蛋白芯片文献解读
文献实例一
抗体芯片技术应用于肿瘤侵袭机制研究（Journal of Clinical Investigation 2010）
Emory大学的研究者通过前期实验筛选到RSK2蛋白，认为RSK2与头颈部鳞状细胞癌转移相关。研究者首先使用头颈部鳞状细胞癌细胞系、临床标本进行了RSK2表达水平的检测，继而进行了RSK2活性抑制实验，RNAi实验，以及stable knock down实验证明了RSK2与肿瘤细胞的转移相关，在此基础上，研究者将RSK2 knock out的头颈部鳞状细胞癌细胞系接种到裸鼠体内，观察到敲除后的细胞系不影响肿瘤形成，但是影响了肿瘤的转移。
A：高转移能力细胞系M4e RSK2 stable knock down；
B：淋巴结vimentin免疫组化检测；
C： RSK2敲除后肿瘤形成未受影响；D：肿瘤Ki－67免疫组化检测。
那么RSK2影响肿瘤转移的机制是什么呢？Western Blot检测发现与转移相关的Erk、Akt、Stat3都没有变化，那么其他激酶呢？
A：M4e细胞RSK2敲除前后Erk、Akt、Stat3磷酸化水平没有变化；
B： MAPK 信号通路以及RSK2在信号通路中的位置
根据已有的研究，RSK2与MAPK通路密切相关，因此研究者选择了MAPK pathway的磷酸化水平检测芯片（包括185种抗体，约90个磷酸化位点），对RSK2敲除前后的M4e细胞进行了检测，以磷酸化水平变化15％为标准，筛选到C-MET、FAK、C-JUN、CREB、HSP27等蛋白质磷酸化水平下调。
其中C-MET与FAK处在通路图的上游，而且是酪氨酸激酶，而RSK2是丝氨酸苏氨酸激酶，WB的结果验证了芯片结果是可靠的，作者分析，这两个蛋白的磷酸化之所以发生改变，应该是RSK2敲除后，肿瘤细胞的转移前信号通路重新响应的结果；
而对于CREB蛋白，已知RSK2可以调节与激活CREB蛋白，作者在不同细胞系中又进行了CREB敲除、RSK2过表达与CREB敲除、野生型以及突变型CREB过表达实验，发现CREB的侵袭能力依靠RSK2表达与激活ser133磷酸化；
研究者发现的另一个非常值得关注的成员是HSP27蛋白，它有可能是RSK2的一种新的底物。为了探索RSK2与HSP27的关系，进行了体外测定实验，在野生型、S15、S78、S82突变型的HSP28中分别加入RSK2，实验发现RSK2可以磷酸化野生型的S78、S82位点，而不影响S15位点。这说明HSP27是RSK2的一种新的磷酸化底物；
作者进而进行了一系列转染、突变等试验，证实HSP27的侵袭能力需要RSK27激活S78、S82磷酸化；
A：RNAi Hsp27显著影响细胞的侵袭能力；
B：只有稳定转染Hsp27 S78与S82双磷酸化突变的细胞侵袭能力显著提高。
同时，作者直接观察了HSP27与肌动蛋白关系。发现RSK2敲除后，肌动蛋白无法维持其结构，当转染S78、S82磷酸化双突变的HSP27后，肌动蛋白的结构获得稳定；
以上说明RSK2通过调节HSP27磷酸化来维持肌动蛋白的稳定性。
讨论中，作者对抗体芯片的应用特别指出，抗体芯片是一种高通量的手段，磷酸化与非磷酸化抗体的配对对实验数据的处理带来了便利，芯片的应用不仅可以鉴定出潜在的底物，还可以发现新的相互作用，例如C-MET和RSK2；最后一点，芯片提供了一种发现与要研究的蛋白相关的新的信号通路以及全面理解其作用机制的手段。
文献实例二
抗体芯片技术与Western Blot等技术的比较（Proteomics Clinical Applications 2009）
美国NIS与NIH的研究者对于信号通路抗体芯片技术进行了探索与评价，研究者选用了HepG2细胞模型，采用胰岛素干预的方法，使用抗体芯片、Luminex悬浮芯片技术以及Western Blot的方法对干预后胰岛素信号通路的磷酸化水平进行了检测，并对结果进行了比较与评价。
研究中采用的不同技术具有以下特点：
· 胰岛素受体信号通路抗体芯片，包括97种抗体，识别大约24种蛋白的43个磷酸化位点；
· 悬浮芯片Luminex，包括7种抗体，其中6种识别磷酸化位点，1种为GAPDH抗体，作为内参；
· Western Blot，使用7种抗体，可检测4种磷酸化位点。
主要结果如下：
| Luminex | FMB Array | |||||
| Insulin | PMA | PMA+Insulin | Insulin | PMA | PMA+Insulin | |
| Akt (Ser473) | 1.21 | 1.17 | 1.07 | 2.03 | 1.37 | 1.24 |
| FOXO1a (Ser256) | 1.23 | 1.17 | 1.17 | 1.35 | 0.84 | 0.87 |
| IRS-1 (Ser307) | 0.47 | 1.47 | 1.1 | 0.81 | 2.73 | 1.63 |
| p44/42 MAP Kinase (Thr202) | 1.23 | 1.13 | 1.13 | 1.51 | 0.72 | 1.03 |
