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Haris, Parvez I. "Can infrared spectroscopy provide information on protein–protein interactions?" Biochemical Society Transactions 38, no. 4 (July 26, 2010): 940–46. http://dx.doi.org/10.1042/bst0380940.
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For most biophysical techniques, characterization of protein–protein interactions is challenging; this is especially true with methods that rely on a physical phenomenon that is common to both of the interacting proteins. Thus, for example, in IR spectroscopy, the carbonyl vibration (1600–1700 cm−1) associated with the amide bonds from both of the interacting proteins will overlap extensively, making the interpretation of spectral changes very complicated. Isotope-edited infrared spectroscopy, where one of the interacting proteins is uniformly labelled with 13C or 13C,15N has been introduced as a solution to this problem, enabling the study of protein–protein interactions using IR spectroscopy. The large shift of the amide I band (approx. 45 cm−1 towards lower frequency) upon 13C labelling of one of the proteins reveals the amide I band of the unlabelled protein, enabling it to be used as a probe for monitoring conformational changes. With site-specific isotopic labelling, structural resolution at the level of individual amino acid residues can be achieved. Furthermore, the ability to record IR spectra of proteins in diverse environments means that isotope-edited IR spectroscopy can be used to structurally characterize difficult systems such as protein–protein complexes bound to membranes or large insoluble peptide/protein aggregates. In the present article, examples of application of isotope-edited IR spectroscopy for studying protein–protein interactions are provided.
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Chaud, Saula Goulart, Admar Costa de Oliveira, and Paulo César Ocheuze Trivelin. "Nitrogen 15 abundance in protein fractions of beans fertilized with (15NH4)2SO4." Scientia Agricola 59, no. 4 (December 2002): 777–80. http://dx.doi.org/10.1590/s0103-90162002000400023.
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Studies evaluating the protein nutritive value of beans labelled with 15N, ussing nitrogen balance and the quantitation of faecal and urinary endogenous nitrogen, determined by isotopic dilution, have been extensively used. The objective of this research was to verify if the isotopic labelling of raw, freeze dried beans (Phaseolus vulgaris L., cultivar Piratã 1) with 1.394 atoms%15N, resulted in the same abundance of the whole flour and of the protein fractions extracted from the beans with 0.5 mol L-1 NaCl. The isotopic abundance found in the whole bean flour, in the protein extract, in the globulin and albumin fractions were respectively: 1.394 ± 0.011; 1.403 ± 0.012; 1.399 ± 0.007 and 1.399 ± 0.028 atoms % of 15N, presenting no difference (P > 0.05). However, a difference was found (P < 0.05) between the above mentioned abundances and the isotopic abundance found in the nitrogen of the proteins in the extraction residue, which was 0.969 ± 0.084. Since the abundances did not differ, the protein nutritive indexes, such as digestibility and biological value, determined from the nitrogen balance and corrected for isotopic dilution, would not be affected by extracting the proteins from the beans with 0.5 mol L¹ NaCl. If working with the nitrogen balance of the residual proteins after extraction and even with the whole flours, these indexes could present incorrect values, since the isotopic labelling of the residual proteins was less than that of the protein fractions.
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Muona, Mikko, A. Sesilja Aranko, and Hideo Iwai. "Segmental Isotopic Labelling of a Multidomain Protein by Protein Ligation by Protein Trans-Splicing." ChemBioChem 9, no. 18 (December 15, 2008): 2958–61. http://dx.doi.org/10.1002/cbic.200800604.
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Clifton, Luke A. "Unravelling the structural complexity of protein–lipid interactions with neutron reflectometry." Biochemical Society Transactions 49, no. 4 (July 9, 2021): 1537–46. http://dx.doi.org/10.1042/bst20201071.
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Neutron reflectometry (NR) is a large-facility technique used to examine structure at interfaces. In this brief review an introduction to the utilisation of NR in the study of protein–lipid interactions is given. Cold neutron beams penetrate matter deeply, have low energies, wavelengths in the Ångstrom regime and are sensitive to light elements. High differential hydrogen sensitivity (between protium and deuterium) enables solution and sample isotopic labelling to be utilised to enhance or diminish the scattering signal of individual components within complex biological structures. The combination of these effects means NR can probe buried structures such as those at the solid–liquid interface and encode molecular level structural information on interfacial protein–lipid complexes revealing the relative distribution of components as well as the overall structure. Model biological membrane sample systems can be structurally probed to examine phenomena such as antimicrobial mode of activity, as well as structural and mechanistic properties peripheral/integral proteins within membrane complexes. Here, the example of the antimicrobial protein α1-purothionin binding to a model Gram negative bacterial outer membrane is used to highlight the utilisation of this technique, detailing how changes in the protein/lipid distributions across the membrane before and after the protein interaction can be easily encoded using hydrogen isotope labelling.
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RYCHEN, GUIDO, DIDIER MPASSI, STEPHAN JURJANZ, MICHEL MERTES, IRENE LENOIR-WIJNKOOP, JEAN MICHEL ANTOINE, and FRANÇOIS LAURENT. "15N as a marker to assess portal absorption of nitrogen from milk, yogurt and heat-treated yogurt in the growing pig." Journal of Dairy Research 69, no. 1 (February 2002): 95–101. http://dx.doi.org/10.1017/s0022029901005374.
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Milk and yogurt constitute a major source of dietary protein. The nutritive value of dietary proteins is linked to subsequent postprandial amino acid availability in the portal blood (Rérat, 1988). Portal absorption of nutrients cannot be studied in humans, but pigs provide a valid model for studying protein digestion in humans (Rowan et al. 1994).Since stable isotopes are suitable to distinguish the exogenous from endogenous protein fraction in the intestinal lumen, intrinsic isotopic labelling of milk proteins has been considered a useful technique for nutritional investigations (Gaudichon et al. 1995; Gaudichon et al. 1999; Mahé et al. 1994). Recently, the use of 15N-labelled milk proteins were used to distinguish exogenous from endogenous N fractions in the human intestine after ingestion of 15N-milk or 15N-yogurt (Gaudichon et al. 1995). These authors pointed out that the jejunal flux of 15N was different for milk and yogurt. It is known that milk proteins and lactose undergo preliminary hydrolysis during lactic fermentation (Tamine & Deeth, 1980). It is also suggested that lactic fermentation enhances the nutritional value of milk proteins (Vass et al. 1984).
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THABET, AHMED, JOHANNES SCHMIDT, SVEN BAUMANN, WALTHER HONSCHA, MARTIN VON BERGEN, ARWID DAUGSCHIES, and BERIT BANGOURA. "Resistance towards monensin is proposed to be acquired in a Toxoplasma gondii model by reduced invasion and egress activities, in addition to increased intracellular replication." Parasitology 145, no. 3 (September 5, 2017): 313–25. http://dx.doi.org/10.1017/s0031182017001512.
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SUMMARYMonensin (Mon) is an anticoccidial polyether ionophore widely used to control coccidiosis. The extensive use of polyether ionophores on poultry farms resulted in widespread resistance, but the underlying resistance mechanisms are unknown in detail. For analysing the mode of action by which resistance against polyether ionophores is obtained, we induced in vitro Mon resistance in Toxoplasma gondii-RH strain (MonR-RH) and compared it with the sensitive parental strain (Sen-RH). The proteome assessment of MonR-RH and Sen-RH strains was obtained after isotopic labelling using stable isotope labelling by amino acid in cell culture. Relative proteomic quantification between resistant and sensitive strains was performed using liquid chromatography-mass spectrometry/mass spectrometry. Overall, 1024 proteins were quantified and 52 proteins of them were regulated. The bioinformatic analysis revealed regulation of cytoskeletal and transmembrane proteins being involved in transport mechanisms, metal ion-binding and invasion. During invasion, actin and microneme protein 8 (MIC8) are seem to be important for conoid extrusion and forming moving junction with host cells, respectively. Actin was significantly upregulated, while MIC8 was downregulated, which indicate an invasion reduction in the resistant strain. Resistance against Mon is not a simple process but it involves reduced invasion and egress activity of T. gondii tachyzoites while intracellular replication is enhanced.
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LÓPEZ-HELLÍN, Joan, Ricardo GONZALO, Mónica TEJEDA, Montserrat CARRASCAL, Maya R. VILÀ, Joaquín ABIÁN, and Elena GARCÍA-ARUMÍ. "Transcriptomic and proteomic analysis of liver and muscle alterations caused by surgical stress in rats." Clinical Science 108, no. 2 (January 21, 2005): 167–78. http://dx.doi.org/10.1042/cs20040144.
