Improvements for the visualization of low-molecular weight protein and peptides of human tears using MALDI

Many low-molecular weight proteins and peptides in human tears are potentially bioactive proteins but the range and number of these is yet to be fully characterized. A number of different sample preparation techniques were used to maximize the visualization of peptides from reflex tears. Samples were pretreated using precipitation and filtration techniques prior to analyses using MALDI-TOF MS. Peptides were searched for between 700 to 4000 m/z. Sample dilution in several different buffer systems followed by filtration with a 30-kDa cutoff filter and C18 reverse phase microcolumn purification produced significantly (p = 0.049) more peaks in tears than other methods used to prepare tears prior to MALDI-TOF MS. This study has established a technique for optimizing the visualization of naturally occurring peptides in tears.     

Differential proteomic analysis during the vegetative phase change and the floral transition in Malus domestica

In order to identify the proteomic changes of apple (Malus domestica Borkh.) during the vegetative phase change and the floral transition, leaf protein of juvenile, adult vegetative and reproductive phase in a seedling ('Jonathan' × 'Golden Delicious') was extracted and analyzed by 2-D electrophoresis and Matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Seventy two gel spots with significant expression differences between ontogenetic phases were obtained. Five protein spots were only detected in leaves of juvenile phase and 11 were not; 17 spots were found exclusively in adult vegetative leaves; and only one spot solely appeared in reproductive leaves while 12 did not. Twenty six of the differentially expressed proteins identified were involved in photosynthesis. Seven enzymes were related to respiration and carbohydrate metabolism. Fifteen other proteins also presented qualitative or quantitative differences among developmental phases. The spatial distribution of one differentially expressed protein, serine hydroxymethyltransferase, was confirmed by enzyme linked immunosorbent assay and immunohistochemistry. These results strongly support the idea that the vegetative phase change and the floral transition are regulated independently during developmental process.     

三种前处理在基质辅助激光解析电离飞行时间质谱鉴定假丝酵母菌属中的应用比较

目的 比较3种前处理方法在基质辅助激光解析电离飞行时间质谱(MALDI TOF MS)鉴定假丝酵母菌属中的结果可靠性。 方法 以ITS测序鉴定结果为金标准,对临床分离的66株假丝酵母分别采用传统直涂法、改良直涂法和甲酸-乙腈蛋白提取法进行前处理,MALDI TOF MS鉴定,比较3种方法的Biotyper Log值,分析质谱图的差异。 结果 传统直涂法、改良直涂法和甲酸-乙腈提取法对66株假丝酵母的属水平鉴定率分别为48.5%、50.0%和97.0%,Biotyper Log均值分别为1.628、1.674和2.010,其中甲酸-乙腈提取法对66株假丝酵母的种水平鉴定率为53.0%。甲酸-乙腈提取法得到的质谱图比另2种方法的质谱图离子峰更加密集,图像更复杂,鉴定结果可信度更高。 结论 甲酸-乙腈蛋白提取法对假丝酵母菌属的鉴定成功率和可靠性明显高于传统直涂法和改良直涂法,对临床假丝酵母菌病的准确诊断具有重要价值。     

Proteomic comparison between interphase and metaphase of isolated chloroplasts of Cyanidioschyzon merolae (Cyanidiophyceae,Rhodophyta)

With the completion of the Cyanidioschyzon merolae genome project, detailed analysis of organelle proteins with mass spectrometry has now become possible. Chloroplasts of the unicellular red alga Cyanidioschyzon merolae De Luca, Taddei et Varano (Rhodophyta, Cyanidiophyceae) were isolated from synchronized culture and the chloroplast lysates of both interphase and metaphase cells were prepared and subjected to 2D‐polyacrylamide gel electrophoresis. A total of 355 spots (170 identical spots) were recognized and quantified and then analyzed using mass spectrometry. A total of 105 proteins were identified, including 18 proteins for posttranslational functions, 17 photosynthesis‐related proteins, 10 carbohydrate‐related proteins, 15 proteins of unknown functions, and eight proteins predicted to be contaminated from other organelles. On the basis of spot quantity, photosynthesis‐related proteins were most dominant (45.3% in interphase and 56.4% in metaphase). In particular, the proteins forming phycobilisomal complexes were abundant. Comparison of interphase with metaphase revealed that CMG086C (aminomethyltransferase) notably increased in interphase. CMN235C (similar to chlorophyll a/b‐binding protein, CP24) increased in metaphase in agreement with a previously performed microarray analysis. Both CMQ295C (cell division protein FtsH) and CMS004C (plastid division protein FtsZ) increased in interphase. Seven proteins were detected to be interphase‐specific, and 12 proteins were metaphase‐specific. Proteins of unknown functions were poorly characterized by homology search, although thioredoxin‐like domains were predicted in several proteins.     

