  
作者: 修复者    时间: 2016-10-25 21:43

我喜欢这个贴子  
作者: 海小鱼    时间: 2016-10-31 14:43

@,@..是什么意思呀?  
作者: abc987    时间: 2016-11-3 12:27

不错啊! 一个字牛啊!  
作者: 咖啡功夫猫    时间: 2016-11-9 09:35

我等你哟!  
作者: wq90    时间: 2016-12-18 19:09

干细胞美容
作者: 追风    时间: 2016-12-26 09:18

帮你顶,人还是厚道点好  
作者: 旅美学者    时间: 2016-12-28 12:23

今天没事来逛逛,看了一下,感觉相当的不错。  
作者: 糊涂小蜗牛    时间: 2017-1-5 19:10

ips是诱导多能干细胞induced pluripotent stem cells iPS
作者: 快乐小郎    时间: 2017-1-21 13:43

这个贴不错!!!!!  
作者: 咕咚123    时间: 2017-1-25 00:42

先顶后看  
作者: kaikai    时间: 2017-2-6 00:27

我的啦嘿嘿  
作者: HongHong    时间: 2017-2-8 04:02

几头雾水…  
作者: highlight    时间: 2017-2-24 09:10

呵呵,支持一下哈  
作者: kaikai    时间: 2017-3-3 01:57

干细胞分化技术
作者: 坛中酒    时间: 2017-3-9 11:02

干细胞之家
作者: 分子工程师    时间: 2017-3-22 12:43

祝干细胞之家 越办越好~~~~~~~~~`  
作者: chinagalaxy    时间: 2017-4-7 12:54

自己知道了  
作者: 橙味绿茶    时间: 2017-4-19 12:43

支持你就顶你  
作者: whyboy    时间: 2017-5-23 21:21

谁能送我几分啊  
作者: hmhy    时间: 2017-6-13 13:25

干细胞研究重在基础
作者: 舒思    时间: 2017-6-15 14:18

先顶后看  
作者: hmhy    时间: 2017-7-5 13:01

谁能送我几分啊  
作者: 墨玉    时间: 2017-7-23 17:20

我喜欢这个贴子  
作者: 多来咪    时间: 2017-7-24 21:16

琴棋书画不会,洗衣做饭嫌累。  
作者: 我学故我思    时间: 2017-8-19 02:29

我在顶贴~!~  
作者: lalala    时间: 2017-8-20 12:01

声明一下:本人看贴和回贴的规则,好贴必看,精华贴必回。  
作者: foxok    时间: 2017-8-20 16:43

几头雾水…  
作者: 小小C    时间: 2017-8-21 22:10

必须顶  
作者: 追风    时间: 2017-9-3 21:03

很好!很强大!  
作者: 修复者    时间: 2017-9-12 06:09

不错不错,我喜欢看  
作者: 橙味绿茶    时间: 2017-9-18 13:27

一个子 没看懂  
作者: biopxl    时间: 2017-9-19 10:53

干细胞之家
作者: 依旧随遇而安    时间: 2017-10-4 17:30

干细胞行业  
作者: 追风    时间: 2017-10-8 14:18

顶顶更健康,越顶吃的越香。  
作者: feixue66    时间: 2017-11-30 14:35

勤奋真能造就财富吗?  
作者: yunshu    时间: 2017-12-1 01:47

不错!  
作者: wq90    时间: 2017-12-3 01:10

我的啦嘿嘿  
作者: 咖啡功夫猫    时间: 2017-12-13 03:34

给我一个女人,我可以创造一个民族;给我一瓶酒,我可以带领他们征服全世界 。。。。。。。。。  
作者: MIYAGI    时间: 2017-12-18 10:35

谢谢分享了!  
作者: 老农爱科学    时间: 2018-1-9 04:11

楼上的话等于没说~~~  
作者: vsill    时间: 2018-1-16 07:01

我来了~~~~~~~~~ 闪人~~~~~~~~~~~~~~~~  
作者: keanuc    时间: 2018-1-26 15:43

今天没事来逛逛  
作者: 桦子    时间: 2018-2-3 15:43

加油啊!!!!顶哦!!!!!支持楼主,支持你~  
作者: 张佳    时间: 2018-2-7 10:10

我帮你 喝喝  
作者: xiaomage    时间: 2018-3-1 22:55

说的不错  
作者: 黄山    时间: 2018-3-6 22:54

这贴?不回都不行啊  
作者: dataeook    时间: 2018-3-13 12:54

看贴回复是好习惯  
作者: changfeng    时间: 2018-4-5 19:48

严重支持!
作者: 再来一天    时间: 2018-4-8 09:01

爷爷都是从孙子走过来的。  
作者: renee    时间: 2018-4-12 00:54

端粒酶研究
作者: ringsing    时间: 2018-5-3 05:01

挺好啊  
作者: 老农爱科学    时间: 2018-5-13 16:40

照你这么说真的有道理哦 呵呵 不进沙子馁~~~  
作者: s06806    时间: 2018-5-20 02:22

小生对楼主之仰慕如滔滔江水连绵不绝,海枯石烂,天崩地裂,永不变心.  
作者: 丸子    时间: 2018-6-27 00:26

干细胞与基因技术
作者: 心仪    时间: 2018-6-27 17:01

顶你一下,好贴要顶!  
作者: 昕昕    时间: 2018-7-11 11:58

希望可以用些时间了~````  
作者: apple0    时间: 2018-8-7 06:15

今天临床的资料更新很多呀
作者: 加菲猫    时间: 2018-8-24 22:57

哦...............  
作者: 我学故我思    时间: 2018-9-8 04:29

干细胞治疗糖尿病  
作者: 锦锦乐道    时间: 2018-9-22 22:49

我又回复了  
作者: ringsing    时间: 2018-9-24 12:18

干细胞行业  
作者: bioprotein    时间: 2018-10-3 19:11

在线等在线等  
作者: ringsing    时间: 2018-10-19 05:49

好人一个  




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