Evaluation of the accuracy of protein quantification using isotope TMPP‐labeled peptides

N‐succinimidyloxycarbonylmethyl tris(2,4,6‐trimethoxyphenyl) phosphonium bromide (TMPP‐Ac‐OSu) reacts rapidly, mildly, and specifically with the N‐terminals of proteins and peptides. Thus, it can be developed as an ideal isotope‐coded tag to be used in quantitative proteomics. Here, we present a strategy for light and heavy TMPP‐based quantitative proteomic analysis, in which peptides in a mixture can be quantified using an on‐tip TMPP derivatization approach. To demonstrate the accuracy of this strategy, light and heavy TMPP‐labeled peptides were combined at different ratios and subsequently analyzed by LC‐MS/MS. The MS spectra and scatter plots show that peptide and protein ratios were both consistent with the mixed ratios. We observed a linear correlation between protein ratios and the predicted ratios. In comparison with SILAC method, the TMPP labeling method produced similarly accurate quantitative results with low CVs. In conclusion, our results suggest that this isotope‐coded TMPP method achieved accurate quantification and compatibility with IEF‐based separation. With the inherent advantages of TMPP derivatization, we believe that it holds great promise for future applications in quantitative proteomics analysis.     

Characterization of acute neurotoxic effects of trimethylolpropane phosphate via neuronal network biosensors.

We have utilized cultured neuronal networks grown on microelectrode arrays to demonstrate rapid, reliable detection of a toxic compound, trimethylolpropane phosphate (TMPP). Initial experiments, which were performed blind, demonstrated rapid classification of the compound as a convulsant, a finding consistent with previous whole animal neurobehavioral studies. TMPP (2-200 microM) reorganized network spike activity into synchronous, quasi-periodic burst episodes. Integrated burst amplitudes invariably increased, reflecting higher spike frequencies within each burst. The variability of network burst parameters, quantified as coefficients of variation (CVs), was decreased. Mean CVs for burst duration, interburst interval, and burst rate were lowered by 42+/-13, 58+/-5.5, and 62+/-1.8%, respectively (mean+/-SEM, n=8 cultures, 197 channels). These changes in network activity paralleled the effects induced by bicuculline, a known disinhibitory and seizure-inducing drug, and confirmed classification of TMPP as a potential epileptogenic compound. Simple pharmacological tests permit exploration of mechanisms underlying observed activity shifts. The EC(50) for GABA inhibition of network activity was increased from 2.8 to 7.0 microM by 20 microM TMPP and to 20.5 microM by 200 microM TMPP. Parallel dose-response curves suggest that TMPP acts by a competitive antagonism of GABA inhibition, and are consistent with reported patch-clamp analysis of TMPP-induced reduction of inhibitory postsynaptic current amplitudes. The potency of TMPP in generating epileptiform activity in vitro was comparable to concentrations reported for in vivo studies. TMPP and bicuculline produced both increases and decreases in burst rate depending on native spontaneous bursting levels. These results demonstrate a need for multivariate analysis of network activity changes to yield accurate predictions of compound effects.     

Power of positive thinking in quantitative proteomics

Derivatization of proteins with specific isotope reagents has been widely explored for quantitative proteomics where the relative abundances of proteins present in different complex samples are compared by MS. This represents an interesting arena for innovation, where protein chemistry and MS are associated for the best of both worlds. Among the numerous reagents developed, those that introduce a permanent positive charge, such as (N‐succinimidyloxycarbonylmethyl)‐tris(2,4,6‐trimethoxyphenyl)phosphonium bromide (TMPP), increase the ionizability of their targets and thus improve the sensitivity of the approach. TMPP labeling also modifies the hydrophobicity and changes the peptide fragmentation pattern. Because TMPP reacts preferably with the N‐termini of proteins and peptides, its use has been explored for proteogenomics and de novo protein sequencing. In this issue of Proteomics, Shen et al. (Proteomics 2015, 15, 2903–2909) show that accurate quantitation of proteins can be obtained with light/heavy TMPP‐labeling of peptides, which can be easily prepared and desalted in a homemade C8‐SCX‐C8 stagetip, and then monitored by nano‐LC‐MS/MS analysis. Their results demonstrate enhanced sequence coverage compared with other approaches. Combined with an efficient enrichment procedure, the higher sensitivity of this “positive attitude” reagent may facilitate much deeper investigations into the quantitative proteomics of complex samples.     