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The metabolic response to injury includes major alterations in protein metabolism; however, little is known about alterations in the synthesis of individual proteins and their role in the stress response. Our aim was to study how individual proteins in liver and muscle are altered by abdominal surgery. Changes produced in mRNA and proteins by abdominal surgery were studied in rats using RAP (random arbitrary priming)-PCR, to investigate mRNA alterations, and standard or isotopic (with in vivo radioactive labelling of proteins) two-dimensional electrophoresis/MS proteomic analyses, to study differential expression of proteins. Many of the differentially expressed proteins identified in blood were specifically synthesized by the liver to participate in the stress response. The hepatic proteins (antioxidant proteins, serine protease inhibitors, acute-phase proteins and transport proteins) were secreted into the bloodstream to produce a systemic action, indicating the central role of the liver in the stress response. Overexpressed proteins identified in liver were associated with the glycolytic processes and the folding of nascent proteins, confirming the high metabolic activity of the liver after surgery. The role of skeletal muscle protein as an amino acid donor to fuel the processes involved in the stress response was shown by the decrease in high-molecular-mass myofibrillar proteins. Combined use of the three techniques studied, differential RAP-PCR and standard and isotopic proteome analysis, provided complementary information on the differentially expressed proteins in a rat model of surgical stress.
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Feldberg, R. S., D. A. Iannitti, and D. E. Cochrane. "Histidine decarboxylase from rat mast cells. Enhanced recovery in cell-free extracts and isotopic labelling." Biochemical Journal 249, no. 1 (January 1, 1988): 297–300. http://dx.doi.org/10.1042/bj2490297.
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A procedure for obtaining rat mast-cell histidine decarboxylase in greater than 50% yield in cell-free extracts was developed. The enzyme was found in the supernatant fractions from a 3,500 g and a 105,000 g centrifugation step and was demonstrated to be sensitive to inhibition by alpha-fluoromethylhistidine but not by phenylalanine. Although the enzyme shows a half-life of only 3 h in cell-free extract, the initial high recovery of activity allowed for active-site labelling of the enzyme by [3H]histidine and NaBH4. Labelled protein migrated on non-denaturing polyacrylamide-gradient-gel electrophoresis as a 55,000 Da species.
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Bequette, B. J., F. R. C. Backwell, M. S. Dhanoa, A. Walker, A. G. Calder, D. Wray-Cahen, J. A. Metcalf, et al. "Kinetics of blood free and milk casein-amino acid labelling in the dairy goat at two stages of lactation." British Journal of Nutrition 72, no. 2 (August 1994): 211–20. http://dx.doi.org/10.1079/bjn19940025.
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The kinetics of blood free amino acids (AA) transfer into milk casein were compared in goats (n 4) at 61 (SE5)d (Expt 1; post-peak, 4.51 (SE 0.26) kg milk/d) and at 180 (SE 6) d (Expt 2; late, 2.36 (SE 0.16) kg milk/d) of lactation during non-primed, continuous (Expt 1, 12 h; Expt 2, 16 h) intravenous infusions of mixtures of L-[1-13C]leucine and L-[1-13C]phenylalanine with either L-[1-13C]valine (Expt 1) or L-[5-13Cmethionine (Expt 2). The 13C enrichments of blood free and casein-bound AA were fitted to a single exponential model to estimate isotopic plateaux and the fractional rate constant for milk casein labelling. Milk protein output and its contribution to whole-body flux was higher in Expt 1 (post-peak) than in Expt 2 (late lactation), but the kinetics of 13C labelling of the casein-bound AA were similar for all AA tracers in both experiments. At both stages of lactation the delay (6–8 h) between the attainment of isotopic plateau for the blood free AA and the corresponding attainment of plateau for the casein-bound AA indicated that the blood free pool was not the immediate precursor pool for milk casein biosynthesis. Plateau enrichments of casein-bound AA were generally higher than those for the corresponding blood free AA in both experiments. These results indicate that the relative contributions of different AA sources to the immediate precursor pool for milk casein biosynthesis are similar at different stages of lactation despite major changes in the partitioning of whole-body flux towards milk protein output. Non-milk protein fluxes were also similar in post-peak and late lactation.
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Chang, Shih Chieh, Charles A. Galea, Eleanor W. W. Leung, Rajeev B. Tajhya, Christine Beeton, Michael W. Pennington, and Raymond S. Norton. "Expression and isotopic labelling of the potassium channel blocker ShK toxin as a thioredoxin fusion protein in bacteria." Toxicon 60, no. 5 (October 2012): 840–50. http://dx.doi.org/10.1016/j.toxicon.2012.05.017.
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Fan, Nai-Jun, Chun-Fang Gao, Chang-Song Wang, Jing-Jing Lv, Guang Zhao, Xin-Hua Sheng, Xiu-Li Wang, Dong-Hui Li, Qing-Yin Liu, and Jian Yin. "Discovery and Verification of Gelsolin as a Potential Biomarker of Colorectal Adenocarcinoma in a Chinese Population: Examining Differential Protein Expression using an Itraq Labelling-Based Proteomics Approach." Canadian Journal of Gastroenterology 26, no. 1 (2012): 41–47. http://dx.doi.org/10.1155/2012/645218.
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Despite the wide range of available colorectal cancer (CRC) screening tests, less than 50% of cases are detected at early stages. However, the identification of differentially expressed proteins or novel protein biomarkers in CRC may have some utility and, ultimately, improve patient care and survival. Proteomics combined with mass spectroscopy and liquid chromatography are emerging as powerful tools that have led to the discovery of potential markers in cancer biomarker discovery in several types of cancers. This article describes a novel technology that uses isotopic reagents to tag selected proteins that show a consistent pattern of differential expression in CRC.OBJECTIVE: To identify and validate potential biomarkers of colorectal adenocarcinoma using a proteomic approach.METHODS: Multidimensional liquid chromatography/mass spectrometry was used to analyze biological samples labelled with isobaric mass tags for relative and absolute quantitation to identify differentially expressed proteins in human colorectal adenocarcinoma and paired normal mucosa for the discovery of cancerous biomarkers. Cancerous and noncancerous samples were compared using online and offline separation. Protein identification was performed using mass spectrometry. The downregulation of gelsolin protein in colorectal adenocarcinoma samples was confirmed by Western blot analysis and validated using immunohistochemistry.RESULTS: A total of 802 nonredundant proteins were identified in colorectal adenocarcinoma samples, 82 of which fell outside the expression range of 0.8 to 1.2, and were considered to be potential cancer-specific proteins. Immunohistochemistry revealed a complete absence of gelsolin expression in 86.89% of samples and a reduction of expression in 13.11% of samples, yielding a sensitivity of 86.89% and a specificity of 100% for distinguishing colorectal adenocarcinoma from normal tissue.CONCLUSIONS: These findings suggest that decreased expression of gelsolin is a potential biomarker of colorectal adenocarcinoma.
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Morgado, Leonor, Ana P. Fernandes, Joana M. Dantas, Marta A. Silva, and Carlos A. Salgueiro. "On the road to improve the bioremediation and electricity-harvesting skills of Geobacter sulfurreducens: functional and structural characterization of multihaem cytochromes." Biochemical Society Transactions 40, no. 6 (November 21, 2012): 1295–301. http://dx.doi.org/10.1042/bst20120099.
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Extracellular electron transfer is one of the physiological hallmarks of Geobacter sulfurreducens, allowing these bacteria to reduce toxic and/or radioactive metals and grow on electrode surfaces. Aiming to functionally optimize the respiratory electron-transfer chains, such properties can be explored through genetically engineered strains. Geobacter species comprise a large number of different multihaem c-type cytochromes involved in the extracellular electron-transfer pathways. The functional characterization of multihaem proteins is particularly complex because of the coexistence of several microstates in solution, connecting the fully reduced and oxidized states. NMR spectroscopy has been used to monitor the stepwise oxidation of each individual haem and thus to obtain information on each microstate. For the structural study of these proteins, a cost-effective isotopic labelling of the protein polypeptide chains was combined with the comparative analysis of 1H-13C HSQC (heteronuclear single-quantum correlation) NMR spectra obtained for labelled and unlabelled samples. These new methodological approaches allowed us to study G. sulfurreducens haem proteins functionally and structurally, revealing functional mechanisms and key residues involved in their electron-transfer capabilities. Such advances can now be applied to the design of engineered haem proteins to improve the bioremediation and electricity-harvesting skills of G. sulfurreducens.
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Mitri, E., L. Barbieri, L. Vaccari, and E. Luchinat. "15N isotopic labelling for in-cell protein studies by NMR spectroscopy and single-cell IR synchrotron radiation FTIR microscopy: a correlative study." Analyst 143, no. 5 (2018): 1171–81. http://dx.doi.org/10.1039/c7an01464c.
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Bresson, Jean L., Brigitte Bader, André Mariotti, Francis Roccicchioli, Claude Ricour, Charles Sachs, and Jean Rey. "36 ENERGY-SOURCE RELATED DIFFERENCES IN WHOLE BODY PROTEIN METABOLISM MEASURED IN PARENTERALLY FED INFANTS, BY COMBINED STABLE ISOTOPIC LABELLING AND INDIRECT CALORIMETRY." Pediatric Research 24, no. 3 (September 1988): 411. http://dx.doi.org/10.1203/00006450-198809000-00059.
