Proteomic Analysis of Osmotic Stress-Responsive Proteins in Sugarcane Leaves

Osmotic stress-related proteins in sugarcane were identified using proteomics approach based on two-dimensional polyacrylamide gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS). Sugarcane settlings were subjected to osmotic stress in the nutrient solution containing 10% (w/v) PEG 6000 for 14 h. Total proteins were extracted from leaves, and separated by 2-DE. Four typical spots exhibited significant changes in PEG treatment compared to control, which were identified using MALDI-TOF-MS successfully. The drought inducible 22 kDa protein and Rubisco small subunit were up-regulated while isoflavone reductase-like (IRLs, related to antioxidant defense system) protein and delta chain of ATP synthase were down-regulated by the osmotic stress. Analysis of the results showed that the most differential proteins under osmotic stress were acidic, unstable and transmembrane proteins, enriched with hydrophobic amino acids such as leucine and alanine which are extremely important for structural stabilization of proteins by hydrophobic interaction. However, the drought inducible 22 kDa protein was a hydrophile and non-transmembrane protein enriched with glutamic acid. These results provide new insight into the part of regulatory mechanism of adaptations to osmotic stress through differential expression of specific proteins and implicate several previously unrecognized proteins to osmotic stress.     

Proteomic Analysis of High Temperature Stress-Responsive Proteins in Chrysanthemum Leaves

Chrysanthemum is one of the most important ornamental flowers in the world, and temperature has a significant influence on its field production. In the present study, differentially expressed proteins were investigated in the leaves of Dendranthema grandiflorum ‘Jinba’ under high temperature stress using label-free quantitative proteomics techniques. The expressed proteins were comparatively identified and analyzed. A total of 1,463 heat-related, differentially expressed proteins were successfully identified by Liquid Chromatography-tandem Mass Spectrometry (LC-MS/MS), and 1,463 heat-related, differentially expressed proteins were successfully identified by mass spectrometry after a high temperature treatment. Among these, 701 proteins were upregulated and 762 proteins were downregulated. The in-depth bioinformatics analysis of these differentially expressed proteins revealed that these were involved in energy metabolism pathways, protein metabolism, and heat shock. In the present study, the investigators determined the changes in the levels of some proteins, and their expression at the protein and molecular levels in chrysanthemum to help reveal the mechanism of heat resistance in chrysanthemum. Furthermore, the present study elucidated some of the proteins correlated to heat resistance in chrysanthemum, and their expression changes at the protein and molecular levels to help reveal the mechanism of heat resistance in this flower species. These results provide a theoretical basis for the selection of new heat resistant varieties of chrysanthemum in the field.     

Proteomic Analysis of Rice Plasma Membrane-associated Proteins in Response to Chitooligosaccharide Elicitors

Chitooligomers or chitooligosaccharides （COS） are elicitors that bind to the plasma membrane （PM） and elicit various defense responses. However, the PM-bound proteins involved in elicitor-mediated plant defense responses still remain widely unknown. In order to get more information about PM proteins involved in rice defense responses, we conducted PM proteomic analysis of the rice suspension cells elicited by COS. A total of 14 up- or downregulated protein spots were observed on 2-D gels of PM fractions at 12 h and 24 h after COS incubation. Of them, eight protein spots were successfully identified by MS （mass spectrography） and predicted to be associated to the PM and function in plant defense, including a putative PKN/PRK1 protein kinase, a putative pyruvate kinase isozyme G, a putative zinc finger protein, a putative MAR-binding protein MFP1, and a putative calcium-dependent protein kinase. Interestingly, a COS-induced pM5-like protein was identified for the first time in plants, which is a transmembrane nodal modulator in transforming growth factor-β（TGFβ） signaling in vertebrates. We also identified two members of a rice polyprotein family, which were up-regulated by COS. Our study would provide a starting point for functionality of PM proteins in the rice basal defense.     