Pten^＋/＋MEFs与Pten^-/-MEFs细胞系的差异表达蛋白质组分析

目的：研究PTEN缺失细胞的蛋白表达规律。方法：用双向电泳技术比较Pten^＋/＋MEFs与Pten^-/-EFs细胞的蛋白表达差异;用基质辅助激光解吸／电离飞行时间质谱（MALDI-TOF-MS）对差异蛋白进行质谱分析;用肽质量指纹图谱检索数据库对差异蛋白进行鉴定;用Northern印迹和Western印迹验证蛋白的差异表达。结果：与Pten^＋/＋MEFs细胞相比。Pten^-/-MEFs细胞有明显的差异性蛋白表达谱,Pten^-/-MEFs细胞中表达下降的蛋白有铜锌超氧化物歧化酶、过氧化物氧化还原酶5和6等,表达增加的蛋白有低分子量蛋白酪氨酸磷酸酶和丝切蛋白（cofflin）1。结论：PTEN的缺失引起细胞内多种蛋白表达改变,这些表达改变的蛋白可能与PTEN缺失后细胞癌变相关。     

铜绿假单胞菌的MALDI-TOF-MS检测方法的建立

目的 建立利用基质辅助激光解吸电离飞行时间质谱仪( MALDI-TOF-MS)对铜绿假单胞菌的快速检测方法.方法 通过MALDI-TOF-MS法对铜绿假单胞菌进行检测分析,并与生化鉴定方法相比较.结果 MALDI-TOF-MS对铜绿假单胞菌的检测后得到肽指纹图片及相关质谱数据,建立MALDI-TOF-MS对铜绿假单胞菌的快速检测方法.结论 MALDI-TOF-MS方法检测铜绿假单胞菌准确快速、操作简单等特点,可发展成为食品检验铜绿假单胞菌的重要(辅助)工具.     

Chemical derivatization of phosphoserine and phosphothreonine containing peptides to increase sensitivity for MALDI-based analysis and for selectivity of MS/MS analysis

Protein phosphorylation is one of the most important and common ways of regulating protein function in cells. However, phosphopeptides are difficult to analyse, ionising poorly under standard MALDI conditions. Several methods have been developed to deal with the low sensitivity and specificity of phosphopeptide analysis. Here, we show an approach using a simple one-step beta-elimination/Michael addition reaction for the derivatization of phosphoserine and phosphothreonine. The substitution of the negatively charged phosphate group by a positively charged S-ethylpyridyl group greatly improves the ionisation of the modified peptides, especially in MALDI MS, increasing the sensitivity of the analysis. The modification allows the formation of a unique fragment ion at m/z 106 under mild collisional activation conditions, which can be used for parent (precursor) ion scanning in order to improve both the sensitivity and the selectivity of the analysis. The optimisation of the approach is described for a standard model peptide and protein and then applied to phosphorylation analysis in two biologically derived proteins purified from different experimental systems.     