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Darling, P., L. J. Wykes, R. Clarke, A. Papageorgiou, and P. B. Pencharz. "Utilization of Non-Protein Nitrogen in Whey-Dominant Formulae by Low-Birth Weight Infants." Clinical Science 84, no. 5 (May 1, 1993): 543–48. http://dx.doi.org/10.1042/cs0840543.
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1. The effects of increasing non-protein nitrogen intake on nitrogen balance and α-amino nitrogen flux rate using [15N]glycine were examined in 30 low-birthweight appropriate-for-gestational-age infants (birthweight 1.5-2.0 kg). The compositions of the three whey-dominant formulae were similar except for the ratios of non-protein nitrogen/protein nitrogen, which were 6.5:93.5, 11.4:88.6 and 17.5:82.5. 2. Infants in the three diet groups each received similar total nitrogen intakes (395 mg of N day−1 kg−1, SD 2.6; n = 3). Protein nitrogen and non-protein nitrogen intakes were different as expected. Energy absorption (449 kJ day−1 kg−1, SD 13; n = 3) did not differ significantly between groups. A similar weight gain was observed in all groups. 3. Nitrogen absorption (76%, SD 4; n = 3) was not significantly different between groups. Apparent urea balance was significantly increased and became positive in the group receiving the formula with the higher proportion of non-protein nitrogen and urea nitrogen. Nitrogen retention, however, was significantly depressed in this group, indicating decreased efficiency of nitrogen utilization at this level of non-protein nitrogen despite an enhanced urea salvage. 4. The enrichment of the 15N label in urinary urea at isotopic steady state was significantly reduced in infants receiving the highest urea-containing formula, presumably due to the dilution of 15N-labelled urea by dietary urea. No difference, however, was found in the enrichment of the 15N label in urinary ammonia. Rates of α-amino nitrogen flux, protein synthesis and protein breakdown calculated from the ammonia labelling did not differ significantly between groups. 5. The source of dietary nitrogen affects the enrichment of urinary nitrogenous end-products and must be taken into account when interpreting results of protein turnover studies using constant infusion of [15N]glycine. 6. Based on the nitrogen balance results, the nonprotein nitrogen content of formulae should not account for more than 12% of total nitrogen.
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Belanche, A., M. R. F. Lee, J. M. Moorby, and C. J. Newbold. "Comparison of ryegrass and red clover on the fermentation pattern, microbial community and efficiency of diet utilisation in the rumen simulation technique (Rusitec)." Animal Production Science 53, no. 10 (2013): 1052. http://dx.doi.org/10.1071/an12183.
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An in vitro experiment was designed to investigate the effects of incubating two forages with a different energy/nitrogen (N) ratio [perennial ryegrass (GR) vs red clover (RC)] on the efficiency of N utilisation by rumen microbes. Second-cut forages were incubated in artificial rumen fermenters (n = 8). Ryegrass represented a supply of quickly available N and energy for the rumen microorganism which led to a rapid fermentation and bacterial growth 2–4 h after feeding. Ryegrass also promoted greater numbers of anaerobic fungi, methanogens and cellulolytic bacteria, which tended to increase neutral detergent fibre disappearance, gas production, volatile fatty acid and methane production than observed using RC diets. On the contrary, RC provided slowly degradable N and energy, which led to a relatively slow bacterial growth (4–8 h after feeding). In terms of diet utilisation, RC diets promoted a higher N outflow (mainly as undegraded-N) and efficiency of microbial protein synthesis per organic matter disappeared. Even so, microbial protein yield was similar on both diets indicating a better N capture by microorganisms fed GR than in those fed RC diets. The use of 15N-labelled forages demonstrated that this high ammonia incorporation by bacteria-fed GR occurred mainly during the early fermentation coinciding with the highest bacterial growth. In conclusion, this experiment demonstrated that the use of isotopic labelling combined with molecular techniques provided an insight into forage utilisation by the rumen microbes; GR diets led to a better efficiency of N utilisation compared with RC; moreover the lower N outflow on GR diets may be partially compensated for a higher proportion of microbial protein leaving the system and the greater volatile fatty acid production. These findings seem to indicate that RC grazing may increase the N pollution compared with GR without substantial improvements on the rumen function, however this must be confirmed in vivo.
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Dietze, Jörn, Alienke van Pijkeren, Anna-Sophia Egger, Mathias Ziegler, Marcel Kwiatkowski, and Ines Heiland. "Natural isotope correction improves analysis of protein modification dynamics." Analytical and Bioanalytical Chemistry 413, no. 30 (October 27, 2021): 7333–40. http://dx.doi.org/10.1007/s00216-021-03732-7.
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AbstractStable isotope labelling in combination with high-resolution mass spectrometry approaches are increasingly used to analyze both metabolite and protein modification dynamics. To enable correct estimation of the resulting dynamics, it is critical to correct the measured values for naturally occurring stable isotopes, a process commonly called isotopologue correction or deconvolution. While the importance of isotopologue correction is well recognized in metabolomics, it has received far less attention in proteomics approaches. Although several tools exist that enable isotopologue correction of mass spectrometry data, the majority is tailored for the analysis of low molecular weight metabolites. We here present PICor which has been developed for isotopologue correction of complex isotope labelling experiments in proteomics or metabolomics and demonstrate the importance of appropriate correction for accurate determination of protein modifications dynamics, using histone acetylation as an example.
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Sasikala, P. S., C. H. Harsha, Jeevisha Bajaj, Rakesh Sharma, Akhilesh Pandey, and Sudhir Krishna. "Quantitative Proteomic Profiling Unravels Dynamic Changes in the Myeloma Cell Proteome Treated with Valproic Acid (VPA)." Blood 118, no. 21 (November 18, 2011): 1847. http://dx.doi.org/10.1182/blood.v118.21.1847.1847.
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Abstract Abstract 1847 Multiple Myeloma (MM) is a post germinal center B-cell malignancy characterized by abnormal proliferation of terminally differentiated plasma cells in the bone marrow. Despite significant advances in multiple myeloma treatment, the disease remains incurable. Earlier studies have shown that HDAC inhibitors are effective in inducing antitumor effects in many hematological malignancies, but the exact mechanism through which they act is unclear. Since Valproic acid (a FDA approved HDAC inhibitor to treat epilepsy) is cost effective and suitable for old patients, we investigated VPA in this study. Our study aims to unravel the mechanism by which VPA induces antitumor effects in MM cell lines using both biochemical and proteomics approaches. In our experiments, MM cell lines (RPMI8226, NCI H929 and MM1R) when treated with VPA showed a block in the G1-S transition (decrease in the S-phase and G2M-phase, increase in the Sub-G0 phase) and decrease in the cell viability in a dose and time dependent fashion. Also, Annexin V staining by FACS and PARP cleavage by immunoblot analysis confirmed the induction of apoptosis through Caspases. We then analyzed the expression pattern of several genes, including those involved in transcription, cell cycle regulation and signaling pathway by quantitative RT PCR and by immunoblot analysis. Our study showed an induction of H3 acetylation, p21 and an increase in the NOTCH1 target genes (Hes1 and Hey1) in the myeloma cells treated with VPA. To further understand the key signaling pathways involved in the pathogenesis of MM and also to characterize how VPA regulates growth arrest, we investigated the differentially expressed proteome in the RPMI 8226 cell line treated with and without VPA using SILAC (Stable Isotope Labelling with Amino acids in Cell culture) based quantitative proteomics approach. Briefly, one population of RPMI 8226 cells was grown in medium with heavy (isotopic) amino acids (13C6 L-Lysine and 13C6 L-Arginine), while the other population was grown in medium containing naturally abundant isotopic form of (normal) amino acids (12C6 L-Lysine and 12C6 L-Arginine). Cells grown in heavy medium were left untreated, while the cells grown in light medium were treated with 1mM VPA for 24 hrs. Cell lysates were pooled, resolved on SDS-PAGE, protein bands excised, trypsin digested and analyzed on LTQ orbitrap velos mass spectrometer. Using SILAC approach, we identified and quantified 2,032 proteins in myeloma cells treated with VPA. We found that several proteins including protein kinases, Cell cycle regulators, transcription factors, membrane proteins, mitochondrial proteins and metabolic enzymes were regulated by VPA. Checking for the presence of known cell surface markers for plasma cell in the proteome data, we found Syndecan (CD138) with 3 unique peptides and noted that its expression was decreased upon VPA treatment. This was confirmed by FACS analysis. Interestingly, we also found that CCND2, an important regulator of plasma cell development and the one that is often implicated in myeloma pathogenesis, was significantly down regulated by VPA treatment. This was confirmed by Immunoblot analysis in a dose and time dependent manner. We also found that VPA is more effective in regulating CCND2 promoter activity in combination with Dexamethazone. Based on the above results, we then reasoned if NOTCH1 induction could regulate CCND2 expression. Interestingly, our study showed that active form of intracellular NOTCH1 down regulated CCND2 promoter activity. NOTCH1 being a membrane bound transcriptional activator, we then hypothesized that increased NOTCH1 signaling pathway could down regulate CCND2 expression by inducing a transcriptional repressor. Our preliminary results showed that NOTCH1 mediated Hes1 induction repressed the promoter activity of CCND2. Overall, our global quantitative proteomic analysis demonstrates that Valproic acid treatment induces dynamic changes in the myeloma proteome. In addition, we have shown that VPA may control the proliferation of myeloma cells at least in part via a NOTCH-Hes1-CCND2 regulatory axis. These results provide an invaluable starting point to design and use Valproic acid in combination with Dexamethazone and/or with Bortezomib as an effective therapy for myeloma. Disclosures: No relevant conflicts of interest to declare.