温敏核不育水稻花药蛋白质组初步分析

采用固相pH梯度 SDS聚丙烯酰胺双向凝胶电泳对温敏核不育水稻 96 4 2S可育与不育条件下减数分裂期花药总蛋白进行了分离 ,通过银染显色 ,获得了分辨率和重复性较好的双向电泳图谱 .PDQuest 2DE图像分析软件可识别约 10 0 0个蛋白质点 .蛋白质点在 2D胶上的重复性为 :沿等电聚焦方向偏差为 1 4 5± 0 2 3mm(n =8) ,沿SDS PAGE方向偏差为 :1 15± 0 17mm(n =8) .对两种育性不同样品的 2D胶上部分共有的蛋白质点 ,采用基质辅助激光解析电离飞行时间质谱 (matrixassistedlaserdesorption ionizationtimeofflightmassspctrometry ,MALDI TOF MS)进行了肽质谱指纹图分析 .通过采用PeptIdent软件对SWISS PROT数据库的查询 ,有 5 0个蛋白质点在数据库得到归属鉴定 .对育性不同的2种样品 2D较上明显差异的蛋白质点进行了分析鉴定 .在不育变化为可育的过程中 ,明显表达上调的蛋白质点包括几丁质酶 ,酸性磷酸酶 ,胞浆激酶 ,谷蛋白前体 ,以及ESTSC72 61蛋白 ,明显下调的蛋白质包括β expansin前体 ,谷氨酸氨甲酰转移酶和 1种未知功能的蛋白质     

Proteomic Analysis of Cd-Responsive Proteins in Solanum torvum

Proteomic changes induced by Cd have been described in plants in different scenarios. However, there has been no proteomic study on Cd toxicity, including any low Cd-accumulating species. Here, we investigate the response of a low Cd-accumulating species, Solanum torvum, to Cd toxicity at the root proteomic level using two-dimensional gel electrophoresis (2-DGE). The root 2-DGE map consisted of at least 927 reproducible protein spots, of which 45 were classified as differentially expressed proteins based on three replicated separations. MALDI-TOF MS analysis identified 19 of these spots, and MALDI-TOF/TOF MS analysis identified 8 of the spots. The eight proteins identified were two S-adenosylmethionine (SAM) synthetases, actin, an ATP synthase subunit, two tubulin proteins, alcohol dehydrogenase (ADH), and 14-3-3 protein 4. These proteins are involved in phytohormone synthesis, defense responses, energy metabolism, and cytoskeleton construction. Thus, our proteomic analysis revealed that Cd stress promotes an increase in the abundance of proteins involved in antioxidant defenses and anti-stress protection.     

Comparative Proteomic Analysis Reveals Nitrogen Fertilizer Increases Spikelet Number per Panicle in Rice by Repressing Protein Degradation and 14-3-3 Proteins

The spikelet number per panicle is established in the early stages of panicle development. Nitrogen fertilizer application before panicle initiation is known to increase spikelet number, which is one of the most important traits in rice productivity determination. However, the basic proteomic mechanism remains poorly understood. The present study shows that nitrogen fertilizer significantly increased spikelet number and grain yield in rice. Proteomic variations were further analyzed in young panicles at the secondary panicle branch initiation and spikelet meristem initiation under nitrogen fertilizer treatment. Proteomic analysis identified 63 proteins with significant differential accumulation in young panicles under nitrogen fertilizer treatment. Proteolysis represents the largest functional category, which suggests that protein degradation is an important pathway in the response to nitrogen fertilizer. Importantly, nitrogen fertilizer significantly reduced 14-3-3 proteins, which interact with key enzymes associated with carbon and nitrogen metabolism, and the rice FT homologue Hd3a. Real-time PCR revealed that Hd3a signaling is also repressed by nitrogen fertilizer in leaves. This study contributes to a better understanding of the regulation of nitrogen fertilizers in the flowering pathway leading to panicle development. The identification of novel genes provides new insight into the profound impacts of nitrogen fertilizer on panicle development in rice.     

甜杨低温响应microRNAs的克隆与分析

MicroRNAs (miRNAs)作为一类21碱基左右的非编码小RNAs,参与植物生长发育的调控,并在植物对生物与非生物胁迫的应答过程中发挥重要作用.本研究依据miRNA高度保守特点,利用已公布的毛果杨(Populus trichocarpa)基因组序列设计引物,从甜杨(Populus suaveolens)基因组中克隆获得了12个miRNA基因座序列.序列比对结果表明,这些miRNA基因均为毛果杨低温响应miRNA基因的同源序列.同时,以低温(0℃)处理0～48 h的甜杨幼苗为试材,通过半定量RT-PCR法对miRNA基因的成熟体序列在不同处理时间下的表达谱进行分析,结果显示,大多数miRNA成熟体序列在甜杨低温胁迫下的表达模式与其在毛果杨中的表达极为相似,由此可推测这些保守性miRNAs可能在甜杨和毛果杨两物种对低温胁迫的应答反应中发挥相似的功能,而miR168a、miR168b和miR475a在两物种间表达现象的差异,表明它们可能通过调控多种靶基因而发挥不同作用.本文结果将为进一步研究甜杨基因功能提供基础.     