A rapid and easy protein N‐terminal profiling strategy using (N‐Succinimidyloxycarbonylmethyl)tris(2,4,6‐trimethoxyphenyl)phosphonium bromide (TMPP) labeling and StageTip

Protein N‐terminal profiling is crucial when characterizing biological functions and provides proteomic evidences for genome reannotations. However, most of the current N‐terminal enrichment approaches involve multiple chemical derivatizations and chromatographic separation processes which are time consuming and can contribute to N‐terminal peptide losses. In this study, a fast, one‐step approach utilizing (N‐Succinimidyloxycarbonylmethyl)tris(2,4,6‐trimethoxyphenyl)phosphonium bromide (TMPP) derivatization and StageTip separation was developed to enhance N‐terminal peptide enrichment and analysis. Based on the characteristics of TMPP‐derivatized samples, such as a higher hydrophobicity and increased likelihood to produce a and b ions in collision‐induced dissociation or HCD fragmentation modes, first the SDS‐PAGE was optimized to increase protein loading and gel entry and to remove unbound TMPP. Then, this process was combined with a simplified StageTip separation and a new scoring criterion (considering a, b and y ions) to identify more TMPP‐modified N‐terminal spectra. When utilizing a low amount of starting material (～20 μg protein), a total of 581 yeast N‐terminal peptides were identified, with 485 of them being TMPP modified, in only about one third of the general experimental time. It is hoped that the workflow constructed herein will provide a fast and practical strategy for N‐terminomic studies.     

Phosphoproteome exploration reveals a reformatting of cellular processes in response to low sterol biosynthetic capacity in Arabidopsis

Sterols are membrane-bound isoprenoid lipids that are required for cell viability and growth. In plants, it is generally assumed that 3-hydroxy-3-methylglutaryl-CoA-reductase (HMGR) is a key element of their biosynthesis, but the molecular regulation of that pathway is largely unknown. In an attempt to identify regulators of the biosynthetic flux from acyl-CoA toward phytosterols, we compared the membrane phosphoproteome of wild-type Arabidopsis thaliana and of a mutant being deficient in HMGR1. We performed a N-terminal labeling of microsomal peptides with a trimethoxyphenyl phosphonium (TMPP) derivative, followed by a quantitative assessment of phosphopeptides with a spectral counting method. TMPP derivatization of peptides resulted in an improved LC-MS/MS detection due to increased hydrophobicity in chromatography and ionization efficiency in electrospray. The phosphoproteome coverage was 40% higher with this methodology. We further found that 31 proteins were in a different phosphorylation state in the hmgr1-1 mutant as compared with the wild-type. One-third of these proteins were identified based on novel phosphopeptides. This approach revealed that phosphorylation changes in the Arabidopsis membrane proteome targets major cellular processes such as transports, calcium homeostasis, photomorphogenesis, and carbohydrate synthesis. A reformatting of these processes appears to be a response of a genetically reduced sterol biosynthesis.     

Chemistry of tris(2,4,6-trimethoxyphenyl)phosphine with rhodium(I) and iridium(I) olefin complexes

