Protein fingerprinting with digital sequences of linear protein subsequence volumes: a computational study.

Proteins in a proteome can be identified from a sequence of K integers equal to the digitized volumes of subsequences with L residues from the primary sequence of a stretched protein. Exhaustive computations on the proteins of Helicobacter pylori (UniProt id UP000000210) with L and K in the range 4-8 show that approx. 90% of the proteins can be identified uniquely in this manner. This computational result can be translated into practice with a nanopore, an emerging technology that does not require analyte immobilization, proteolysis or labeling. Unlike other methods, most of which focus on a specific target protein, nanopore-based methods enable the identification of multiple proteins from a sample in a single run. Recent work by Kennedy, Kolmogorov and associates shows that the blockade current due to a protein molecule translocating through a nanopore is roughly proportional to one or more contiguous residues. The present study points to a modified version in which the volumes of subsequences (rather than of single residues) may be obtained by integrating the blockade current due to L contiguous residues. The advantages arising from this include lower detector bandwidth, elimination of the homopolymer problem and reduced noise. Because an identifier is based on near as well as distant (up to 2KL-L) residues, this approach uses more global information than an approach based on single residues and short-range correlations. The results of the study, which are available in a data supplement, are discussed in detail. Potential implementation issues are addressed.

Proteomic Sample Preparation through Extraction by Unspecific Adsorption on Silica Beads for ArgC-like Digestion.

Sample preparation for mass-spectrometry-based proteomic analyses usually requires intricate, multistep workflows that are often limited in capacity or suffer from sample loss. Here, we introduce a lean adsorption-based protocol (ABP) for the extraction of proteins from fresh cell lysates that enables us to modify and tag protein samples under harsh conditions, such as organic solvents, high salt concentrations, or low pH values. This offers high versatility while also reducing the required steps in the preparation process significantly. Protein identifications are slightly increased compared to traditional acetone precipitation followed by an in-solution digestion (AP/IS) or filter aided sample preparation (FASP) and proved complementary to both methods regarding proteome coverage. When combined with ArgC-like digestion, this approach delivered 5386 uniquely identified proteins, a substantial increase of 18.27% over tryptic digestion (4554), while decreasing spectra complexity due to a lower number of peptide to spectra matches per protein and the number of missed cleaved peptides. In addition, an increased number of identified membrane proteins and histones as well as improved fragmentation and intensity coverage were observed through comprehensive data analysis.

Combined Proteome and Toxicology Approach Reveals the Lethality of Venom Toxins from Jellyfish Cyanea nozakii.

Jellyfish are a type of poisonous cnidarian invertebrate that secrete lethal venom for predation or defense. Human beings often become victims of jellyfish stings accidentally while swimming or fishing and suffer severe pain, itching, swelling, inflammation, shock, and even death. Jellyfish venom is composed of various toxins, and the lethal toxin is the most toxic and hazardous component of the venom, which is responsible for deaths caused by jellyfish stings and envenomation. Our previous study revealed many toxins in jellyfish venom, including phospholipase A2, metalloproteinase, and protease inhibitors. However, it is still unknown which type of toxin is lethal and how it works. Herein a combined toxicology analysis, proteome strategy, and purification approach was employed to investigate the lethality of the venom of the jellyfish Cyanea nozakii. Toxicity analysis revealed that cardiotoxicity including acute myocardial infarction and a significant decrease in both heart rate and blood pressure is the primary cause of death. Purified lethal toxin containing a fraction of jellyfish venom was subsequently subjected to proteome analysis and bioinformation analysis. A total of 316 and 374 homologous proteins were identified, including phospholipase A2-like toxins and metalloprotease-like toxins. Furthermore, we confirmed that the lethality of the jellyfish venom is related to metalloproteinase activity but without any phospholipase A2 activity or hemolytic activity. Altogether, this study not only provides a comprehensive understanding of the lethal mechanism of jellyfish venom but also provides very useful information for the therapeutic or rescue strategy for severe jellyfish stings.

Glycoproteomic Alterations in Drug-Resistant Nonsmall Cell Lung Cancer Cells Revealed by Lectin Magnetic Nanoprobe-Based Mass Spectrometry.