Comparative Analysis of the Endosperm Proteins Separated by 2-D Electrophoresis for Two Cultivars of Hybrid Rice （Oryza sativa L,）

Liangyoupeijiu is a two-parental-line, and Shanyou63 is a three-parental-line hybrid rice （Oryza sativa L.）. Although both belong to the indica subspecies, they have obvious differences with respect to morphology, physiology and grain quality. Variations in endosperm protein compositions were studied by comparing the 2-D electrophoresis （2-DE） maps for these two cultivars of hybrid rice. After matrix-assisted laser desorption/ionization time of flight mass spectrometry （MALDI-TOF/MS） analysis, a 21-kDa precursor of 19- kDa globulin was identified as the major storage protein for both cultivars. Some isoforms of peroxiredoxin and seed maturation protein were found to only exist in Shanyou63, whereas aldose reductase and starch granule-bound starch synthase were only detected in Liangyoupeijiu. These data might provide a foundation for further comparative studies of these two cultivars of hybrid rice.     

Proteome analysis of silk gland proteins from the silkworm, Bombyx mori

The silk gland of Bombyx mori is an organ specialized for the synthesis and secretion of silk proteins. We report here the resolution of silk gland proteins by 2-DE and the identification of many of those proteins. This was accomplished by dissecting the glands into several sections, with each exhibiting more than 400 protein spots by 2-DE, of which 100 spots were excised and characterized by in-gel digestion followed by PMF. Ninety-three proteins were tentatively identified. These were then categorized into groups involved in silk protein secretion, transport, lipid metabolism, defense, etc. Western blotting of a 2-DE gel using an antibody of the carotenoid binding protein confirmed the presence of this protein in the silk gland. Proteins including fibroin L-chain and P25 were found as multiple isoforms, some of which contained differential amounts of phosphate residues as analyzed by on-probe dephosphorylation. The current analysis contributes to our understanding of proteins expressed by the silk gland not only of the model lepidopteran B. mori, but also to proteins from other silk-producing insects such as Philosamia cynthia ricini.     

Proteome analysis of human hepatocellular carcinoma tissues by two-dimensional difference gel electrophoresis and mass spectrometry

Proteome analysis of human hepatocellular carcinoma tissues was conducted using two-dimensional difference gel electrophoresis coupled with mass spectrometry. Paired samples from the normal and tumor region of resected human liver were labeled with Cy3 and Cy5, respectively while the pooled standard sample was labeled with Cy2. After analysis by the DeCyder software, protein spots that exhibited at least a two-fold difference in intensity were excised for in-gel tryptic digestion and matrix-assisted laser desorption/ionization-time of flight mass spectrometry. A total of 6 and 42 proteins were successfully identified from the well- and poorly-differentiated samples, respectively. The majority of these proteins are related to detoxification/oxidative stress and metabolism. Three down-regulated metabolic enzymes, methionine adenosyltransferase, glycine N-methyltransferase, and betaine-homocysteine S-methyltransferase that are involved in the methylation cycle in the liver are of special interest. Their expression levels, especially, methionine adenosyltransferase, seemed to have a major influence on the level of S-adenosylmethionine (AdoMet), a vital intermediate metabolite required for the proper functioning of the liver. Recent work has shown that chronic deficiency in AdoMet in the liver results in spontaneous development of steatohepatitis and hepatocellular carcinoma, and hence the down-regulation of hepatic methionine adenosyltransferase in our hepatocellular carcinoma samples is in line with this observation. Moreover, when a comparison is made between the differentially expressed proteins from our human hepatocellular carcinoma samples and from the liver tissues of knockout mice deficient in methionine adenosyltransferase, there is a fairly good correlation between them.     

Proteomic changes in rat serum, polymorphonuclear and mononuclear leukocytes after chronic nicotine administration

In order to gain information about the effect triggered at the molecular level by nicotine, its neuroimmunomodulatory properties and its impact on the pathogenesis of inflammatory diseases, peripheral blood serum and leukocytes of rat submitted to passive nicotine administration were subjected to proteomic investigation. Serum, polymorphonuclear (PMN) and mononuclear (MN) leukocytes from chronically treated animals and from control animals were analysed by a two-dimensional (2-D) gel electrophoresis/mass spectrometry approach to detect differentially expressed proteins. The nicotine regimen selected is known to have a stimulatory effect on locomotor activity and to produce a sensitisation of the mesolimbic dopamine system mechanism involved in addiction development. After 2-D gel analysis and matching, 36 spots in serum, seven in PMN and five in MN were found to display a statistical difference in their expression and were subjected to matrix-assisted laser desorption/ionization-time of flight-mass spectrometry peptide fingerprinting for protein identification. Fifteen different proteins were identified. The results indicate an overall impact of nicotine on proteins involved in a variety of cellular and metabolic pathways, including acute phase response (suggesting the effect on inflammatory cascades and more in general on the immune system), oxidative stress metabolism and assembly and regulation of cytoskeleton. In particular, the observed changes imply a general reduction in the inflammatory response with a concomitant increased unbalance of the oxidative stress metabolism in the periphery and point to a number of potential noninvasive markers for the central nervous system (CNS) and non-CNS mediated activities of nicotine.     