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Sugoro, I., Y. Maharani, M. Hanani, D. Ansori, H. Wahyudiyanto, A. Rizqikah, Dasumiati, et al. "Labelling of corn as forage for ruminants using isotope 15N." IOP Conference Series: Earth and Environmental Science 902, no. 1 (November 1, 2021): 012010. http://dx.doi.org/10.1088/1755-1315/902/1/012010.
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Abstract In vitro and in vivo testing for ruminant feed efficiency can be done by utilizing the stable isotope Nitrogen-15 (15N) as a tracer. Feed can be traced by labeling the forage using isotope 15N. Feed crops are labeled using an isotope 15N-enriched fertilizer. The critical thing to note is to know the content of isotopes 15N in the part of forage feed plants that have been labeled. This research aims to know the effect of urea fertilizer on the percent of atom excess 15N on corn. Corn are labeled using urea enriched with isotopes 15N in the form of urea fertilizer (10% excess atom 15N) with different doses (0-200% recommended urea dose). As a control used corn plants given urea fertilizer is not labeled 15N. The results showed that corn forage feed was successfully labeled and correlated with the dose of fertilizer. The range of atom excess 15N was 4.28 – 6.99% in corn forage. Biomass production showed no significant difference between the dose of fertilization and control, but neither protein content. Based on data, the corn forage can be used for further testing.
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Hsu, Jue-Liang, and Shu-Hui Chen. "Stable isotope dimethyl labelling for quantitative proteomics and beyond." Philosophical Transactions of the Royal Society A: Mathematical, Physical and Engineering Sciences 374, no. 2079 (October 28, 2016): 20150364. http://dx.doi.org/10.1098/rsta.2015.0364.
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Stable-isotope reductive dimethylation, a cost-effective, simple, robust, reliable and easy-to- multiplex labelling method, is widely applied to quantitative proteomics using liquid chromatography-mass spectrometry. This review focuses on biological applications of stable-isotope dimethyl labelling for a large-scale comparative analysis of protein expression and post-translational modifications based on its unique properties of the labelling chemistry. Some other applications of the labelling method for sample preparation and mass spectrometry-based protein identification and characterization are also summarized. This article is part of the themed issue ‘Quantitative mass spectrometry’.
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Wu, Zhengliang L., and Miroslaw Lech. "Modification degrees at specific sites on heparan sulphate: an approach to measure chemical modifications on biological molecules with stable isotope labelling." Biochemical Journal 389, no. 2 (July 5, 2005): 383–88. http://dx.doi.org/10.1042/bj20041827.
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Chemical modification of biological molecules is a general mechanism for cellular regulation. A quantitative approach has been developed to measure the extent of modification on HS (heparan sulphates). Sulphation on HS by sulphotransferases leads to variable sulphation levels, which allows cells to tune their affinities to various extracellular proteins, including growth factors. With stable isotope labelling and HPLC-coupled MS, modification degrees at various O-sulphation sites could be determined. A bovine kidney HS sample was first saturated in vitro with 34S by an OST (O-sulphotransferase), then digested with nitrous acid and analysed with HPLC-coupled MS. The 34S-labelled oligosaccharides were identified based on their unique isotope clusters. The modification degrees at the sulphotransferase recognition sites were obtained by calculating the intensities of isotopic peaks in the isotope clusters. The modification degrees at 3-OST-1 and 6-OST-1 sites were examined in detail. This approach can also be used to study other types of chemical modifications on biological molecules.
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Blaum, Bärbel S., Ursula Neu, Thomas Peters, and Thilo Stehle. "Spin ballet for sweet encounters: saturation-transfer difference NMR and X-ray crystallography complement each other in the elucidation of protein–glycan interactions." Acta Crystallographica Section F Structural Biology Communications 74, no. 8 (July 26, 2018): 451–62. http://dx.doi.org/10.1107/s2053230x18006581.
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Biomolecular NMR spectroscopy has limitations in the determination of protein structures: an inherent size limit and the requirement for expensive and potentially difficult isotope labelling pose considerable hurdles. Therefore, structural analysis of larger proteins is almost exclusively performed by crystallography. However, the diversity of biological NMR applications outperforms that of any other structural biology technique. For the characterization of transient complexes formed by proteins and small ligands, notably oligosaccharides, one NMR technique has recently proven to be particularly powerful: saturation-transfer difference NMR (STD-NMR) spectroscopy. STD-NMR experiments are fast and simple to set up, with no general protein size limit and no requirement for isotope labelling. The method performs best in the moderate-to-low affinity range that is of interest in most of glycobiology. With small amounts of unlabelled protein, STD-NMR experiments can identify hits from mixtures of potential ligands, characterize mutant proteins and pinpoint binding epitopes on the ligand side. STD-NMR can thus be employed to complement and improve protein–ligand complex models obtained by other structural biology techniques or by purely computational means. With a set of protein–glycan interactions from our own work, this review provides an introduction to the technique for structural biologists. It exemplifies how crystallography and STD-NMR can be combined to elucidate protein–glycan (and other protein–ligand) interactions in atomic detail, and how the technique can extend structural biology from simplified systems amenable to crystallization to more complex biological entities such as membranes, live viruses or entire cells.
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HOUSTON, K. M., S. A. BABAYAN, J. E. ALLEN, and W. HARNETT. "DoesLitomosoides sigmodontissynthesize dimethylethanolamine from choline?" Parasitology 135, no. 1 (September 25, 2007): 55–61. http://dx.doi.org/10.1017/s0031182007003642.
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SUMMARYJuvenile femaleLitomosoides sigmodontissecrete a protein (Juv-p120) highly modified with dimethylethanolamine (DMAE). In an attempt to establish the source of this decoration worms were pulsed with [3H]-choline and [3H]-ethanolamine and the radio-isotope labelled products analysed. Both isotope labels were successfully taken up by the worms, as demonstrated by labelling of phospholipids with [3H]-choline, being predominantly incorporated into phosphatidylcholine and [3H]-ethanolamine into phosphatidylethanolamine. Isotope labelling of phosphatidylethanolamine was particularly striking with the worms taking up ~30 times as much labelled ethanolamine as choline. It was possible to detect faint labelling of Juv-p120 with [3H]-ethanolamine after prolonged exposure periods but, unlike the situation with the phospholipids, it was much more readily labelled with [3H]-choline. When pulsing with [3H]-ethanolamine it was also possible to detect isotope-labelled phosphatidylcholine, which may ultimately account for the low levels of labelling of Juv-p120. Overall our results raise the previously unconsidered but intriguing possibility that inL. sigmodontis, choline may be the precursor of DMAE.
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Joshua, G. W. P., L. J. S. Harrison, and M. M. H. Sewell. "Developmental changes in proteins and glycoproteins revealed by direct radio-iodination of viable Taenia saginata larvae." Parasitology 99, no. 2 (October 1989): 265–74. http://dx.doi.org/10.1017/s0031182000058728.
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SummaryDirect surface I radio-isotope labelling techniques and SDS—PAGE analysis were used to compare the proteins and lentil—lectin adherent glycoproteins of the bovine stage of viable Taenia saginata larvae at three points in their development, the invasive oncospheres, immature (4-week-old) and mature (12 to 16-week-old) cysticerci. Some proteins and glycoproteins were present on all three of the ages of the parasite examined but there were also distinct age-specific proteins and glycoproteins detected on oncospheres and 4-week-old cysticerci and a marked difference between the protein/glycoprotein profiles of the parasite was apparent at these earlier stages of development and the mature cysticerci. The latter were characterized by the presence of high, 160–200 kDa molecular weight, lysine rich, glycoproteins, whereas small 16 and 18 kDa glycoproteins and a reduction-sensitive 23 kDa glycoprotein were first detected on 4-week-old immature cysticerci. Antigenic characterization of the isotope-labelled proteins and glycoproteins by immunoprecipitation against a panel of clinically defined bovine sera combined with SDS–PAGE analysis indicated that relatively few proteins were precipitated by sera from T. saginata-infected cattle as compared to the glycoproteins. However, both protein and glycoprotein antigens of possible protective and/or diagnostic significance were identified from oncospheres and cysticerci.
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Huo, Yilin, Valentina Iadevaia, Zhong Yao, Isabelle Kelly, Sabina Cosulich, Sylvie Guichard, Leonard J. Foster, and Christopher G. Proud. "Stable isotope-labelling analysis of the impact of inhibition of the mammalian target of rapamycin on protein synthesis." Biochemical Journal 444, no. 1 (April 26, 2012): 141–51. http://dx.doi.org/10.1042/bj20112107.