Proteomic Analysis of Microtubule-associated Proteins during Macrophage Activation

Classical activation of macrophages induces a wide range of signaling and vesicle trafficking events to produce a more aggressive cellular phenotype. The microtubule (MT) cytoskeleton is crucial for the regulation of immune responses. In the current study, we used a large scale proteomics approach to analyze the change in protein composition of the MT-associated protein (MAP) network by macrophage stimulation with the inflammatory cytokine interferon-γ and the endotoxin lipopolysaccharide. Overall the analysis identified 409 proteins that bound directly or indirectly to MTs. Of these, 52 were up-regulated 2-fold or greater and 42 were down-regulated 2-fold or greater after interferon-γ/lipopolysaccharide stimulation. Bioinformatics analysis based on publicly available binary protein interaction data produced a putative interaction network of MAPs in activated macrophages. We confirmed the up-regulation of several MAPs by immunoblotting and immunofluorescence analysis. More detailed analysis of one up-regulated protein revealed a role for HSP90β in stabilization of the MT cytoskeleton during macrophage activation.Microtubules (MTs)¹ are major structural components of the cytoskeleton that are intricately involved in cell morphology, motility, division, and intracellular organization and transport. The diverse roles of MTs are dependent on the polymer having the capacity to be both dynamic and static in nature. Individual MTs alternate between growing and shrinking by the rapid attachment and detachment of tubulin subunits at their ends (1, 2). Thus, MTs can continually reorganize and undergo cycles of growing, pausing, and shortening. A number of mechanisms exist to regulate this dynamic equilibrium and involve association of proteins with the MT lattice. MT-associated proteins (MAPs), such as MAP4 and tau, stabilize MTs by binding to the wall thus inhibiting MT disassembly (3, 4). Recently MT plus (+) end-binding proteins have been implicated in stabilizing MTs by associating with cortical proteins to tether the MT end to peripheral target sites (5–7). Stabilized MT subsets are biochemically distinct and acquire posttranslational modifications that can be used to differentiate them from dynamic subsets. For example, posttranslational modifications such as glutamylation (8), detyrosination (8, 9), and acetylation (10) occur on MTs that exhibit increased stability. Stabilized MTs have been implicated in MT transport by allowing increased binding of MT motors (11, 12). Numerous other MAPs have been shown to regulate MT form and function including control of MT nucleation and elongation, MT linkage to and movement of organelles, and modulation of MT growth to allow scaffolding of signal transduction events (13).The extensive MT network provides a large surface area to serve as a platform for the binding of a large number of proteins that is likely heavily influenced by local cellular events and cell type. Traditionally the term MAP referred to proteins that bind directly to tubulin within the MT polymer, and a lot of recent debate and controversy have surrounded the definition of a MAP (14, 15). In this and other reports the definition of MAPs is considered to also include proteins that indirectly or transiently interact with MTs, co-localize with MTs, or influence MT growth dynamics in some way (16). The advent of proteomics has allowed cytoskeleton researchers to resolve the spectrum of MAPs. To date, the MT proteome has been resolved by MS analysis in developmentally important animal and plant models including Xenopus laevis egg extracts (17), Drosophila melanogaster embryos (18), Artemia franciscana embryos (19), Arabidopsis suspension cells (20), and complex mammalian tissues such as rat brain (21). The MT proteome has also been described for specialized MT structures including mitotic spindles (22–24), centrosomes (25, 26), and cilia (27, 28).Macrophages are key regulators of the immune system connecting innate and specific immune responses. Lipopolysaccharide (LPS), an outer membrane component of Gram-negative bacteria, is a potent activator of monocytes and macrophages. LPS triggers the abundant secretion of many cytokines from macrophages including IL-1 (29), IL-6, (30), and tumor necrosis factor-α (31), which together contributes to the pathophysiology of septic shock. IFN-γ is a proinflammatory cytokine produced by the host in response to intracellular pathogens. IFN-γ binds to IFN-γ receptors on macrophages, and IFN-γ signaling induces the production and/or release of cytokines, like IL-1 or tumor necrosis factor-α, which enhance LPS-mediated effects (32). Thus, the synergy between LPS and inflammatory cytokines such as IFN-γ represents an important regulatory mechanism by which the host tackles a significant, ongoing infection before it activates potent effector responses (33). It has been demonstrated that LPS may cause changes in monocyte cytoskeleton and directly influence assembly of isolated MTs (34). Recently we observed that classical activation of murine resident peritoneal or RAW264.7 macrophages with a combination of IFN-γ and LPS induces an increase in stabilized cytoplasmic MTs (5). A significant effort has been made to unravel the importance of stable MTs in cellular processes over the past few years. With respect to macrophage function, stable MTs could potentially function as tracks for vesicle secretion of cytokines and matrix metalloproteinases necessary to effect the enhanced inflammatory response observed in classically activated macrophages. We recently demonstrated that stable MTs are important for cell spreading as well as the binding of large particles in activated macrophages (5). The stabilization of macrophage interphase MTs is uniquely rapid, thus serving as an ideal model for studying MAPs involved in MT modulation in mammalian cells.The focus of the present study was to identify the MT-associated proteins involved in altering and stabilizing MT structures and also to resolve the spectrum of proteins within the MT proteome of a mammalian cell. To achieve this goal, we used a proteomics approach involving a MAP purification technique based on MT co-sedimentation (35) followed by off-line fractionation and identification of MAPs using LC-MS/MS. Information provided by mass spectrometry analysis allowed us to analyze the changes in MAP abundance during activation of macrophages by IFN-γ/LPS. These studies also provided candidate proteins for selective molecular intervention for chronic inflammatory disorders.     