| NFkB-p65 (Ser536) | 0.48 | 1.5 | 1.08 | 0.42 | 0.92 | 0.96 |
| Raf1 (Ser259) | 1.67 | 1.11 | 1.21 | 2.09 | 1.18 | 1.48 |
悬浮芯片平台、Western blot检测到的位点磷酸化水平调变趋势与抗体芯片检测的结果一致，例如Akt (Ser473)、IRS-1 (Ser307) 、Raf1 (Ser259)、FOXO1a。
对于本次实验获得的结果，作者总结后认为，在以往磷酸化研究中，经常使用质谱的方法，而这种方法经常会遇到富集环节等困难，而且在研究中经常用到的是对已知磷酸化位点的研究，因此抗体芯片技术是一种很好的补充，第二抗体芯片技术自身包括了磷酸化抗体与非磷酸化抗体，根据得到的结果可以直接计算出磷酸化程度，不需要使用内参；第三，抗体芯片技术是一种高通量的手段，相比较于以往的手段WB，能够获得更多的信息，发现一些蛋白可能的新功能，例如这篇文献，作者发现了HSP27与PTEN可能与佛博酯引起的胰岛素抵抗有关。
文献实例三
抗体芯片在寻找药物作用靶点中的作用(Leukemia 2012)
CBL纯合突变在许多白血病中广泛存在，该突变降低泛素连接酶活性从而使SFK(SRC家族激酶)和RTK(受体酪氨酸激酶)活性受影响。CBL纯合突变导致的白血病预后差，需要更好的治疗方法。作者提出鉴定最重要的SFK和RTK作为分子靶点可能为寻找更有效的治疗方法提供参考。
作者首先采用SNP芯片结合测序的方法从30个细胞系中找到CBL RFD R420Q突变细胞系GDM-1 。
a：白血病细胞系中的UPD11q；
b：对GDM-1的UPD11q测序发现位于RFD的R420Q突变。
c：(ARMS)-PCR结果显示GDM-1细胞系中只有CBL R420Q(163bp)而没有WT CBL(216bp)。
流式细胞仪实验，WT CBL异位表达和shRNA敲低实验进一步证实GDM-1细胞系极具CBL纯和突变的代表性(表现出高的KIT表达和GM-CSF高敏感性)，WT CBL异位表达会抑制GDM-1的增殖，但不会抑制野生型CBL细胞系THP-1。
那么，各种RTK和SFK抑制剂对这个GDM-1的抑制作用是怎样的呢？作者用7种抑制剂处理GDM-1，2个control细胞系(THP1和U937)以及CBL杂合突变细胞系(MOLM13)，发现SFK和RTK双重抑制剂Dasatinib(达沙替尼)能特意性抑制GDM-1。
那么，怎么去找出这个抑制剂特异性抑制的蛋白呢？作者用到了FMB公司的包含228个酪氨酸磷酸化位点的Tyrosine Phosphorylation proarray，检测dasatinib ( SFK和RTK双重抑制剂)或sunitinib (RTK抑制剂)处理后的GDM-1细胞裂解液。分别找到44个和56个位点蛋白磷酸化水平降低。
对其进行venn分析后即找到dasatinib特异性抑制的蛋白靶点，包括26种蛋白(29个酪氨酸位点)被Dasatinib特异性抑制，不仅包括了RTK(KIT，FGFR1，M-CSFR)，也有其他磷酸化蛋白(SRC，STAT1，STAT3，p73)。一些蛋白是与活化的KIT和GM-CSF受体下游的级联反应相关。
文献示例四
疾病标志物筛选(Human Cytokin Array)
SatorisInc和Stanford大学的一个研究小组利用RayBiotech的抗体芯片（AAH-CYT）从老年患者的血浆中鉴定出18个生物标志蛋白。用这18个生物标志蛋白来预测阿兹海默症（Alzheimer's disease）与诊断结果相比有高达90%的准确率；而且可以用来鉴别出一些有轻微认知障碍的人，这些人在2-6年后也可能进展为阿兹海默症患者。
这一发现的意义在于对阿兹海默症进行分子水平的检测能够帮助完善对此病症的处理及治疗。
使用Full Moon公司芯片发表的文献：
CBL mutation-related patterns of phosphorylation and sensitivity to tyrosine kinase inhibitors
H Makishima, Y Sugimoto, H Szpurka… - Leukemia, 2012 - nature.com... For detection of 228 site-specific tyrosine phosphorylation profiles with six replicates each, 100 ug of cell lysate was labeled with biotin and hybridized on Tyrosine Phosphorylation ProArray (Full Moon BioSystems, Sunnyvale, CA, USA) using Antibody Array Assay Kit (Full ...