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mTORC1 [mTOR (mammalian target of rapamycin) complex 1] regulates diverse cell functions. mTORC1 controls the phosphorylation of several proteins involved in mRNA translation and the translation of specific mRNAs, including those containing a 5′-TOP (5′-terminal oligopyrimidine). To date, most of the proteins encoded by known 5′-TOP mRNAs are proteins involved in mRNA translation, such as ribosomal proteins and elongation factors. Rapamycin inhibits some mTORC1 functions, whereas mTOR-KIs (mTOR kinase inhibitors) interfere with all of them. mTOR-KIs inhibit overall protein synthesis more strongly than rapamycin. To study the effects of rapamycin or mTOR-KIs on synthesis of specific proteins, we applied pSILAC [pulsed SILAC (stable isotope-labelling with amino acids in cell culture)]. Our results reveal, first, that mTOR-KIs and rapamycin differentially affect the synthesis of many proteins. Secondly, mTOR-KIs inhibit the synthesis of proteins encoded by 5′-TOP mRNAs much more strongly than rapamycin does, revealing that these mRNAs are controlled by rapamycin-insensitive outputs from mTOR. Thirdly, the synthesis of certain other proteins shows a similar pattern of inhibition. Some of them appear to be encoded by ‘novel’ 5′-TOP mRNAs; they include proteins which, like known 5′-TOP mRNA-encoded proteins, are involved in protein synthesis, whereas others are enzymes involved in intermediary or anabolic metabolism. These results indicate that mTOR signalling may promote diverse biosynthetic processes through the translational up-regulation of specific mRNAs. Lastly, a SILAC-based approach revealed that, although rapamycin and mTOR-KIs have little effect on general protein stability, they stabilize proteins encoded by 5′-TOP mRNAs.
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Alqahtani, Ali, Kate Heesom, Jonathan L. Bramson, David Curiel, Hideyo Ugai, and David A. Matthews. "Analysis of purified Wild type and mutant adenovirus particles by SILAC based quantitative proteomics." Journal of General Virology 95, no. 11 (November 1, 2014): 2504–11. http://dx.doi.org/10.1099/vir.0.068221-0.
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We used SILAC (stable isotope labelling of amino acids in cell culture) and high-throughput quantitative MS mass spectrometry to analyse the protein composition of highly purified WT wild type adenoviruses, mutant adenoviruses lacking an internal protein component (protein V) and recombinant adenoviruses of the type commonly used in gene therapy, including one virus that had been used in a clinical trial. We found that the viral protein abundance and composition were consistent across all types of virus examined except for the virus lacking protein V, which also had reduced amounts of another viral core protein, protein VII. In all the samples analysed we found no evidence of consistent packaging or contamination with cellular proteins. We believe this technique is a powerful method to analyse the protein composition of this important gene therapy vector and genetically engineered or synthetic virus-like particles. The raw data have been deposited at proteomexchange, identifer PXD001120.
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Iadevaia, Valentina, Yilin Huo, Ze Zhang, Leonard J. Foster, and Christopher G. Proud. "Roles of the mammalian target of rapamycin, mTOR, in controlling ribosome biogenesis and protein synthesis." Biochemical Society Transactions 40, no. 1 (January 19, 2012): 168–72. http://dx.doi.org/10.1042/bst20110682.
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mTORC1 (mammalian target of rapamycin complex 1) is controlled by diverse signals (e.g. hormones, growth factors, nutrients and cellular energy status) and regulates a range of processes including anabolic metabolism, cell growth and cell division. We have studied the impact of inhibiting mTOR on protein synthesis in human cells. Partial inhibition of mTORC1 by rapamycin has only a limited impact on protein synthesis, but inhibiting mTOR kinase activity causes much greater inhibition of protein synthesis. Using a pulsed stable-isotope-labelling technique, we show that the rapamycin and mTOR (mammalian target of rapamycin) kinase inhibitors have differential effects on the synthesis of specific proteins. In particular, the synthesis of proteins encoded by mRNAs that have a 5′-terminal pyrimidine tract is strongly inhibited by mTOR kinase inhibitors. Many of these mRNAs encode ribosomal proteins. mTORC1 also promotes the synthesis of rRNA, although the mechanisms involved remain to be clarified. We found that mTORC1 also regulates the processing of the precursors of rRNA. mTORC1 thus co-ordinates several steps in ribosome biogenesis.
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BEYNON, Robert J., Deborah M. LEYLAND, Richard P. EVERSHED, Richard H. T. EDWARDS, and Stephen P. COBURN. "Measurement of the turnover of glycogen phosphorylase by GC/MS using stable isotope derivatives of pyridoxine (vitamin B6)." Biochemical Journal 317, no. 2 (July 15, 1996): 613–19. http://dx.doi.org/10.1042/bj3170613.
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The majority of vitamin B6 in the body is in skeletal muscle, bound as the cofactor pyridoxal 5´-phosphate to one abundant protein, glycogen phosphorylase. Previous work has established that radiolabelled vitamin B6 can be used as a turnover label for glycogen phosphorylase. In this study, a stable isotope derivative of pyridoxine {dideuterated pyridoxine; 3-hydroxy-4-(hydroxymethyl)-5-[hydroxymethyl-2H2]-2-methylpyridine} ([2H2]PN) has been used as a metabolic tracer to study the kinetics of labelling of the body pools of vitamin B6 in mice. A non-invasive method was developed in which the isotope abundance of the urinary excretory product of vitamin B6 metabolism, 4-pyridoxic acid, was analysed by GC/MS. The change in isotope abundance of urinary 4-pyridoxic acid following administration of [2H2]PN reflects the kinetics of labelling of the body pools of vitamin B6, and yields, non-invasively, the rate of degradation of glycogen phosphorylase.
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Joshua, G. W. P., L. J. S. Harrison, and M. M. H. Sewell. "Protein antigens in the cyst fluid of Taenia saginata cysticerci." Parasitology 100, no. 3 (June 1990): 463–67. http://dx.doi.org/10.1017/s003118200007877x.
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SUMMARYTaenia saginata cyst fluid proteins from 4, 8, 12 and 16-week-old cysticerci were analysed by a combination of direct I radio-isotope labelling, immunoprecipitation using a panel of sera from infected cattle infected with T. saginata and SDS–PAGE. Protein antigens of 12, 14, 16, 20 and 26 kDa were identified in all of the cyst fluids examined. These were immunogenic and were precipitated by serum taken from cattle from 8 weeks after infection onwards and were therefore considered to be of diagnostic potential. A 185 kDa protein antigen found only in the cyst fluid of 4-week-old cysticerci and a 43 kDa protein antigen first detected in cyst fluid from 8-week-old cysticerci were also identified but were considered to be of more limited diagnostic potential due to their restricted presence. An apparently non-immunogenic 67 kDa protein, found in all the cyst fluids examined, may have been host serum albumin.
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Chiotellis, Aristeidis, Hazem Ahmed, Thomas Betzel, Matthias Tanriver, Christopher J. White, Haewon Song, Sara Da Ros, Roger Schibli, Jeffrey W. Bode, and Simon M. Ametamey. "Chemoselective 18F-incorporation into pyridyl acyltrifluoroborates for rapid radiolabelling of peptides and proteins at room temperature." Chemical Communications 56, no. 5 (2020): 723–26. http://dx.doi.org/10.1039/c9cc08645e.
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A new prosthetic group is reported for quantitative 18F-labelling of peptides and proteins based on the chemoselective ligation of potassium acyltrifluoroborates (KATs) and hydroxylamines without any detectable 18F/19F isotope exchange at the KAT moiety.
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Huo, Yilin, Valentina Iadevaia, and Christopher G. Proud. "Differing effects of rapamycin and mTOR kinase inhibitors on protein synthesis." Biochemical Society Transactions 39, no. 2 (March 22, 2011): 446–50. http://dx.doi.org/10.1042/bst0390446.
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mTOR (mammalian target of rapamycin) forms two distinct types of complex, mTORC (mTOR complex) 1 and 2. Rapamycin inhibits some of the functions of mTORC1, whereas newly developed mTOR kinase inhibitors interfere with the actions of both types of complex. We have explored the effects of rapamycin and mTOR kinase inhibitors on general protein synthesis and, using a new stable isotope-labelling method, the synthesis of specific proteins. In HeLa cells, rapamycin only had a modest effect on total protein synthesis, whereas mTOR kinase inhibitors decreased protein synthesis by approx. 30%. This does not seem to be due to the ability of mTOR kinase inhibitors to block the binding of eIFs (eukaryotic initiation factors) eIF4G and eIF4E. Analysis of the effects of the inhibitors on the synthesis of specific proteins showed a spectrum of behaviours. As expected, synthesis of proteins encoded by mRNAs that contain a 5′-TOP (5′-terminal oligopyrimidine tract) was impaired by rapamycin, but more strongly by mTOR kinase inhibition. Several proteins not known to be encoded by 5′-TOP mRNAs also showed similar behaviour. Synthesis of proteins encoded by ‘non-TOP’ mRNAs was less inhibited by mTOR kinase inhibitors and especially by rapamycin. The implications of our findings are discussed.