红海榄根部盐胁迫反应的比较蛋白质组学分析

红海榄(Rhizophora stylosa)是一种典型的红树林盐生植物.本研究利用蛋白组学技术对淡栽(R0)和3% NaCl盐栽(R3)处理后的红海榄根部总蛋白进行了比较研究.双向电泳图谱的结果表明,R0和R3分别有981和972个蛋白点,蛋白点主要集中在分子量28～70 kD,等电点4.0～8.5之间. R0和R3之间差异明显的有15个蛋白点.其中,8个蛋白的表达量在R0中表达增高（10倍）,而在R3中相对下降.另外,7个蛋白的表达量在R0中较低,而在R3中表达量显著增高.对这15个蛋白点进行肽质量指纹图谱分析,10个蛋白点找到匹配蛋白.功能预测分析发现,在盐水栽培上调的蛋白质一般与逆境胁迫有关,淡水栽培上调的蛋白质一般与基本代谢有关.这些研究结果为进一步研究红海榄的耐盐机理提供了有意义的线索.     

Proteomic Analysis of Membrane-Associated Proteins from Rat Liver Autophagosomes

Proteins associated with membranes from purified rat liver autophagosomes were separated by two-dimensional (2D) gel electrophoresis (zoom gels, pI 4-7 and 6-9), silver-stained and identified by MALDI-TOF mass spectrometry. Among >1,500 detectable protein spots, 58 (derived from 39 different known proteins) were at least twofold (and significantly) enriched in autophagosomal membranes relative to cytoplasmic membranes. All of these membrane-associated proteins were also present in the cytosol, many of them being truncated enzyme variants that would be expected to serve a binding rather than an enzymatic function.     

Proteomic Analysis of Salt Stress Responses in Rice Shoot

To gain a better understanding of the mechanism of rice (Oryza sativa L.) in response to salt stress, we performed a proteomics analysis of rice in response to 250 mM NaCl treatment using shoots of 3-day-old nascent seedlings. The changes of protein patterns were monitored with two-dimensional gel electrophoresis. Of 57 protein spots showing changes in abundance in response to salt stress, 52 were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The identified proteins were classified into eight functional categories. Several novel salt stress-responsive proteins, including protein synthesis inhibitor I, photosystem II stability/assembly factor HCF136, trigger factor-like protein and cycloartenol-C24-methyltransferase are upregulated upon salt stress. In order to figure out the different and similar molecular mechanism among salt and other stresses, regulation of some salt responsive proteins under other abiotic stress (cold and dehydration) and abscisic acid application was also analyzed. The possible molecular mechanism of rice seedlings in response to salinity and other stresses were discussed.     