Rapid identification of monospecific monoclonal antibodies using a human proteome microarray
JS Jeong, L Jiang, E Albino, J Marrero… - Molecular & Cellular Proteomics, 2012 - ASBMB... microarrays on Full Moon slides (Full Moon BioSystems, Sunnyvale, CA).
Current Knowledge on Microarray Technology-An Overview
CK Naidu… - Tropical Journal of Pharmaceutical Research, 2012... Clontech Antibody Microarrays 380 & 500 Screens 380 or 507 antibodies in one day. Full Moon BiosystemsPhospho Explorer S1 Antibody Array Consists of 562 well-characterized
phospho-specific antibodies, a subset of the Phospho Explorer Array (PEX100). ...
Increased prevalence of regulatory T cells in the lung cancer microenvironment: a role of thymic stromal lymphopoietin
H Li, H Zhao, J Yu, Y Su, S Cao, X An… - Cancer Immunology, 2011 - Springer... DCs, LPS-DCs, and imDCs was analyzed using phospho-specific protein microarray (Full Moon Biosystems, Inc., CA ... The antibody microarray was blocked with blocking solution at room temperature for 45 min ... The array was incubated with the bio- tin-labeled cell lysates for 1-2 ...
Cardioprotective Effects of Salvianolic Acid A on Myocardial Ischemia-Reperfusion Injury In Vivo and In Vitro
F Huaying, Y Liu, F Fenghua, X Hui… - Evidence-Based …, 2011 - hindawi.com Salvianolic acid A (SAA), one of the major active components of Danshen that is a traditional Chinese medicine, has been reported to possess protective effect in cardiac diseases and antioxidative activity. This study aims to investigate the cardioprotection of SAA in vivo and ...
Cellular Iron Depletion Stimulates the JNK and p38 MAPK Signaling Transduction Pathways, Dissociation of ASK1-Thioredoxin, and Activation of ASK1
Y Yu… - Journal of Biological Chemistry, 2011 - ASBMB... As an internal control for protein loading, membranes were also probed for -actin. MAPK Phospho-antibody Array. A phospho-specific MAPK antibody array (Full Moon Biosystems,Sunnyvale, CA) was utilized as described previously (32). ...
Inhibition of eEF-2 kinase sensitizes human glioma cells to TRAIL and down-regulates Bcl-xL expression
Y Zhang, Y Cheng, L Zhang, X Ren… - Biochemical and biophysical research communications, 2011.. á-Tubulin antibody was obtained from Santa Cruz (Santa Cruz, CA, USA); Antibody Microarray Assay Kit was obtained from Full Moon Biosystems (Sunnyvale, CA, USA). All of the cell culture media and other reagents were from Invitrogen (Carlsbad, CA, USA).
Cocaine hijacks ó1 receptor to initiate induction of activated leukocyte cell adhesion molecule: implication for increased monocyte adhesion and migration in the CNS
H Yao, K Kim, M Duan, T Hayashi… -Journal of Neuroscience 2011,... PDGF antibody array. PDGF antibody array was performed according to the manufacturer's instructions (Full Moon Biosystems). Briefly, confluent cultures of HBMECs treated with cocaine (10 ìm) or untreated were used for isolation of lysates. ...
Targeting GPCR-mediated p70S6K activity may improve head and neck cancer response to cetuximab
NE Bhola, SM Thomas, M Freilino, S Joyce… - Clinical Cancer Research, 2011... Phosphoprotein arrays were obtained from FullMoonBiosystems. ... Furthermore, our antibody array showed that phospho-IRS (Ser312) was increased 2-fold in EGFR siRNA-transfected cells ...
Mesenchymal Activin-A Overcomes Defective Human Trisomy 21 Trophoblast Fusion
P Gerbaud, G Pidoux, J Guibourdenche… - Endocrinology, 2011... Louis, MO; signaling pathway antagonists LY294002 (440202), G? 6976 (6976), and Y27632 (688000) from Calbiochem La Jolla, CA; TGF? signaling phos- pho-antibody array (PTG176) from Full Moon Biosystems, Inc., Sunnyvale, CA; and RayBio arrays (AAH-CYT-8) from ...