Identification and characterization of proteins interacting with SIRT1 and SIRT3: implications in the anti‐aging and metabolic effects of sirtuins

Sirtuins are a family of NAD⁺‐dependent protein deacetylases that regulate cellular functions through deacetylation of a wide range of protein targets. Overexpression of Sir2, the first gene discovered in this family, is able to extend the life span in various organisms. The anti‐aging effects of human homologues of sirtuins, SIRT1‐7, have also been suggested by animal and human association studies. However, the precise mechanisms whereby sirtuins exert their anti‐aging effects remain elusive. In this study, we aim to identify novel interacting partners of SIRT1 and SIRT3, two human sirtuins ubiquitously expressed in many tissue types. Our results demonstrate that SIRT1 and SIRT3 are localized within different intracellular compartments, mainly nuclei and mitochondria, respectively. Using affinity purification and MALDI‐TOF/TOF‐MS/MS analysis, their potential interacting partners have been identified from the enriched subcellular fractions and specific interactions confirmed by co‐immunoprecipitation and Western blotting experiment. Further analyses suggest that overexpression of SIRT1 or SIRT3 in HEK293 cells could induce hypoacetylation and affect the intracellular localizations and protein stabilities of their interacting partners. Taken together, the present study has identified a number of novel SIRT protein interacting partners, which might be critically involved in the anti‐aging and metabolic regulatory activities of sirtuins.     

Proteomics approach to identify wound-response related proteins from rice leaf sheath

In order to avoid the complex conditions of the intact plant for simple analysis of proteins in wound-response stress, we used the detached rice leaf sheath which is a very active part of the rice seedling. Proteins were extracted from rice leaf sheath at 0, 12, 24, 48 h after cutting and separated by two-dimensional (2-D) polyacrylamide gel electrophoresis. Changes in differentially displayed proteins were found in leaf sheaths after cutting in the 0-48 h time course. Ten proteins were up-regulated, while 19 proteins were down-regulated compared with those on the four 2-D gels. Among them, 14 proteins were analyzed by N-terminal, or internal amino acid sequence. The clear functions of nine proteins could be identified. Six proteins did not yield amino acid sequence information due to their blocked N-termini. Furthermore, 11 proteins were determined by matrix-assisted laser desorption/ionization-time of flight mass spectrometry, and identified protein database matching. It was shown that the down-regulated proteins were calreticulin (nos. 5, 6), histone H1 (no. 15) and hemoglobin (no. 17), putative peroxidase (no. 19); the up-regulated proteins were Bowman-Birk trypsin inhibitor (no. 23), putative receptor-like protein kinase (nos. 24, 25), calmodulin-related protein (no. 26), small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (no. 27), mannose-binding rice lectin (nos. 28, 29). Among all the above proteins, four (nos. 23, 24, 25, 26) have been confirmed to be wound-response proteins. The others cannot be excluded as also being related to wound-responses, such as the signal transduction-related proteins (nos. 5, 6), photosynthesis-related protein (no. 27), and stress-response proteins (nos. 19, 28, 29). This is the first time protein changes in response to wounding in rice leaf sheath have been shown.     