Understanding the functional role of glycosylation-mediated pathogenesis requires deep characterization of glycoproteome, which remains extremely challenging due to the inherently complex nature of glycoproteins. We demonstrate the utility of lectin-magnetic nanoprobe (MNP@lectin) coupled to Orbitrap HCD-CID-MS/MS for complementary glycotope-specific enrichment and site-specific glycosylation analysis of the glycoproteome. By three nanoprobes, MNP@ConA, MNP@AAL, and MNP@SNA, our results revealed the first large-scale glycoproteome of nonsmall cell lung cancer (NSCLC) with 2290 and 2767 nonredundant glycopeptides confidently identified (Byonic score ≥100) in EGFR-TKI-sensitive PC9 and -resistant PC9-IR cells, respectively, especially with more fucosylated and sialylated glycopeptides in PC9-IR cells. The complementary enrichment was demonstrated with only five glycopeptides commonly enriched in three MNP@lectins. Glycotope specificity of 79 and 62% for enrichment was achieved using MNP@AAL and MNP@SNA, respectively. Label-free quantitation revealed predominant fucosylation in PC9-IR cells, suggesting its potential role associated with NSCLC resistance. Moreover, without immunoprecipitation, this multilectin nanoprobe allows the sensitive identification of 51 glycopeptides from 10 of 12 reported sites from onco-protein EGFR. Our results not only demonstrated a sensitive approach to study the vastly under-represented N-glycoprotome but also may pave the way for a glycoproteomic atlas to further explore the site-specific function of glycoproteins associated with drug resistance in NSCLC.

Protein profile of mouse ovarian follicles grown in vitro.

STUDY QUESTION: Could the follicle proteome be mapped by identifying specific proteins that are common or differ between three developmental stages from the secondary follicle (SF) to the antrum-like stage? SUMMARY ANSWER: From a total of 1401 proteins identified in the follicles, 609 were common to the three developmental stages investigated and 444 were found uniquely at one of the stages. WHAT IS KNOWN ALREADY: The importance of the follicle as a functional structure has been recognized; however, up-to-date the proteome of the whole follicle has not been described. A few studies using proteomics have previously reported on either isolated fully-grown oocytes before or after meiosis resumption or cumulus cells. STUDY DESIGN, SIZE, DURATION: The experimental design included a validated mice model for isolation and individual culture of SFs. The system was chosen as it allows continuous evaluation of follicle growth and selection of follicles for analysis at pre-determined developmental stages: SF, complete Slavjanski membrane rupture (SMR) and antrum-like cavity (AF). The experiments were repeated 13 times independently to acquire the material that was analyzed by proteomics. PARTICIPANTS/MATERIALS, SETTING, METHODS: SFs (n = 2166) were isolated from B6CBA/F1 female mice (n = 42), 12 days old, from 15 l. About half of the follicles isolated as SF were analyzed as such (n = 1143) and pooled to obtain 139 µg of extracted protein. Both SMR (n = 359) and AF (n = 124) were obtained after individual culture of 1023 follicles in a microdrop system under oil, selected for analysis and pooled, to obtain 339 µg and 170 µg of protein, respectively. The follicle proteome was analyzed combining isoelectric focusing (IEF) fractionation with 1D and 2D LC-MS/MS analysis to enhance protein identification. The three protein lists were submitted to the 'Compare gene list' tool in the PANTHER website to gain insights on the Gene Ontology Biological processes present and to Ingenuity Pathway Analysis to highlight protein networks. A label-free quantification was performed with 1D LC-MS/MS analyses to emphasize proteins with different expression profiles between the three follicular stages. Supplementary western blot analysis (using new biological replicates) was performed to confirm the expression variations of three proteins during follicle development in vitro. MAIN RESULTS AND THE ROLE OF CHANCE: It was found that 609 out of 1401 identified proteins were common to the three follicle developmental stages investigated. Some proteins were identified uniquely at one stage: 71 of the 775 identified proteins in SF, 181 of 1092 in SMR and 192 of 1100 in AF. Additional qualitative and quantitative analysis highlighted 44 biological processes over-represented in our samples compared to the Mus musculus gene database. In particular, it was possible to identify proteins implicated in the cell cycle, calcium ion binding and glycolysis, with specific expressions and abundance, throughout in vitro follicle development. LARGE SCALE DATA: Data are available via ProteomeXchange with identifier PXD006227. LIMITATIONS, REASONS FOR CAUTION: The proteome analyses described in this study were performed after in vitro development. Despite fractionation of the samples before LC-MS/MS, proteomic approaches are not exhaustive, thus proteins that are not identified in a group are not necessarily absent from that group, although they are likely to be less abundant. WIDER IMPLICATIONS OF THE FINDINGS: This study allowed a general view of proteins implicated in follicle development in vitro and it represents the most complete catalog of the whole follicle proteome available so far. Not only were well known proteins of the oocyte identified but also proteins that are probably expressed only in granulosa cells. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by the Portuguese Foundation for Science and Technology, FCT (PhD fellowship SFRH/BD/65299/2009 to A.A.), the Swedish Childhood Cancer Foundation (PR 2014-0144 to K.A.R-.W.) and Stockholm County Council to K.A.R-.W. The authors of the study have no conflict of interest to report.