Proteomic Analysis of Specific Proteins in the Root of Salt-Tolerant Barley

Comparative two-dimensional electrophoresis showed six proteins, which were significantly produced in the root of salt-tolerant barley. These proteins were identified as stress/defense-related proteins that do not scavenge reactive oxygen species directly, suggesting that salt-tolerant barley develops not only an antioxidative system, but also physical and biochemical changes to cope with salt stress.     

Characteristics of Nitrate Reductase-inactivating Proteins Obtained from Corn Roots and Rice Cell Cultures

Nitrate reductase (NR)-inactivating proteins from corn roots (Wf-9 × 38-11) and rice cell suspension cultures were tested against a partially purified NR obtained from corn leaves (W64A × W182E). The corn protein was purified 921-fold and the rice protein, 1,660-fold using standard purification procedures. Approximate molecular weight values were 75,000 for the corn protein, and 150,000 for the rice protein as determined by Sephadex G-100 gel filtration. The Sephadex-treated proteins were characterized by electrophoresis on polyacrylamide gels. With a running pH of 9.4 the corn protein remained at the origin whereas the rice protein migrated with an R_F value of 0.49. With a running pH of 4.0 the corn protein migrated with an R_F value of 0.25. With the corn protein the activities of NR inactivation and hydrolysis of azocasein were detected in the same protein band. The rice protein, however, had no associated protease activity. From sodium dodecyl sulfate gel electrophoresis, there was one major protein band with an estimated molecular weight of 66,000 in corn protein. In rice protein four bands were observed with estimated molecular weights of 73,000, 66,000, 62,500, and 58,500, respectively.     

Proteomic Analysis of Rice Seedlings Under Cold Stress

Low temperature can greatly restrict the growth and development of rice. The rice seedlings show growth retardation, lamina wrap, and part of blade even died under the condition of low temperature. In order to get more information about cold stress responses in rice, two dimensional electrophoresis and bioinformatics analysis of mass spectrometry were used to preliminary survey the cold tolerance of cold sensitive line 9311 and cold resistance variety Fujisaka 5 under cold stress. Two dimensional electrophoresis maps of 9311 and Fujisaka 5 were established under cold treatment. With analysis of bioinformation, the proteins were found involve in many aspects of rice development. The largest category of proteins is functioning on metabolism. By comparing the proteins from the two varieties, it can be found that most proteins from 9311 were down-regulated and were up-regulated in Fujisaka 5. The results showed that the membrane composition and structure were damaged, metabolism changed dramatically and rice defense system was activated under the cold stimulation. Fifty-nine proteins related to the resistance of cold stress were identified in our study, and we have investigated and classified all of their biological functions. The importance of our study are providing some conduct for the research of rice resistant to cold stress, supporting auxiliary technique for rice varieties and widening the search field of cold tolerance in plants.     

人原发性肺腺癌转移相关分子的定量蛋白质组学研究

癌细胞转移是人原发性肺腺癌(lung adenocarcinoma, AdC)死亡率高和预后差的主要原因．为了筛选潜在的肺腺癌转移相关分子标志物,依据临床诊断选取无转移的肺腺癌组织和有转移的肺腺癌组织作为研究对象,首先采用激光捕获显微切割技术(laser capture microdissection, LCM)对两组肺腺癌组织中的癌细胞进行纯化,再利用荧光差异凝胶电泳技术(two-dimensional differential in-gel electrophoresis, 2D-DIGE)分离无转移肺腺癌组和有转移肺腺癌组的癌细胞总蛋白,通过Decyder软件分析两组差异表达的蛋白质点,质谱(mass spectrometry, MS)对差异表达的蛋白质点进行鉴定,Western blot验证部分差异蛋白annexin A1, annexin A2, annexin A3, B23和 S100A9的表达．建立了LCM 纯化的无转移和有转移的肺腺癌组织癌细胞的2D-DIGE图谱,质谱鉴定了20个非冗余差异蛋白质,其中12个蛋白质在有转移肺腺癌组中较无转移肺腺癌组表达上调,8个蛋白质在有转移肺腺癌组中表达下调．Western blot验证分析显示,差异蛋白annexin A1,annexin A2,annexin A3和 S100A9的表达水平在有转移肺腺癌中较无转移肺腺癌增高,B23的表达水平在有转移肺腺癌中较无转移肺腺癌降低．免疫组化进一步证实S100A9在有转移的肺腺癌中较无转移的肺腺癌中表达上调．首次应用LCM技术联合2D-DIGE及MS技术分析鉴定出肺腺癌转移相关蛋白质,为研究肺腺癌的转移分子机制、筛选预测肺腺癌转移的分子标志物奠定了基础．     