Direct mass spectrometric peptide profiling and fragmentation of larval peptide hormone release sites in Drosophila melanogaster reveals tagma-specific peptide expression and differential processing

Regulatory peptides represent a diverse group of messenger molecules. In insects, they are produced by endocrine cells as well as secretory neurones within the CNS. Many regulatory peptides are released as hormones into the haemolymph to regulate, for example, diuresis, heartbeat or ecdysis behaviour. Hormonal release of neuropeptides takes place at specialized organs, so-called neurohaemal organs. We have performed a mass spectrometric characterization of the peptide complement of the main neurohaemal organs and endocrine cells of the Drosophila melanogaster larva to gain insight into the hormonal communication possibilities of the fruit fly. Using matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) and MALDI-TOF-TOF tandem mass spectrometry, we detected 23 different peptides of which five were unpredicted by previous genome screenings. We also found a hitherto unknown peptide product of the capa gene in the ring gland and transverse nerves, suggesting that it might be released as hormone. Our results show that the peptidome of the neurohaemal organs is tagma-specific and does not change during metamorphosis. We also provide evidence for the first case of differential prohormone processing in Drosophila.     

Lipoproteomics I: mapping of proteins in low-density lipoprotein using two-dimensional gel electrophoresis and mass spectrometry

The molecular mechanisms underlying the relationship between low-density lipoprotein (LDL) and the risk of atherosclerosis are not clear. Therefore, detailed information about the protein composition of LDL may contribute to reveal its role in atherogenesis and the mechanisms that lead to coronary disease in humans. Here, we sought to map the proteins in human LDL by a proteomic approach. LDL was isolated by two-step discontinuous density-gradient ultracentrifugation and the proteins were separated with two-dimensional gel electrophoresis and identified with peptide mass fingerprinting, using matrix assisted laser desorption/ionization-time of flight-mass spectrometry and with amino acid sequencing using electrospray ionization tandem mass spectrometry. These procedures identified apo B-100, apo C-II, apo C-III (three isoforms), apo E (four isoforms), apo A-I (two isoforms), apo A-IV, apo J and apo M (three isoforms not previously described). In addition, three proteins that have not previously been identified in LDL were found: serum amyloid A-IV (two isoforms), calgranulin A, and lysozyme C. The identities of apo M, calgranulin A, and lysozyme C were confirmed by sequence information obtained after collision-induced dissociation fragmentation of peptides characteristic for these proteins. Moreover, the presence of lysozyme C was further corroborated by demonstrating enriched hydrolytic activity in LDL against Micrococcus lysodeikticus. These results indicate that in addition to the dominating apo B-100, LDL contains a number of other apolipoproteins, many of which occur in different isoforms. The demonstration, for the first time, that LDL contains calgranulin A and lysozyme C raises the possibility that LDL proteins may play hitherto unknown role(s) in immune and inflammatory reactions of the arterial wall.     

Proteomic profiling of eggs from a hybrid abalone and its parental lines: Haliotis discus hannai Ino and Haliotis gigantea

Proteomic analysis was performed on the eggs of hybrid abalone and their corresponding parental lines. A total of 915 ± 19 stained protein spots were detected from Haliotis discus hannai♀ × H. discus hannai♂ (DD), 935 ± 16 from H. gigantea♀ × H. gigantea♂ (GG) and 923 ± 13 from H. gigantea♀ × H. discus hannai♂ (GD). The spots from DD and GD were clustered together. The distance between DD and GG was maximal by hierarchical cluster analysis. A total of 112 protein gel spots were identified; of these, 59 were abalone proteins. The proteins were involved in major biological processes including energy metabolism, proliferation, apoptosis, signal transduction, immunity, lipid metabolism, electron carrier proteins, protein biosynthesis and decomposition, and cytoskeletal structure. Three of 20 differential expression protein spots involved in energy metabolism exhibited as upregulated in GD, 13 spots exhibited additivity, and four spots exhibited as downregulated in the offspring. Eleven protein spots were expressed at the highest level in DD. The proteins involved in stress responses included superoxide dismutase, peroxiredoxin 6, thioredoxin peroxidase and glutathione‐S‐transferase. Two of seven differential expression protein spots involved in response to stress exhibited as upregulated in GD, three exhibited additivity, and two exhibited as downregulated. These results might suggest that proteomic approaches are suitable for the analysis of hybrids and the functional prediction of abalone hybridization.