Equilibration of (2)H labeling between body water and free amino acids: enabling studies of proteome synthesis

Protein synthesis can be estimated by measuring the incorporation of a labeled amino acid into a proteolytic peptide. Although prelabeled amino acids are typically administered, recent studies have tested (2)H(2)O; the assumption is that there is rapid equilibration of (2)H (in body water) with the carbon-bound hydrogens of amino acids before those amino acids are incorporated into a protein(s). We have determined the temporal changes in (2)H labeling of body water and amino acids which should build confidence in (2)H(2)O-based studies of protein synthesis when one aims to measure the (2)H labeling of proteolytic peptides.     

Nonradioactive labeling of probe with digoxigenin by polymerase chain reaction

Probes nonradioactively labeled with the steroid hapten digoxigenin have several intriguing properties, including a high sensitivity equivalent to that of radioactive probes, speed in detection, low hazard potential in handling, and possibility of long-term storage. The use of polymerase chain reaction for labeling probe has been demonstrated to offer various advantages including efficient labeling of fragments as small as 100 bp, direct labeling of genomic DNA, and labeling with subnanogram amounts of input DNA. We therefore investigated whether this technique could be adapted for labeling with a relatively large molecule such as digoxigenin. In this report, we show that the polymerase chain reaction is a very efficient technique for synthesis of digoxigenin-labeled DNA and we present an extremely simple procedure for purification of the non-isotopically labeled fragments.     

Microwave‐assisted 18O labeling of Fmoc‐protected amino acids

Recently, there has been an increased interest in isotopical labeling of peptides. Although there are several techniques allowing for a complete labeling of all carboxyl groups in peptides, regioselective labeling would be beneficial in many situations. Such labeling requires the use of ¹⁸O‐labeled Fmoc amino acids. We have designed a method for such labeling that is an improvement on a technique proposed earlier. The new procedure is suitable for microscale synthesis and could be used in peptide and proteomics laboratories. Although for the majority of tested amino acids our method gives good labeling efficiency, it is time consuming. Therefore, we have decided to use microwave‐assisted procedure. This approach resulted in reduction of reaction time to 15 min and increased reaction efficiency. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

15N cyclotides by whole plant labeling

Cyclotides are 28-37 amino acid peptides incorporating three disulfide bonds and a cyclic backbone. Their cyclic and knotted topology renders them immune to denaturation by heat or organic solvents and highly resistant to proteolysis. They have a range of interesting and potentially useful pharmaceutical properties and have been proposed as scaffolds within which peptides with drug activities can be stabilized for delivery. Some members of the family also have agricultural applications deriving from their potent insecticidal activity. Labeling peptides with the NMR-active and stable 15N isotope facilitates a range of studies by NMR, including structural and dynamics studies and their use as tracers. However, owing to their head-to-tail cyclized peptide backbone labeled cyclotides are not amenable to conventional recombinant labeling strategies. We have developed an approach to overcome this limitation by growing the cyclotide-bearing plant Oldenlandia affinis on nitrogen-free agar media supplemented with 15N salts and obtaining complete labeling at no detriment to plant biomass. We purified the insecticidal cyclotides kalata B1 and kalata B2 as examples and provide heteronuclear single quantum coherence (HSQC) NMR spectra for each. This method of labeling cyclotides involves only a fraction of the cost of uniform labeling by solid-phase peptide synthesis.     

Efficient oxidative folding and site-specific labeling of human hepcidin to study its interaction with receptor ferroportin

Hepcidin is a small disulfide-rich peptide hormone that plays a key role in the regulation of iron homeostasis by binding and mediating the degradation of the cell membrane iron efflux transporter, ferroportin. Since it is a small peptide, chemical synthesis is a suitable approach for the preparation of mature human hepcidin. However, oxidative folding of synthetic hepcidin is extremely difficult due to its high cysteine content and high aggregation propensity. To improve its oxidative folding efficiency, we propose a reversible S-modification approach. Introduction of eight negatively charged sulfonate moieties into synthetic hepcidin significantly decreased its aggregation propensity and, under optimized conditions, dramatically increased the refolding yield. The folded hepcidin displayed a typical disulfide-constrained β-sheet structure and could induce internalization of enhanced green fluorescent protein (EGFP) tagged ferroportin in transfected HEK293 cells. In order to study interactions between hepcidin and its receptor ferroportin, we propose a general approach for site-specific labeling of synthetic hepcidin analogues by incorporation of an l-propargylglycine during chemical synthesis. Following efficient oxidative refolding, a hepcidin analogue with Met20 replaced by l-propargylglycine was efficiently mono-labeled by a red fluorescent dye through click chemistry. The labeled hepcidin was internalized into the transfected cells together with the EGFP-tagged ferroportin, suggesting direct binding between hepcidin and ferroportin. The labeled hepcidin was also a suitable tool to visualize internalization of overexpressed or even endogenously expressed ferroportin without tags. We anticipate that the present refolding and labeling approaches could also be used for other synthetic peptides.     