Proteomic Analysis of Protease Resistant Proteins in the Diabetic Rat Kidney

Glycation induced protein aggregation has been implicated in the development of diabetic complications and neurodegenerative diseases. These aggregates are known to be resistant to proteolytic digestion. Here we report the identification of protease resistant proteins from the streptozotocin induced diabetic rat kidney, which included enzymes in glucose metabolism and stress response proteins. These protease resistant proteins were characterized to be advanced glycation end products modified and ubiquitinated by immunological and mass spectrometry analysis. Further, diabetic rat kidney exhibited significantly impaired proteasomal activity. The functional analysis of identified physiologically important enzymes showed that their activity was reduced in diabetic condition. Loss of functional activity of these proteins was compensated by enhanced gene expression. Aggregation prone regions were predicted by in silico analysis and compared with advanced glycation end products modification sites. These findings suggested that the accumulation of protein aggregates is an inevitable consequence of impaired proteasomal activity and protease resistance due to advanced glycation end products modification.One of the foremost causes of diabetic complications is formation of sugar-derived substances called advanced glycation end products (AGEs),¹ which affect target cell through altered protein structure- function, matrix-matrix/matrix-cell interaction, and by activation of receptor for AGE (RAGE) signaling pathway (1). Although the accumulation of AGEs is a slow process in healthy individuals, their formation is markedly accelerated in diabetes because of hyperglycemia (2). AGE-modified proteins are thermostable and resistant to denaturation. The stability of proteins is believed to be because of additional negative charge (highly oxidized state) brought by AGE modification of proteins, which may contribute to protease resistance (3). Glycation induced protease resistance has been studied in collagen (4–6) and amyloid (7). In addition to glycation, impairment in the proteasomal function may facilitate accumulation of protease resistant protein aggregates in diabetes. Proteasome mediated protein degradation is a central quality control mechanism in the cell. Activity of proteasome is affected during aging (8) and physiological disorders like diabetes (9) resulting in accumulation of ubiquitinated protein aggregates. In muscle extract of diabetic rats, accumulation of toxic glycated proteins was observed because of decreased proteasomal activity (6–9). This proteolytic system is of particular importance in protecting cells against adverse conditions, such as heat shock, glycation, or oxidative stress. However, when the generation of damaged proteins exceeds the capacity of the cell to degrade them, they are progressively accumulated leading to cytotoxicity (10). Severely aggregated, cross-linked, and oxidized proteins are poor substrates for degradation and inhibit the proteasomal activity (11).The kidney is one of the main organs affected in diabetes caused by accumulation of AGEs. Proteins of extracellular matrix, kidney, as well as proteins from circulation, get AGE modified and trapped in the kidney (12). Both intracellular and extracellular AGEs have been observed in the diabetic kidney. Extracellular AGEs interact with the RAGE leading to apoptosis and inflammation (13), whereas intracellular AGEs are formed because of various dicarbonyls. Eventually, both types of the AGEs contribute to kidney damage (14). Furthermore, methyl glyoxal, a highly reactive dicarbonyl covalently modifies the 20S proteasome, decreasing its activity in the diabetic kidney (15). Together AGE modification and decreased proteasomal function may be responsible for the accumulation of protease resistant proteins (PRPs) in the diabetic kidney. In our previous study, we have reported the presence of AGE modified proteins in the kidney of the streptozotocin (STZ) induced diabetic rat (12). The current work is inspired by a DARTS (drug affinity responsive target stability) approach, wherein the drug targets are relatively less susceptible to protease action on drug binding (16). A similar approach was adopted here to identify protease resistant proteins from the diabetic kidney. These proteins were characterized to be AGE modified and ubiquitinated by Western blot analysis and mass spectrometry. Functional characterization and expression analysis of some of the identified proteins was performed to gain insight into the consequences of these modifications in diabetes. Further, aggregation prone regions in these proteins were predicted by the in silico approach. These findings shed light on the role of identified PRPs in diabetic complications.