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Gabel, Frank, Dominique Bicout, Ursula Lehnert, Moeava Tehei, Martin Weik, and Giuseppe Zaccai. "Protein dynamics studied by neutron scattering." Quarterly Reviews of Biophysics 35, no. 4 (November 2002): 327–67. http://dx.doi.org/10.1017/s0033583502003840.
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1. Introduction 3282. Basic concepts of neutron scattering 3292.1 Introduction 3292.2 Neutron-scattering functions 3312.3 Coherent and incoherent neutron scattering. The particular role of hydrogen in incoherent scattering 3322.4 Total elastic scattering, EISF and mean square displacement (MSD) 3332.5 Quasielastic scattering and relaxation function 3342.6 Inelastic scattering and density of states 3353. Experimental aspects and instruments 3353.1 Energy and space resolution 3353.2 General sample aspects 3353.3 Potential effects of D2O on dynamics 3363.4 Experimental 2H (deuterium) labelling 3364. Physics of protein dynamics 3364.1 Models 3364.2 The dynamical transition 3384.3 Effective force constants 3395. Dynamics of hydrated protein powders 3395.1 First experiments on myoglobin 3405.2 Dynamical transitions in other proteins 3405.3 The role of hydration water 3415.4 Influence of the solvent 3445.5 Diffusional motions within proteins by QENS 3465.6 Inelastic neutron scattering and vibrational spectra 3475.7 Conclusions 3516. Membranes 3526.1 Lipid bilayers 3536.2 BR and the purple membrane (PM) 3536.2.1 The dynamical transition in the PM 3536.2.2 QENS from oriented PM 3546.2.3 Hydration dependence of PM motions 3556.2.4 Local dynamics in PM studied by isotope labelling 3566.2.5 Dynamics of different BR conformations 3577. Protein solutions 3587.1 From powders to solutions 3587.2 Water dynamics and solvent dependence of the dynamical transition in proteins 3598. Comparing neutron scattering with other techniques 3599. Biological relevance 3609.1 Dynamics–activity relations 3609.2 Dynamics–stability relations (adaptation to extreme environments) 3609.3 Protein folding 36110. Acknowledgements 36411. References 364This review of protein dynamics studied by neutron scattering focuses on data collected in the last 10 years. After an introduction to thermal neutron scattering and instrumental aspects, theoretical models that have been used to interpret the data are presented and discussed. Experiments are described according to sample type, protein powders, solutions and membranes. Neutron-scattering results are compared to those obtained from other techniques. The biological relevance of the experimental results is discussed. The major conclusion of the last decade concerns the strong dependence of internal dynamics on the macromolecular environment.
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Jennewein, M., A. Hermanne, R. P. Mason, P. E. Thorpe, and F. Rösch. "A new method for the labelling of proteins with radioactive arsenic isotopes." Nuclear Instruments and Methods in Physics Research Section A: Accelerators, Spectrometers, Detectors and Associated Equipment 569, no. 2 (December 2006): 512–17. http://dx.doi.org/10.1016/j.nima.2006.08.088.
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Tan, Chee Fan, Hui San Teo, Jung Eun Park, Bamaprasad Dutta, Shun Wilford Tse, Melvin Khee-Shing Leow, Walter Wahli, and Siu Kwan Sze. "Exploring Extracellular Vesicles Biogenesis in Hypothalamic Cells through a Heavy Isotope Pulse/Trace Proteomic Approach." Cells 9, no. 5 (May 25, 2020): 1320. http://dx.doi.org/10.3390/cells9051320.
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Studies have shown that the process of extracellular vesicles (EVs) secretion and lysosome status are linked. When the lysosome is under stress, the cells would secrete more EVs to maintain cellular homeostasis. However, the process that governs lysosomal activity and EVs secretion remains poorly defined and we postulated that certain proteins essential for EVs biogenesis are constantly synthesized and preferentially sorted to the EVs rather than the lysosome. A pulsed stable isotope labelling of amino acids in cell culture (pSILAC) based quantitative proteomics methodology was employed to study the preferential localization of the newly synthesized proteins into the EVs over lysosome in mHypoA 2/28 hypothalamic cell line. Through proteomic analysis, we found numerous newly synthesized lysosomal enzymes—such as the cathepsin proteins—that preferentially localize into the EVs over the lysosome. Chemical inhibition against cathepsin D promoted EVs secretion and a change in the EVs protein composition and therefore indicates its involvement in EVs biogenesis. In conclusion, we applied a heavy isotope pulse/trace proteomic approach to study EVs biogenesis in hypothalamic cells. The results demonstrated the regulation of EVs secretion by the cathepsin proteins that may serve as a potential therapeutic target for a range of neurological disorder associated with energy homeostasis.
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Tinti, Michele, Maria Lucia S. Güther, Thomas W. M. Crozier, Angus I. Lamond, and Michael A. J. Ferguson. "Proteome turnover in the bloodstream and procyclic forms of Trypanosoma brucei measured by quantitative proteomics." Wellcome Open Research 4 (October 9, 2019): 152. http://dx.doi.org/10.12688/wellcomeopenres.15421.1.
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Background: Cellular proteins vary significantly in both abundance and turnover rates. These parameters depend upon their rates of synthesis and degradation and it is useful to have access to data on protein turnover rates when, for example, designing genetic knock-down experiments or assessing the potential usefulness of covalent enzyme inhibitors. Little is known about the nature and regulation of protein turnover in Trypanosoma brucei, the etiological agent of human and animal African trypanosomiasis. Methods: To establish baseline data on T. brucei proteome turnover, a Stable Isotope Labelling with Amino acids in Cell culture (SILAC)-based mass spectrometry analysis was performed to reveal the synthesis and degradation profiles for thousands of proteins in the bloodstream and procyclic forms of this parasite. Results: This analysis revealed a slower average turnover rate of the procyclic form proteome relative to the bloodstream proteome. As expected, many of the proteins with the fastest turnover rates have functions in the cell cycle and in the regulation of cytokinesis in both bloodstream and procyclic forms. Moreover, the cellular localization of T. brucei proteins correlates with their turnover, with mitochondrial and glycosomal proteins exhibiting slower than average turnover rates. Conclusions: The intention of this study is to provide the trypanosome research community with a resource for protein turnover data for any protein or group of proteins. To this end, bioinformatic analyses of these data are made available via an open-access web resource with data visualization functions.
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Guerrera, Ida Chiara, and Oliver Kleiner. "Application of Mass Spectrometry in Proteomics." Bioscience Reports 25, no. 1-2 (February 4, 2005): 71–93. http://dx.doi.org/10.1007/s10540-005-2849-x.
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Mass spectrometry has arguably become the core technology in proteomics. The application of mass spectrometry based techniques for the qualitative and quantitative analysis of global proteome samples derived from complex mixtures has had a big impact in the understanding of cellular function. Here, we give a brief introduction to principles of mass spectrometry and instrumentation currently used in proteomics experiments. In addition, recent developments in the application of mass spectrometry in proteomics are summarised. Strategies allowing high-throughput identification of proteins from highly complex mixtures include accurate mass measurement of peptides derived from total proteome digests and multidimensional peptide separations coupled with mass spectrometry. Mass spectrometric analysis of intact proteins permits the characterisation of protein isoforms. Recent developments in stable isotope labelling techniques and chemical tagging allow the mass spectrometry based differential display and quantitation of proteins, and newly established affinity procedures enable the targeted characterisation of post-translationally modified proteins. Finally, advances in mass spectrometric imaging allow the gathering of specific information on the local molecular composition, relative abundance and spatial distribution of peptides and proteins in thin tissue sections.
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Wei, Chen-Xuan, Michael Francis Burrow, Michael George Botelho, and W. Keung Leung. "Analysing Complex Oral Protein Samples: Complete Workflow and Case Analysis of Salivary Pellicles." Journal of Clinical Medicine 10, no. 13 (June 25, 2021): 2801. http://dx.doi.org/10.3390/jcm10132801.
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Studies on small quantity, highly complex protein samples, such as salivary pellicle, have been enabled by recent major technological and analytical breakthroughs. Advances in mass spectrometry-based computational proteomics such as Multidimensional Protein Identification Technology have allowed precise identification and quantification of complex protein samples on a proteome-wide scale, which has enabled the determination of corresponding genes and cellular functions at the protein level. The latter was achieved via protein-protein interaction mapping with Gene Ontology annotation. In recent years, the application of these technologies has broken various barriers in small-quantity-complex-protein research such as salivary pellicle. This review provides a concise summary of contemporary proteomic techniques contributing to (1) increased complex protein (up to hundreds) identification using minute sample sizes (µg level), (2) precise protein quantification by advanced stable isotope labelling or label-free approaches and (3) the emerging concepts and techniques regarding computational integration, such as the Gene Ontology Consortium and protein-protein interaction mapping. The latter integrates the structural, genomic, and biological context of proteins and genes to predict protein interactions and functional connections in a given biological context. The same technological breakthroughs and computational integration concepts can also be applied to other low-volume oral protein complexes such as gingival crevicular or peri-implant sulcular fluids.