Bernier M, Paul RK, et al.:Negative Regulation of STAT3-mediated Cellular Respiration by SirT1, Journal of Biological Chemistry 2011, 286(22):19270-9
EFEMP1 suppresses malignant glioma growth and exerts its action within the tumor extracellular compartment
H Yuanjie, P Peter, S Eric, Z Qinghua, N Jodi… - Molecular Cancer - 2011, 10:123 Cox regression revealed that EFEMP1 is a favorable prognostic marker for patients with GBM. Over-expression of EFEMP1 eliminated tumor development and suppressed angiogenesis, cell proliferation, and VEGFA expression, while the converse was true with ...
-Convergence of the Mammalian Target of Rapamycin Complex 1-and Glycogen Synthase Kinase 3-ignaling Pathways Regulates the Innate Inflammatory
H Wang, J Brown, Z Gu, CA Garcia… - Journal of Immunology 2011, 186:5217-5226... Phospho-Ab microarray and CREB nuclear binding assay. The Human mTOR Signaling Phospho-Specific Antibody Microarray was purchased from and analyzed by Full Moon Biosystem. More than 130 highly specific and characterized ... Gomez JC, Doerschuk CM: Rac2 in murine pulmonary microvascular endothelial cells (ECs),FASEB J. 24: 777.11
Proteome alterations in Primary Open angle Glaucoma Aqueous Humour
AIzzotti, MG Longobardi, C Cartiglia… - Journal of Proteome Research, 2010... 500 (Clontech, CA, USA) and Explorer Antibody Microarray (Full Moon BioSystems, Inc, Sunnyvale,CA, USA). ... n°7030444, Clontech, CA, USA), and the second one was coated with 656 proteins (Explorer Antibody Microarray, Full Moon BioSystems, Inc., Sunnyvale, CA, USA). ...
Affinity Peptidomics: Peptide Selection and Affinity Capture on Hydrogels and Microarrays
F Zhang, ADulneva, J Bailes… - Methods in Molecular Biology, 2010... of micro-manufacturing technologies, commercial antibody arrays do not contain more than just a few hundred antibodies, eg 224 and 725 antibody arrays (PanoramaTMAb Microarrays, Sigma-Aldrich) or 656 antibody arrays (Spring Bio- science, or Full Moon BioSystems). ...
p90 ribosomal S6 kinase 2 promotes invasion and metastasis of human head and neck squamous cell carcinoma cells
S Kang, S Elf, K Lythgoe, T Hitosugi… - Journal of Clinical Investigation 2010, 120(4):1165-1177 Abstract Head and neck squamous cell carcinoma (HNSCC) is one of the most common types of human cancer and frequently metastasizes to LNs. Identifying metastasis-promoting factors is of immense clinical interest, as the prognosis for patients with even a single ...
Sensing the insulin signaling pathway with an antibody array
HJ He, Y Zong, M Bernier… - Proteomics Clinical Applications, 2009 - 3:1440-1450 The development of insulin resistance and type 2 diabetes is determined by various factors, including defects within the insulin signaling pathway. Mediators of insulin resistance operate through activation of various protein kinase C isoforms, IêB kinase (IKK), and/ ...
PGC-1-related coactivator modulates mitochondrial-nuclear crosstalk through endogenous nitric oxide in a cellular model of oncocytic thyroid tumours
M Raharijaona, S Le Pennec, J Poirier… - PLoS One, 2009 Background The PGC-1 related coactivator (PRC), which shares structural and functional features with PGC-1á, is believed to regulate several metabolic pathways as well as mitochondrial biogenesis. Its involvement in the early programming of cell proliferation ...
Nuclear targeting of 6-phosphofructo-2-kinase (PFKFB3) increases proliferation via cyclin-dependent kinases
A Yalcin, BF Clem, A Simmons, A Lane… - Journal of Biological Chemistry, 2009 Abstract The regulation of metabolism and growth must be tightly coupled to guarantee the efficient use of energy and anabolic substrates throughout the cell cycle. Fructose 2, 6-bisphosphate (Fru-2, 6-BP) is an allosteric activator of 6-phosphofructo-1-kinase (PFK-1), ...
The glycolytic inhibitor 2-deoxyglucose activates multiple prosurvival pathways through IGF1R
D Zhong, L Xiong, T Liu, X Liu, X Liu… - Journal of Biological Chemistry, 2009 Abstract Recent molecular studies indicate that aerobic glycolysis plays an important role in tumorigenesis and is a valid target for cancer therapy. Although 2-deoxyglucose (2-DG) is well characterized as a glycolytic inhibitor, we recently discovered that it activates a ...
LKB1 is necessary for Akt-mediated phosphorylation of proapoptotic proteins
D Zhong, X Liu, FR Khuri, SY Sun, PM Vertino… - Cancer research, 2008 Abstract LKB1 plays the role of tumor suppressor, opposite to Akt, by negatively regulating mammalian target of rapamycin through the activation of AMP-activated protein kinase and TSC signaling. We have discovered a novel, potentially oncogenic role for LKB1 as a ...
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