Quantitative Secretome Analysis of Activated Jurkat Cells Using Click Chemistry-Based Enrichment of Secreted Glycoproteins.

Quantitative secretome analyses are a high-performance tool for the discovery of physiological and pathophysiological changes in cellular processes. However, serum supplements in cell culture media limit secretome analyses, but serum depletion often leads to cell starvation and consequently biased results. To overcome these limiting factors, we investigated a model of T cell activation (Jurkat cells) and performed an approach for the selective enrichment of secreted proteins from conditioned medium utilizing metabolic marking of newly synthesized glycoproteins. Marked glycoproteins were labeled via bioorthogonal click chemistry and isolated by affinity purification. We assessed two labeling compounds conjugated with either biotin or desthiobiotin and the respective secretome fractions. 356 proteins were quantified using the biotin probe and 463 using desthiobiotin. 59 proteins were found differentially abundant (adjusted p-value ≤0.05, absolute fold change ≥1.5) between inactive and activated T cells using the biotin method and 86 using the desthiobiotin approach, with 31 mutual proteins cross-verified by independent experiments. Moreover, we analyzed the cellular proteome of the same model to demonstrate the benefit of secretome analyses and provide comprehensive data sets of both. 336 proteins (61.3%) were quantified exclusively in the secretome. Data are available via ProteomeXchange with identifier PXD004280.

Proteomic Profiles Reveal the Function of Different Vegetative Tissues of Moringa oleifera.

Moringa oleifera is a rich source of bioactive compounds and is widely used in traditional medicine and food for its nutritional value; however, the protein and peptide components of different tissues are rarely discussed. Here, we describe the first investigation of M. oleifera proteomes using mass spectrometry and bioinformatics methods. We aimed to elucidate the protein profiles of M. oleifera leaves, stem, bark, and root. Totally 202 proteins were identified from four vegetative organs. We identified 101 proteins from leaves, 51 from stem, 94 from bark and 67 from root, finding that only five proteins existed in both four vegetative parts. The calculated pI of most of the proteins is distributed in 5-10 and the molecular weight distributed below 100 kDa. Functional classification analysis revealed that proteins which are involved in catalytic activities are the most abundant both in leaves, stem, bark and root. Identification of several heat shock proteins in four vegetative tissues might be adaptive for resistance to high temperature environmental stresses of tropical or subtropical areas. Some enzymes involved in antioxidant processes were also identified in M. oleifera leaves, stem, bark and root. Among the four tissues studies here, leaves protein content and molecular diversity were the highest. The identification of the flocculating protein MO2.1 and MO2.2 in the bark and root provides clue to clarify the antimicrobial molecular mechanisms of root and bark. This study provides information on the protein compositions of M. oleifera vegetative tissues that will be beneficial for potential drug and food supplement development and plant physiology research.

Proteome analysis of Pueraria mirifica tubers collected in different seasons.