Equilibration of H labeling between body water and free amino acids: Enabling studies of proteome synthesis

Protein synthesis can be estimated by measuring the incorporation of a labeled amino acid into a proteolytic peptide. Although prelabeled amino acids are typically administered, recent studies have tested ²H₂O; the assumption is that there is rapid equilibration of ²H (in body water) with the carbon-bound hydrogens of amino acids before those amino acids are incorporated into a protein(s). We have determined the temporal changes in ²H labeling of body water and amino acids which should build confidence in ²H₂O-based studies of protein synthesis when one aims to measure the ²H labeling of proteolytic peptides.     

Proteolytic 18O labeling by peptidyl-Lys metalloendopeptidase for comparative proteomics

The potential capabilities of a new proteolytic 18O labeling method employing peptidyl-Lys metalloendopeptidase (Lys-N) have been demonstrated for use in comparative proteomics. Conditions (pH>or=9.5) have been found such that Lys-N incorporates only a single 18O atom into the carboxyl terminus of each proteolytically generated peptide. This 18O labeling method has a major advantage over current protelytic 18O labeling methods that generate a mixture of isotopic isoforms resulting from the incorporation of one or two 18O atoms into each peptide species by the proteases (trypsin, Lys-C, or Glu-C) used. We demonstrate that the single 18O atom incorporation property of Lys-N overcomes the major problem of the current proteolytic 18O labeling methods and provides accurate quantification results for isotopically labeled peptides.     

A chemoselective method for site-specific immobilization of peptides via aminooxy group

Site-specific modification of peptides and proteins is an important area of basic research for preparation of well-defined biosensors and probes. The unique properties of aminooxy group present an opportunity for chemoselective site-specific immobilization of peptides to prepare well-defined biosensors. We have prepared FLAG peptide derivatives containing L-epsilon-aminooxylysine (L-epsilon-AOLys, 1a) and L-lysine units in their sequence at the C- and N-terminals via solid-phase synthesis. Site-specific modification of peptides through aminooxy group was demonstrated in the preparation of biosensors and selective conjugation in the preparation of biotinylated probes. Effect of the incorporation of L-epsilon-AOLys (1a) into the peptide sequence and its subsequent labeling on the FLAG epitopic character was measured using a surface plasmon resonance detector. It was found that incorporation of L-epsilon-AOLys (1a) into the FLAG peptide and site-specific immobilization through aminooxy group preserved the integrity of FLAG epitope.     

Quantitative proteome analysis by solid-phase isotope tagging and mass spectrometry

The adaptation of sequences of chemical reactions to a solid-phase format has been essential to the automation, reproducibility, and efficiency of a number of biotechnological processes including peptide and oligonucleotide synthesis and sequencing. Here we describe a method for the site-specific, stable isotopic labeling of cysteinyl peptides in complex peptide mixtures through a solid-phase capture and release process, and the concomitant isolation of the labeled peptides. The recovered peptides were analyzed by microcapillary liquid chromatography and tandem mass spectrometry (microLC-MS/MS) to determine their sequences and relative quantities. The method was used to detect galactose-induced changes in protein abundance in the yeast Saccharomyces cerevisiae. A side-by-side comparison with the isotope-coded affinity tag (ICAT) method demonstrated that the solid-phase method for stable isotope tagging of peptides is comparatively simpler, more efficient, and more sensitive.     

Solid-phase synthesis of C-terminally modified peptides.

In this paper, a straightforward and generic protocol is presented to label the C-terminus of a peptide with any desired moiety that is functionalized with a primary amine. Amine-functional molecules included are polymers (useful for hybrid polymers), long alkyl chains (used in peptide amphiphiles and stabilization of peptides), propargyl amine and azido propyl-amine (desirable for 'click' chemistry), dansyl amine (fluorescent labeling of peptides) and crown ethers (peptide switches/hybrids). In the first part of the procedure, the primary amine is attached to an aldehyde-functional resin via reductive amination. To the secondary amine that is produced, an amino acid sequence is coupled via a standard solid-phase peptide synthesis protocol. Since one procedure can be applied for any given amine-functional moiety, a robust method for C-terminal peptide labeling is obtained.     