Rh(I) and Ir(I) complexes of the type [Rh(cod)(η²-TMPP)]¹⁺ (1) and M(cod)(η²-TMPP-O) (M = Rh (2), Ir (3); cod = cyclooctadiene; TMPP = tris(2,4,6-trimethoxyphenyl)phosphine; TMPP-O = mono-demethylated form of TMPP) have been isolated from reactions of [M(cod)Cl]₂ with M′BF₄ (M′ = Ag⁺, K⁺, Na⁺) followed by addition of the tertiary phosphine ligand. This chemistry is dependent on the identity of the metal, as both the cationic phosphine complex and the neutral phosphino-phenoxide compound are stable for Rh(I), whereas only the latter is stable for Ir(I). The three complexes have been characterized by IR and NMR (¹H and ³¹P) spectroscopies as well as by cyclic voltammetry. The ¹H NMR spectrum of [Rh(cod)(η²-TMPP)]¹⁺ (1) is in accord with the formula and reveals that the TMPP phenyl rings are undergoing rapid exchange between coordinated and non-coordinated modes; the corresponding spectra of 2 and 3 support free rotation about the P---C bonds of the unbound phenyl rings with no fluxionality of the bound demethylated ring. The ³¹P{¹H} NMR spectrum of the neutral species 2 exhibits a significant upfield shift with respect to the analogous cationic compound 1. This shielding is the result of improved electron donation to the metal from a phenoxide group as compared to an ether substituent. In situ addition of CO to the reaction between TMPP and [Rh(cod)Cl]₂ or [Ir(cod)Cl]₂ in the presence of M′BF₄ results in the isolation of the monocarbonyl species [Rh(TMPP)(η²-TMPP)(CO)][BF₄] (5) and the stable dicarbonyl compound [Ir(TMPP)₂(CO)₂][BF₄] (4), respectively. Single crystal X-ray data for

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. The geometry of 4 is square planar, with essentially ideal angles for the mutually trans disposed phosphine and carbonyl ligands, as found in earlier studies for the analogous Rh dicarbonyl compound. The ¹H NMR spectrum of 4 supports the assignment of magnetically equivalent phosphorus nuclei in solution. The results of this study indicate that cyclooctadiene is a particularly strong ligand for monovalent late transition metals ligated by TMPP, to the extent that it is inert with respect to substitution in the absence of π-acceptor ligands such as carbon monoxide.     

Deep N-terminomics of Mycobacterium tuberculosis H37Rv extensively correct annotated encoding genes

Mycobacterium tuberculosis (MTB) is a severe causing agent of tuberculosis (TB). Although H37Rv, the type strain of M. tuberculosis was sequenced in 1998, annotation errors of encoding genes have been frequently reported in hundreds of papers. This phenomenon is particularly severe at the 5′ end of the genes. Here, we applied a TMPP [(N-Succinimidyloxycarbonylmethyl) tris (2,4,6-trimethoxyphenyl) phosphonium bromide] labeling combined with StageTip separating strategy on M. tuberculosis H37Rv to characterize the N-terminal start sites of its annotated encoding genes. Totally, 1047 proteins were identified with 2058 TMPP labeled N-terminal peptides from all the 2625 mass spectrometer (MS) sequenced proteins. Comparative genomics analysis allowed the re-annotation of 43 proteins' N-termini in H37Rv and 762 proteins in Mycobacteriaceae. All revised N-termini start sites were distributed in 5’-UTR of annotated genes due to over-annotation of previous N-terminal initiation codon, especially the ATG. In addition, we identified and verified a novel gene Rv1078A in +3 frame different from the annotated gene Rv1078 in +2 frame. Altogether, our findings contribute to the better understanding of N-terminal of H37Rv and other species from Mycobacteriaceae that can assist future studies on biological study.     

Enriching C-terminal peptide from endopeptidase ArgC digest for protein C-terminal analysis

This Letter describes a method for enriching C-terminal peptide of protein for C-terminal sequence analysis. This method employs endopeptidase ArgC digestion and C-terminal peptide enrichment using m-aminophenylboronic acid–agarose as an arginine-capture material. The selectively recovered C-terminal peptide incorporates no artificial derivatization. Therefore, the widely used functional groups (e.g. α-NH₂ and α-COOH) can be used for any necessary transformation. In this research, a TMPP mass tag was attached to the α-NH₂ group to clarify the amino acid sequence of the C-terminal peptide.     