Pueraria mirifica-derived tuberous powder has been long-term consumed in Thailand as female hormone-replacement traditional remedies. The protein profiles of tubers collected in different seasons were evaluated. Phenol extraction, 2D-PAGE, and mass spectrometry were employed for tuberous proteome analysis. Out of the 322 proteins detected, over 59% were functionally classified as being involved in metabolism. The rest proteins were involved in defense, protein synthesis, cell structure, transportation, stress, storage, and also unidentified function. The proteins were found to be differentially expressed with respect to harvest season. Importantly, chalcone isomerase, isoflavone synthase, cytochrome p450, UDP-glycosyltransferase, and isoflavone reductase, which are all involved in the biosynthesis pathway of bioactive isoflavonoids, were most abundantly expressed in the summer-collected tubers. This is the first report on the proteomic patterns in P. mirifica tubers in relevant with seasonal variation. The study enlights the understanding of variance isoflavonoid production in P. mirifica tubers.

Proteomic Analysis of Lonicera japonica Thunb. Immature Flower Buds Using Combinatorial Peptide Ligand Libraries and Polyethylene Glycol Fractionation.

Lonicera japonica Thunb. flower is a well-known medicinal plant that has been widely used for the treatment of human disease. To explore the molecular mechanisms underlying the biological activities of L. japonica immature flower buds, a gel-free/label-free proteomic technique was used in combination with combinatorial peptide ligand libraries (CPLL) and polyethylene glycol (PEG) fractionation for the enrichment of low-abundance proteins and removal of high-abundance proteins, respectively. A total of 177, 614, and 529 proteins were identified in crude protein extraction, CPLL fractions, and PEG fractions, respectively. Among the identified proteins, 283 and 239 proteins were specifically identified by the CPLL and PEG methods, respectively. In particular, proteins related to the oxidative pentose phosphate pathway, signaling, hormone metabolism, and transport were highly enriched by CPLL and PEG fractionation compared to crude protein extraction. A total of 28 secondary metabolism-related proteins and 25 metabolites were identified in L. japonica immature flower buds. To determine the specificity of the identified proteins and metabolites for L. japonica immature flower buds, Cerasus flower buds were used, which resulted in the abundance of hydroxymethylbutenyl 4-diphosphate synthase in L. japonica immature flower buds being 10-fold higher than that in Cerasus flower buds. These results suggest that proteins related to secondary metabolism might be responsible for the biological activities of L. japonica immature flower buds.

Heat-Treatment-Responsive Proteins in Different Developmental Stages of Tomato Pollen Detected by Targeted Mass Accuracy Precursor Alignment (tMAPA).

Recently, we have developed a quantitative shotgun proteomics strategy called mass accuracy precursor alignment (MAPA). The MAPA algorithm uses high mass accuracy to bin mass-to-charge (m/z) ratios of precursor ions from LC-MS analyses, determines their intensities, and extracts a quantitative sample versus m/z ratio data alignment matrix from a multitude of samples. Here, we introduce a novel feature of this algorithm that allows the extraction and alignment of proteotypic peptide precursor ions or any other target peptide from complex shotgun proteomics data for accurate quantification of unique proteins. This strategy circumvents the problem of confusing the quantification of proteins due to indistinguishable protein isoforms by a typical shotgun proteomics approach. We applied this strategy to a comparison of control and heat-treated tomato pollen grains at two developmental stages, post-meiotic and mature. Pollen is a temperature-sensitive tissue involved in the reproductive cycle of plants and plays a major role in fruit setting and yield. By LC-MS-based shotgun proteomics, we identified more than 2000 proteins in total for all different tissues. By applying the targeted MAPA data-processing strategy, 51 unique proteins were identified as heat-treatment-responsive protein candidates. The potential function of the identified candidates in a specific developmental stage is discussed.

Comparison of Different Sample Preparation Protocols Reveals Lysis Buffer-Specific Extraction Biases in Gram-Negative Bacteria and Human Cells.