Expression,stabilization and purification of membrane proteins via diverse protein synthesis systems and detergents involving cell-free associated with self-assembly peptide surfactants

G-protein coupled receptors (GPCRs) are involved in regulating most of physiological actions and metabolism in the bodies, which have become most frequently addressed therapeutic targets for various disorders and diseases. Purified GPCR-based drug discoveries have become routine that approaches to structural study, novel biophysical and biochemical function analyses. However, several bottlenecks that GPCR-directed drugs need to conquer the problems including overexpression, solubilization, and purification as well as stabilization. The breakthroughs are to obtain efficient protein yield and stabilize their functional conformation which are both urgently requiring of effective protein synthesis system methods and optimal surfactants. Cell-free protein synthesis system is superior to the high yields and post-translation modifications, and early signs of self-assembly peptide detergents also emerged to superiority in purification of membrane proteins. We herein focus several predominant protein synthesis systems and surfactants involving the novel peptide detergents, and uncover the advantages of cell-free protein synthesis system with self-assembling peptide detergents in purification of functional GPCRs. This review is useful to further study in membrane proteins as well as the new drug exploration.     

Synthesis of potent and selective inhibitors of human plasma kallikrein

The synthesis and in vitro enzyme inhibition profile of a series of novel trifluoromethylketone (TFMK) inhibitors of human plasma kallikrein (PK) are described. We have developed an efficient method for the construction of peptide TFMKs that provides the final product devoid of compromised stereochemical integrity. Many of these compounds are potent inhibitors of PK and exhibit reduced inhibition of tissue kallikrein (TK) and plasmin (HP).     

A sensitive method for determining the phosphorylation status of natriuretic peptide receptors: cGK-Ialpha does not regulate NPR-A

Natriuretic peptide receptor A (NPR-A) and natriuretic peptide receptor B (NPR-B) are transmembrane guanylyl cyclases that catalyze the synthesis of cGMP in response to natriuretic peptides. Phosphorylation and dephosphorylation regulate these receptors and have been traditionally studied by (32)PO(4) labeling of transfected cells. However, this approach cannot be used to determine the phosphorylation state of receptors isolated from unlabeled sources. Here, we use Pro-Q Diamond and SYPRO Ruby dyes to quantify the phosphorylation status and protein levels, respectively, of natriuretic peptide receptors from tissues and cells. Strong Pro-Q Diamond signals for NPR-A and NPR-B were obtained when receptors were isolated from lung tissue, liver tissue and overexpressing cells. The level of NPR-A Pro-Q staining was also high in kidney but was much lower in heart tissue. In contrast, the SYPRO Ruby protein signal was weaker and more variable. In a direct comparison, Pro-Q Diamond staining was as sensitive as but more specific than the (32)PO(4) labeling method. The two approaches were highly correlated (R(2) = 0.98). We exploited these techniques to measure the effect of cGMP-dependent protein kinase Ialpha on the phosphate content and guanylyl cyclase activity of NPR-A. Neither value was significantly affected in cells overexpressing cGK-Ialpha or in tissues from mice lacking cGK-I. We conclude that cGK-I does not regulate the cyclase activity or phosphorylation state of NPR-A. Furthermore, we find that Pro-Q Diamond staining is a sensitive method for measuring the phosphate levels of natriuretic peptide receptors, but protein levels are best detected by Western blot analysis, not SYPRO Ruby staining.     

Plasma proteome dynamics: analysis of lipoproteins and acute phase response proteins with 2H2O metabolic labeling

Understanding the pathologies related to the regulation of protein metabolism requires methods for studying the kinetics of individual proteins. We developed a (2)H(2)O metabolic labeling technique and software for protein kinetic studies in free living organisms. This approach for proteome dynamic studies requires the measurement of total body water enrichments by GC-MS, isotopic distribution of the tryptic peptide by LC-MS/MS, and estimation of the asymptotical number of deuterium incorporated into a peptide by software. We applied this technique to measure the synthesis rates of several plasma lipoproteins and acute phase response proteins in rats. Samples were collected at different time points, and proteins were separated by a gradient gel electrophoresis. (2)H labeling of tryptic peptides was analyzed by ion trap tandem mass spectrometry (LTQ MS/MS) for measurement of the fractional synthesis rates of plasma proteins. The high sensitivity of LTQ MS in zoom scan mode in combination with (2)H label amplification in proteolytic peptides allows detection of the changes in plasma protein synthesis related to animal nutritional status. Our results demonstrate that fasting has divergent effects on the rate of synthesis of plasma proteins, increasing synthesis of ApoB 100 but decreasing formation of albumin and fibrinogen. We conclude that this technique can effectively measure the synthesis of plasma proteins and can be used to study the regulation of protein homeostasis under physiological and pathological conditions.     