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Zerbe, Oliver, Christian Baumann, Matthias Schuster, Kerstin Moehle, Kathryn K. Oi, and Erich Michel. "Peptides in BioNMR Research." CHIMIA International Journal for Chemistry 75, no. 6 (June 30, 2021): 505–7. http://dx.doi.org/10.2533/chimia.2021.505.
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Heteronuclear NMR in combination with isotope labelling is used to study folding of polypeptides induced by metals in the case of metallothioneins, binding of the peptidic allosteric modulator ρ-TIA to the human G-protein coupled α1b adrenergic receptor, the development of therapeutic drugs that interfere with the biosynthesis of the outer membrane of Gram-negative bacteria, and a system in which protein assembly is induced upon peptide addition. NMR in these cases is used to derive precise structural data and to study the dynamics.
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Babeu, Jean-Philippe, Samuel D. Wilson, Élie Lambert, Dominique Lévesque, François-Michel Boisvert, and François Boudreau. "Quantitative Proteomics Identifies DNA Repair as a Novel Biological Function for Hepatocyte Nuclear Factor 4α in Colorectal Cancer Cells." Cancers 11, no. 5 (May 5, 2019): 626. http://dx.doi.org/10.3390/cancers11050626.
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Hepatocyte nuclear factor 4α (HNF4α) is a transcription factor that acts as a master regulator of genes for several endoderm-derived tissues, including the intestine, in which it plays a central role during development and tumorigenesis. To better define the mechanisms by which HNF4α can influence these processes, we identified proteins interacting with HNF4α using stable isotope labelling with amino acids in cell culture (SILAC)-based quantitative proteomics with either immunoprecipitation of green fluorescent protein (GFP) or with proximity-dependent purification by the biotin ligase BirA (BioID), both fused to HNF4α. Surprisingly, these analyses identified a significant enrichment of proteins characterized with a role in DNA repair, a so far unidentified biological feature of this transcription factor. Several of these proteins including PARP1, RAD50, and DNA-PKcs were confirmed to interact with HNF4α in colorectal cancer cell lines. Following DNA damage, HNF4α was able to increase cell viability in colorectal cancer cells. Overall, these observations identify a potential role for this transcription factor during the DNA damage response.
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Atasoglu, C., and A. Y. Guliye. "Use of stable isotopes to measurede novosynthesis and turnover of amino acid-C and -N in mixed micro-organisms from the sheep rumenin vitro." British Journal of Nutrition 91, no. 2 (February 2004): 253–61. http://dx.doi.org/10.1079/bjn20031040.
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Protein synthesis and turnover in ruminal micro-organisms were assessed by stable-isotope methods in order to follow independently the fate of amino acid (AA)-C and -N in different AA. Rumen fluid taken from sheep receiving a grass hay–concentrate diet were strained and incubatedin vitrowith starch–cellobiose–xylose in the presence of NH3and 5 g algal protein hydrolysate (APH)/l, in incubations where the labels were15NH3, [15N]APH or [13C]APH. Total15N incorporation was calculated from separate incubations with15NH3and [15N]APH, and net N synthesis from the increase in AA in protein-bound material. The large difference between total and net AA synthesis indicated that substantial turnover of microbial protein occurred, averaging 3·5 %/h. Soluble AA-N was incorporated on average more extensively than soluble AA-C (70v.50 % respectively,P=0·001); however, incorporation of individual AA varied. Ninety percent of phenylalanine-C was derived from the C-skeleton of soluble AA, whereas the incorporation of phenylalanine-N was 72 %. In contrast, only 15 % aspartate-C + asparagine-C was incorporated, while 45 % aspartate-N+asparagine-N was incorporated. Deconvolution analysis of mass spectra indicated substantial exchange of carboxyl groups in several AA before incorporation and a condensation of unidentified C2and C4intermediates during isoleucine metabolism. The present results demonstrate that differential labelling with stable isotopes is a way in which fluxes of AA synthesis and degradation, their biosynthetic routes, and separate fates of AA-C and -N can be determined in a mixed microbial population.
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Slupsky, Carolyn M., Lisa N. Gentile, and Lawrence P. McIntosh. "Assigning the NMR spectra of aromatic amino acids in proteins: analysis of two Ets pointed domains." Biochemistry and Cell Biology 76, no. 2-3 (May 1, 1998): 379–90. http://dx.doi.org/10.1139/o98-017.
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The measurement of interproton nuclear Overhauser enhancements (NOEs) and dihedral angle restraints of aromatic amino acids is a critical step towards determining the structure of a protein. The complete assignment of the resonances from aromatic rings and the subsequent resolution and identification of their associated NOEs, however, can be a difficult task. Shown here is a strategy for assigning the 1H, 13C, and 15N signals from the aromatic side chains of histidine, tryptophan, tyrosine, and phenylalanine using a suite of homo- and hetero-nuclear scalar and NOE correlation experiments, as well as selective deuterium isotope labelling. In addition, a comparison of NOE information obtained from homonuclear NOE spectroscopy (NOESY) and 13C-edited NOESY - heteronuclear single quantum correlation experiments indicates that high-resolution homonuclear two-dimensional NOESY spectra of selectively deuterated proteins are invaluable for obtaining distance restraints to the aromatic residues.Key words: NMR assignment, aromatic residue, transcription factor, NOE, dihedral angle.
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Vahkal, Brett, Jamie Kraft, Emanuela Ferretti, Minyoung Chung, Jean-François Beaulieu, and Illimar Altosaar. "Review of Methodological Approaches to Human Milk Small Extracellular Vesicle Proteomics." Biomolecules 11, no. 6 (June 3, 2021): 833. http://dx.doi.org/10.3390/biom11060833.
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Proteomics can map extracellular vesicles (EVs), including exosomes, across disease states between organisms and cell types. Due to the diverse origin and cargo of EVs, tailoring methodological and analytical techniques can support the reproducibility of results. Proteomics scans are sensitive to in-sample contaminants, which can be retained during EV isolation procedures. Contaminants can also arise from the biological origin of exosomes, such as the lipid-rich environment in human milk. Human milk (HM) EVs and exosomes are emerging as a research interest in health and disease, though the experimental characterization and functional assays remain varied. Past studies of HM EV proteomes have used data-dependent acquisition methods for protein detection, however, improvements in data independent acquisition could allow for previously undetected EV proteins to be identified by mass spectrometry. Depending on the research question, only a specific population of proteins can be compared and measured using isotope and other labelling techniques. In this review, we summarize published HM EV proteomics protocols and suggest a methodological workflow with the end-goal of effective and reproducible analysis of human milk EV proteomes.
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Akay, Alper, Ashley Craig, Nicolas Lehrbach, Mark Larance, Ehsan Pourkarimi, Jane E. Wright, Angus Lamond, Eric Miska, and Anton Gartner. "RNA-binding protein GLD-1/quaking genetically interacts with the mir-35 and the let- 7 miRNA pathways in Caenorhabditis elegans." Open Biology 3, no. 11 (November 2013): 130151. http://dx.doi.org/10.1098/rsob.130151.
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Messenger RNA translation is regulated by RNA-binding proteins and small non-coding RNAs called microRNAs. Even though we know the majority of RNA-binding proteins and microRNAs that regulate messenger RNA expression, evidence of interactions between the two remain elusive. The role of the RNA-binding protein GLD-1 as a translational repressor is well studied during Caenorhabditis elegans germline development and maintenance. Possible functions of GLD-1 during somatic development and the mechanism of how GLD-1 acts as a translational repressor are not known. Its human homologue, quaking (QKI), is essential for embryonic development. Here, we report that the RNA-binding protein GLD-1 in C. elegans affects multiple microRNA pathways and interacts with proteins required for microRNA function. Using genome-wide RNAi screening, we found that nhl-2 and vig-1 , two known modulators of miRNA function, genetically interact with GLD-1. gld-1 mutations enhance multiple phenotypes conferred by mir-35 and let-7 family mutants during somatic development. We used stable isotope labelling with amino acids in cell culture to globally analyse the changes in the proteome conferred by let-7 and gld-1 during animal development. We identified the histone mRNA-binding protein CDL-1 to be, in part, responsible for the phenotypes observed in let-7 and gld-1 mutants. The link between GLD-1 and miRNA-mediated gene regulation is further supported by its biochemical interaction with ALG-1, CGH-1 and PAB-1, proteins implicated in miRNA regulation. Overall, we have uncovered genetic and biochemical interactions between GLD-1 and miRNA pathways.