We evaluated different in-solution and FASP-based sample preparation strategies for absolute protein quantification. Label-free quantification (LFQ) was employed to compare different sample preparation strategies in the bacterium Pseudomonas aeruginosa and human embryonic kidney cells (HEK), and organismal-specific differences in general performance and enrichment of specific protein classes were noted. The original FASP protocol globally enriched for most proteins in the bacterial sample, whereas the sodium deoxycholate in-solution strategy was more efficient with HEK cells. Although detergents were found to be highly suited for global proteome analysis, higher intensities were obtained for high-abundant nucleic acid-associated protein complexes, like the ribosome and histone proteins, using guanidine hydrochloride. Importantly, we show for the first time that the observable total proteome mass of a sample strongly depends on the sample preparation protocol, with some protocols resulting in a significant underestimation of protein mass due to incomplete protein extraction of biased protein groups. Furthermore, we demonstrate that some of the observed abundance biases can be overcome by incorporating a nuclease treatment step or, alternatively, a correction factor for complementary sample preparation approaches.

Quantitative Neuropeptidome Analysis Reveals Neuropeptides Are Correlated with Social Behavior Regulation of the Honeybee Workers.

Neuropeptides play vital roles in orchestrating neural communication and physiological modulation in organisms, acting as neurotransmitters, neuromodulators, and neurohormones. The highly evolved social structure of honeybees is a good system for understanding how neuropeptides regulate social behaviors; however, much knowledge on neuropeptidomic variation in the age-related division of labor remains unknown. An in-depth comparison of the brain neuropeptidomic dynamics over four time points of age-related polyethism was performed on two strains of honeybees, the Italian bee (Apis mellifera ligustica, ITb) and the high royal jelly producing bee (RJb, selected for increasing royal jelly production for almost four decades from the ITb in China). Among the 158 identified nonredundant neuropeptides, 77 were previously unreported, significantly expanding the coverage of the honeybee neuropeptidome. The fact that 14 identical neuropeptide precursors changed their expression levels during the division of labor in both the ITb and RJb indicates they are highly related to task transition of honeybee workers. These observations further suggest the two lines of bees employ a similar neuropeptidome modification to tune their respective physiology of age polyethism via regulating excretory system, circadian clock system, and so forth. Noticeably, the enhanced level of neuropeptides implicated in regulating water homeostasis, brood pheromone recognition, foraging capacity, and pollen collection in RJb signify the fact that neuropeptides are also involved in the regulation of RJ secretion. These findings gain novel understanding of honeybee neuropeptidome correlated with social behavior regulation, which is potentially important in neurobiology for honeybees and other insects.

According to the CPLL proteome sheriffs, not all aperitifs are created equal!

Combinatorial peptide ligand libraries (CPLLs) have been adopted for investigating the proteome of a popular aperitif in Northern Italy, called "Amaro Branzi", stated to be an infusion of a secret herbal mixture, of which some ingredients are declared on the label, namely Angelica officinalis, Gentiana lutea and orange peel, sweetened by a final addition of honey. In order to assess the genuineness of this commercial liqueur, we have prepared extracts of the three vegetable ingredients, assessed their proteomes, and compared them to the one found in the aperitif. The amaro's proteome was identified via prior capture with CPLLs at two different pH values (2.2 and 4.8). Via mass spectrometry analysis of the recovered fractions, after elution of the captured populations in 4% boiling SDS, we could confirm the presence of the following: six proteins originating from honey, 11 from orange peels, 29 from G. lutea and 46 from A. officinalis (including shared species), plus 33 species which could not be attributed to the other secret ingredients, due to paucity of genomic data on plant proteins, for a total of 93 unique gene products (merging shared proteins). This fully confirmed the genuineness of the product. Considering that most of these species could be present in trace amounts, undetectable by conventional techniques, the CPLL methodology, due to its ability to enhance the signal of trace components up to 3 to 4 orders of magnitude, could represent a powerful tool for investigating the genuineness and natural origin of commercial beverages in order to protect consumers from adulterated products.

Shotgun proteomic analysis of porcine colostrum and mature milk.