Implications of 15N-metabolic labeling for automated peptide identification in Arabidopsis thaliana

We report the first metabolic labeling of Arabidopsis thaliana for proteomic investigation, demonstrating efficient and complete labeling of intact plants. Using a reversed-database strategy, we evaluate the performance of the MASCOT search engine in the analysis of combined natural abundance and 15N-labeled samples. We find that 15N-metabolic labeling appears to increase the ambiguity associated with peptide identifications due in part to changes in the number of isobaric amino acids when the isotopic label is introduced. This is reflected by changes in the distributions of false positive identifications with respect to MASCOT score. However, by determining the nitrogen count from each pair of labeled and unlabeled peptides we may improve our confidence in both heavy and light identifications.     

Measuring protein synthesis using metabolic ²H labeling, high-resolution mass spectrometry, and an algorithm

We recently developed a method for estimating protein dynamics in vivo with heavy water ((2)H(2)O) using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) [16], and we confirmed that (2)H labeling of many hepatic free amino acids rapidly equilibrated with body water. Although this is a reliable method, it required modest sample purification and necessitated the determination of tissue-specific amino acid labeling. Another approach for quantifying protein kinetics is to measure the (2)H enrichments of body water (precursor) and protein-bound amino acid or proteolytic peptide (product) and to estimate how many copies of deuterium are incorporated into a product. In the current study, we used nanospray linear trap Fourier transform ion cyclotron resonance mass spectrometry (LTQ FT-ICR MS) to simultaneously measure the isotopic enrichment of peptides and protein-bound amino acids. A mathematical algorithm was developed to aid the data processing. The most notable improvement centers on the fact that the precursor/product labeling ratio can be obtained by measuring the labeling of water and a protein (or peptide) of interest, thereby minimizing the need to measure the amino acid labeling. As a proof of principle, we demonstrate that this approach can detect the effect of nutritional status on albumin synthesis in rats given (2)H(2)O.     

Evaluation of new base-labile 2-(4-nitrophenylsulfonyl) ethoxycarbonyl (Nsc)-amino acids for solid-phase peptide synthesis.

The 2-(4-nitrophenylsulfonyl)ethoxycarbonyl (Nsc) group is a new base-labile protecting group for solid-phase peptide synthesis, completely interchangeable with the fluorenylmethoxycarbonyl (Fmoc) protecting group, but with certain advantages. In this paper, we report a methodology with Nalpha-Nsc-protected amino acids for the synthesis of some melanotropins important to our research, namely, gamma-melanocyte-stimulating hormone (gamma-MSH), its [Nle3]-analogue, and a cyclic alpha-MSH/beta-MSH hybrid. We developed an efficient protocol for the synthesis of the cyclic MSH analogue that yielded this peptide in >98% purity. The gamma-MSH synthesis, which gave problems with both the Boc and Fmoc strategies, yielded the desired peptide by Nsc-chemistry but was accompanied by side products. Finally, the Nle3-gamma-MSH analogue was synthesized more efficiently using the Fmoc strategy, suggesting that Nsc-chemistry might not be the best methodology for certain sequences.     

Following tracks of hemichannels

It has been suggested that plasma membrane-bound hemichannels perform physiological and pathophysiological functions per se. Such functions require the presence of hemichannels on the cell surface and their accessibility to the extracellular environment for at least some limited period of time. We have previously shown that hemichannels can be labeled by means of antibodies directed to an external loop domain of connexin (Cx) 43. We now provide evidence that trafficking of hemichannel vesicles can be visualized upon binding of a labeled homophilic peptide corresponding to a region of the first extracellular loop (EL1) of Cx43. In vivo imaging was performed after labeling hemichannels from the extracellular site with a mimetic peptide tagged with a fluorochrome (Alexa-546). Using a Cx43-CFP transfected HeLa cell line for incubation with the mimetic peptide, a significant number of double-labeled vesicles were found inside the cells. This double labeling indicates that a portion of Cx43 within the cell had accessed the cell surface as hemichannels where it bound to the peptide and was subsequently endocytosed. Pulse labeling with the peptide showed a decrease in the number of dual-labeled vesicles over time, indicating degradation and/or concurrent recycling of hemichannel vesicles.