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Ruiz, Lorena, Yohann Couté, Borja Sánchez, Clara G. de los Reyes-Gavilán, Jean-Charles Sanchez, and Abelardo Margolles. "The cell-envelope proteome of Bifidobacterium longum in an in vitro bile environment." Microbiology 155, no. 3 (March 1, 2009): 957–67. http://dx.doi.org/10.1099/mic.0.024273-0.
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Host–bacteria interactions are often mediated via surface-associated proteins. The identification of these proteins is an important goal of bacterial proteomics. To address how bile can influence the cell-envelope proteome of Bifidobacterium longum biotype longum NCIMB 8809, we analysed its membrane protein fraction using stable isotope labelling of amino acids in cell culture (SILAC). We were able to identify 141 proteins in the membrane fraction, including a large percentage of the theoretical transporters of this species. Moreover, the envelope-associated soluble fraction was analysed using different subfractionation techniques and differential in-gel fluorescence electrophoresis (DIGE). This approach identified 128 different proteins. Some of them were well-known cell wall proteins, but others were highly conserved cytoplasmic proteins probably displaying a ‘moonlighting’ function. We were able to identify 11 proteins in the membrane fraction and 6 proteins in the envelope-associated soluble fraction whose concentration varied in the presence of bile. Bile promoted changes in the levels of proteins with important biological functions, such as some ribosomal proteins and enolase. Also, oligopeptide-binding proteins were accumulated on the cell surface, which was reflected in a different tripeptide transport rate in the cells grown with bile. The data reported here will provide the first cell-envelope proteome map for B. longum, and may contribute to understanding the bile tolerance of these bacteria.
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Hoskin, S. O., S. Gavet, E. Milne, and G. E. Lobley. "Does glutamine act as a substrate for transamination reactions in the liver of fed and fasted sheep?" British Journal of Nutrition 85, no. 5 (May 2001): 591–97. http://dx.doi.org/10.1079/bjn2001332.
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The present study investigated the relative importance of glutamine as a transamination source in the ovine liver by examination of the labelling of amino acids (AA) in the hepatic free pool, mixed liver and plasma proteins of fed and fasted sheep, following infusion of isotopically-labelled glutamine. In a cross-over design four sheep were either fasted for 3 d or fed to 1·2×energy maintenance and finally euthanased. At each intake, the sheep were infused for 6 h with [2-15N]glutamine (150 μmol/h) and samples of total plasma protein isolated. Following the terminal infusion, liver tissue total proteins were prepared and hydrolysed and15N-enrichments in seventeen AA were determined by GC–combustion–isotope-ratio mass spectrometry. All AA were enriched (relative to natural abundance) except lysine and threonine, with the lowest enrichments in phenylalanine and histidine. There was no effect of the fedv.fasted state, except for leucine and isoleucine in liver protein (P<0·05). Enrichments in liver protein were greater than in plasma protein (P<0·01, except proline) and probably reflect the faster turnover rate of hepatic constitutive proteins compared with export proteins. Amination to methionine was greater than that to phenylalanine (P<0·01), suggesting a mechanism for preferentially protecting the former. This factor could be important for ruminant production, as methionine is often considered to be the first limiting AA for animals offered certain silages and conserved forages. Enrichments in all AA (except for glutamine, alanine and aspartate) were less than that for glutamate (P<0·01), and thus transaminations may have occurred with glutamine directly or via glutamate, following the action of hepatic glutaminase.
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Ayala, Isabel, Olivier Hamelin, Carlos Amero, Ombeline Pessey, Michael J. Plevin, Pierre Gans, and Jérôme Boisbouvier. "An optimized isotopic labelling strategy of isoleucine-γ2methyl groups for solution NMR studies of high molecular weight proteins." Chem. Commun. 48, no. 10 (2012): 1434–36. http://dx.doi.org/10.1039/c1cc12932e.
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Zolotarev, Yu A., A. K. Dadayan, E. V. Bocharov, N. F. Myasoedov, Yu A. Borisov, B. V. Vaskovsky, and E. M. Dorokhova. "New development in the tritium labelling of peptides and proteins using solid catalytic isotopic exchange with spillover-tritium." Amino Acids 24, no. 3 (April 1, 2003): 325–33. http://dx.doi.org/10.1007/s00726-002-0404-7.
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makkar, sarbjeet, rohana Liyanage, Jackson Lay, and Rath Narayan. "Proteomic analysis of macrophages activated with salmonella lipopolysaccharide (INC2P.413)." Journal of Immunology 194, no. 1_Supplement (May 1, 2015): 55.7. http://dx.doi.org/10.4049/jimmunol.194.supp.55.7.
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Abstract Macrophages play pivotal role in immunity. They are activated by pathogen derived molecules such as lipopolysaccharides (LPS) which triggers the production of various proteins that drive and resolve inflammation. There are many studies on the effect of LPS at the genome level but the knowledge of its proteomic effects is limited. To determine the proteomic effects of LPS, we used Stable Isotope Labelling of Amino acids in Cell culture (SILAC) with a chicken macrophage cell line HTC, stimulated with Salmonella typhimurium LPS. The heavy (H) (13C-lysine) labelled cells were treated with LPS and the regular or light lysine (L) grown cells treated as controls. After 16h of stimulation, the proteins were extracted, quantified, and an equal amount from both H and L cells mixed. The protein mixtures were subjected to reduction/alkylation, and trypsin digestion followed by high throughput liquid chromatography/ tandem mass spectrometry. We quantified 133 proteins using Skyline software, out of which 13 were upregulated and 11 down regulated. Some of the upregulated proteins included HSP 70 and 90, DNA helicases, prostaglandin D synthase and CCL5 ligand. The down regulated proteins were ubiquitin, proteasome, and proteins related to protease activity. In conclusion this study suggests that activation of macrophages with LPS results in differential expressions of proteins involved in cytoskeleton remodeling, cell migration and cell signaling pathways involved in immune response.
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Bequette, B. J., C. Backwell, G. E. Lobley, and J. C. MacRae. "Milk protein precursors in lactating goats." Proceedings of the British Society of Animal Production (1972) 1992 (March 1992): 2. http://dx.doi.org/10.1017/s0308229600021279.
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With the failure of current nutritional schemes for dairy ruminants to predict yields of milk and milk components, and perceptions of milk's nutritional value following recommendations to reduce dietary fat intake, an integrated approach to feeding and metabolism needs to be developed. Such a system must therefore be ‘metabolite’ based.An ability to predict changes in milk constituent output in response to alterations in nutrition requires, in the first instance, the identification of specific precursors for milk component synthesis in the lactating mammary gland. Arteriovenous differences across the mammary gland indicate that blood free amino acids (AA) are either taken-up by the gland in excess, equal to, or in insufficient amounts compared to their output in milk (1). Isotope labelling experiments have indicated that, in addition to AA free in blood, the mammary gland utilises a substantial amount of AA derived from constitutive mammary gland protein breakdown (2). The present experiment was designed to investigate the AA precursors and kinetics of milk protein synthesis and to confirm and extend the latter observations.
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Fichtner, A., H. Bohnenberger, O. Elakad, A. Richter, C. Lenz, C. Oing, P. Ströbel, S. Kueffer, D. Nettersheim, and F. Bremmer. "Proteomic profiling of cisplatin-resistant and cisplatin-sensitive germ cell tumour cell lines using quantitative mass spectrometry." World Journal of Urology 40, no. 2 (January 27, 2022): 373–83. http://dx.doi.org/10.1007/s00345-022-03936-1.
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Abstract Purpose Advanced testicular germ cell tumours (GCT) generally have a good prognosis owing to their unique sensitivity towards cisplatin-based chemotherapies. However, cisplatin-resistant GCT have a poor outcome. Further studies are mandatory to better understand resistance mechanisms and develop therapeutic strategies for refractory GCTs. Methods Protein levels in cisplatin-resistant GCT cell lines of NTERA-2, NCCIT and 2102EP were analyzed by quantitative proteomic mass spectrometry (MS) in combination with stable isotope labelling by amino acids in cell culture (SILAC). Differentially abundant protein markers of acquired cisplatin resistance were validated by Western blotting. Comprehensive bioinformatical annotation using gene set enrichment analyses (GSEA) and STRING interaction analysis were performed to identify commonly affected pathways in cisplatin resistance and the data were compared to the GCT cohort of the ‘The Cancer Genome Atlas’. Results A total of 4375 proteins were quantified by MS, 144 of which were found to be differentially abundant between isogenic resistant and sensitive cell line pairs (24 proteins for NTERA-2, 60 proteins for NCCIT, 75 proteins for 2102EP). Western blotting confirmed regulation of key resistance-associated proteins (CBS, ANXA1, LDHA, CTH, FDXR). GSEA revealed a statistically significant enrichment of DNA repair-associated proteins in all three resistant cell lines and specific additional processes for individual cell lines. Conclusion High resolution MS combined with SILAC is a powerful tool and 144 significantly deregulated proteins were found in cisplatin-resistant GCT cell lines. Our study provides the largest proteomic in vitro library for cisplatin resistance in GCT, yet, enabling further studies to develop new treatment options for patients with refractory GCT.