The epitheliochorial nature of the porcine placenta prevents the transfer of maternal immunity. Therefore, ingestion of the colostrum immediately after birth is crucial for neonatal piglets to acquire passive immunity from the sow. We performed a shotgun proteomic analysis of porcine milk to reveal in detail the protein composition of porcine milk. On the basis of the Swiss-Prot database, 113 and 118 proteins were identified in the porcine colostrum and mature milk, respectively, and 50 of these proteins were common to both samples. Some immune-related proteins, including interleukin-18 (IL-18), were unique to the colostrum. The IL-18 concentration in the colostrum and mature milk of four sows was measured to validate the proteomic analysis, and IL-18 was only detected in the colostrum (191.0 ± 53.9 pg/mL) and not in mature milk. In addition, some proteins involved in primary defense, such as azurocidin, which has never been detected in any other mammal's milk, were also identified in the colostrum.

An adaptive alignment algorithm for quality-controlled label-free LC-MS.

Label-free quantification using precursor-based intensities is a versatile workflow for large-scale proteomics studies. The method however requires extensive computational analysis and is therefore in need of robust quality control during the data mining stage. We present a new label-free data analysis workflow integrated into a multiuser software platform. A novel adaptive alignment algorithm has been developed to minimize the possible systematic bias introduced into the analysis. Parameters are estimated on the fly from the data at hand, producing a user-friendly analysis suite. Quality metrics are output in every step of the analysis as well as actively incorporated into the parameter estimation. We furthermore show the improvement of this system by comprehensive comparison to classical label-free analysis methodology as well as current state-of-the-art software.

Mass spectrometric evaluation of neuropeptidomic profiles upon heat stabilization treatment of neuroendocrine tissues in crustaceans.

Tissue heat stabilization is a vital component in successful mammalian neuropeptidomic studies. Heat stabilization using focused microwave irradiation, conventional microwave irradiation, boiling, and treatment with the Denator Stabilizor T1 have all proven effective in arresting post-mortem protein degradation. Although research has reported the presence of protein fragments in crustacean hemolymph when protease inhibitors were not added to the sample, the degree to which post-mortem protease activity affects neuropeptidomic tissue studies in crustacean species has not been investigated in depth. This work examines the need for Stabilizor T1 or boiling tissue stabilization methods for neuropeptide studies of Callinectes sapidus (blue crab) pericardial organ tissue. Neuropeptides in stabilized and nonstabilized tissue were extracted using acidified methanol or N,N-dimethylformamide (DMF) and analyzed by MALDI-TOF and nanoLC-ESI-MS/MS platforms. Post-mortem fragments did not dramatically affect MALDI analysis in the range m/z 650-1600, but observations in ESI MS/MS experiments suggest that putative post-mortem fragments can mask neuropeptide signal and add spectral complexity to crustacean neuropeptidomic studies. The impact of the added spectral complexity did not dramatically affect the number of detected neuropeptides between stabilized and nonstabilized tissues. However, it is prudent that neuropeptidomic studies of crustacean species include a preliminary experiment using the heat stabilization method to assess the extent of neuropeptide masking by larger, highly charged molecular species.

Beneficial effects of omega-3 fatty acids in the proteome of high-density lipoprotein proteome.

BACKGROUND: Omega-3 poly-unsaturated fatty acids (ω-3 PUFAs) have demonstrated to be beneficial in the prevention of cardiovascular disease, however, the mechanisms by which they perform their cardiovascular protection have not been clarified. Intriguingly, some of these protective effects have also been linked to HDL. The hypothesis of this study was that ω-3 PUFAs could modify the protein cargo of HDL particle in a triglyceride non-dependent mode. The objective of the study was to compare the proteome of HDL before and after ω-3 PUFAs supplemented diet. METHODS: A comparative proteomic analysis in 6 smoker subjects HDL before and after a 5 weeks ω-3 PUFAs enriched diet has been performed. RESULTS: Among the altered proteins, clusterin, paraoxonase, and apoAI were found to increase, while fibronectin, α-1-antitrypsin, complement C1r subcomponent and complement factor H decreased after diet supplementation with ω-3 PUFAs. Immunodetection assays confirmed these results. The up-regulated proteins are related to anti-oxidant, anti-inflammatory and anti-atherosclerotic properties of HDL, while the down-regulated proteins are related to regulation of complement activation and acute phase response. CONCLUSIONS: Despite the low number of subjects included in the study, our findings demonstrate that ω-3 PUFAs supplementation modifies lipoprotein containing apoAI (LpAI) proteome and suggest that these protein changes improve the functionality of the particle.

In-depth analysis of low abundant proteins in bovine colostrum using different fractionation techniques.

Bovine colostrum is well known for its large content of bioactive components and its importance for neonatal survival. Unfortunately, the colostrum proteome is complicated by a wide dynamic range, because of a few dominating proteins that hamper sensitivity and proteome coverage achieved on low abundant proteins. Moreover, the composition of colostrum is complex and the proteins are located within different physical fractions that make up the colostrum. To gain a more exhaustive picture of the bovine colostrum proteome and gather information on protein location, we performed an extensive pre-analysis fractionation of colostrum prior to 2D-LC-MS/MS analysis. Physical and chemical properties of the proteins and colostrum were used alone or in combination for the separation of proteins. ELISA was used to quantify and verify the presence of proteins in colostrum. In total, 403 proteins were identified in the nonfractionated colostrum (NF) and seven fractions (F1-F7) using six different fractionation techniques. Fractionation contributed with 69 additional proteins in the fluid phase compared with NF. Different fractionation techniques each resulted in detection of unique subsets of proteins. Whey production by high-speed centrifugation contributed most to detection of low abundant proteins. Hence, prefractionation of colostrum prior to 2D-LC-MS/MS analysis expanded our knowledge on the presence and location of low abundant proteins in bovine colostrum.

Comparative proteomic analysis of auxin-induced embryogenic and nonembryogenic tissues of the solanaceous tree Cyphomandra betacea (Tamarillo).

Cyphomandra betacea (tamarillo) is a tree that produces edible, highly nutritional fruits. In tamarillo, somatic embryogenesis (SE) is achieved through a two-step process starting with the formation of an embryogenic tissue on an auxin-rich medium and further development of embryos, following tissue transfer to an auxin-free medium. During the induction stage, both embryogenic (EC) and nonembryogenic calli (NEC) arise from the same explant (immature leaves or mature zygotic embryos) in the presence of either picloram or 2,4-dichlorophenoxyacetic acid. In an attempt to find somatic embryogenic-specific proteins, a comparative analysis of the proteome of tamarillo's EC and NEC was performed. Analysis of 2-DE gels revealed ca. 150 differentially expressed proteins, from which 22 have been identified by LC-MS/MS. Proteins exclusively or predominantly expressed in EC included metabolism-related proteins, such as enolases or treonine synthases, and also heat-shock and ribosomal proteins. Pathogenesis-related proteins were found mainly in NEC. A number of additional differentially expressed proteins involved in various functional categories were also identified. A quantitative real time PCR (qPCR) analysis revealed no significant differences at the mRNA level for 11 differentially expressed proteins, with exception of the pathogenesis-related proteins that were up-regulated in NEC. This seems to indicate that a posttranscriptional control might be responsible for the proteomic differences detected.

[Establishment of extraction method and 2-dimensional electrophoresis conditions for root tuber proteome analysis of Rehmannia glutinosa].

OBJECTIVE: To establish an efficient and high resolution 2-DE (two-dimensional electrophoresis) protocol for root tuber proteome analysis of Rehmannia glutinosa. METHOD: Proteins from root tuber of R. glutinosa were extracted by using five different methods and their productivity and profiles were assessed by means of SDS-PAGE and two-dimensional electrophoresis. RESULT: The trichloroacetic acid (TCA)-phenol extraction method was found most effective for the extraction with the highest protein yield, the most spots in protein patterns, and the highest resolution of proteins, and the clearest background could be achieved simultaneously. A 1:5 solution of ampholine pH 3-10 and pH 5-8 for a nonlinear gel and the 170 microg of protein loading dosage obtained maps with more protein spots and higher resolution of separation patterns. CONCLUSION: This study based on the optimized root tuber proteome preparation and the 2-DE protocol gets a high resolution and reproducibility 2-DE image, which will be expected to have excellent applications in proteomics studies of R. glutinosa tuber root.