Synthesis and characterization of peptide grafted porous polymer microstructures

A scanning laser system has been used to generate three-dimensional trimethylolpropane trimethacrylate (TRIM) cross-linked poly(2-hydroxylethyl methacrylate) polymer microstructures through azo-bis(isobutyro)nitrile (AIBN) photopolymerization using a 20 x 0.5 NA microscope objective and 365 nm laser excitation. Macropores are observed to form without the use of porogens in regions of highest light flux. This is attributed to phase separation, which results from differences in monomer reactivity and miscibility. The microstructures were aminated and then protected with the photolabile protective group 6-nitroveratryloxycarbonyl (NVOC). This made it possible to selectively modify the microstructures with the same scanning laser system that was used to fabricate them, resulting in peptide grafted three-dimensional porous microstructures. On the basis of the absorbance of the dibenzofulvene-piperidine, these structures have an amine site density of approximately 0.1 nmol/feature. MALDI-TOF MS was used to characterize peptide photografted microstructures. N-Tris(2,4,6-trimethoxyphenyl)phosphonium (TMPP) labeling of the peptides greatly enhanced detection and allowed post-source decay sequencing of the peptides from the microstructures. The techniques described could be used to generate three-dimensional peptide grafted porous scaffolds for tissue engineering applications.     

Mass spectrometry-based sequencing of protein C-terminal peptide using α-carboxyl group-specific derivatization and COOH capturing

An approach to mass spectrometry (MS)-based sequence analysis of selectively enriched C-terminal peptide from protein is described. This approach employs a combination of the specific derivatization of α-carboxyl group (α-COOH), enzymatic proteolysis using endoproteinase GluC, and enrichment of C-terminal peptide through the use of COOH-capturing material. Highly selective derivatization of α-COOH was achieved by a combination of specific activation of α-COOH through oxazolone chemistry and amidation using 3-aminopropyltris-(2,4,6-trimethoxyphenyl)phosphonium bromide (TMPP-propylamine). This amine component was used to simplify fragmentation in tandem mass spectrometry (MS/MS) measurement, which facilitated manual sequence interpretation. The peptides produced after GluC digestion were then treated with a COOH scavenger to enrich the C-terminal peptide that is only devoid of COOH groups, and the obtained C-terminal peptide was readily sequenced by matrix-assisted laser desorption/ionization (MALDI)-MS/MS due to the TMPP mass tag.     

Deglyco-peplomycin metal complexes on DNA fibers: a role of the sugar moiety for the stability and the orientation of the complexes

Binding structures of metal complexes of deglyco-peplomycin (dPEP) on DNA were investigated by comparing dPEP complexes with those of bleomycin (BLM) using DNA fiber EPR spectroscopy. A low spin species of Fe(III)dPEP observed in the DNA pellet changed irreversibly to several high spin species after the fabrication of the DNA fibers. The g values of the high spin species were different from those of Fe(III)BLM. The high spin species could not be nitrosylated reductively to ON-Fe(II)dPEP, suggesting that some nitrogen atoms coordinated to the Fe(III) were displaced on the DNA fibers. On the other hand, O(2)-Co(II)dPEP remained intact on the fibers similarly to O(2)-Co(II)BLM but with an increased randomness in the orientation on the DNA. In contrast to Cu(II)BLM, a considerable amount of Cu(II)dPEP bound almost randomly on B-form DNA fibers. These results indicated that the sugar moiety in peplomycin or bleomycin is playing an important role in enhancing the stability of the metal-binding domain and in the stereospecificity of the binding on DNA.     

DNA polymerase switching on homotrimeric PCNA at the replication fork of the euryarchaea Pyrococcus abyssi

DNA replication in Archaea, as in other organisms, involves large protein complexes called replisomes. In the Euryarchaeota subdomain, only two putative replicases have been identified, and their roles in leading and lagging strand DNA synthesis are still poorly understood. In this study, we focused on the coupling of proliferating cell nuclear antigen (PCNA)-loading mechanisms with DNA polymerase function in the Euryarchaea Pyrococcus abyssi. PCNA spontaneously loaded onto primed DNA, and replication factor C dramatically increased this loading. Surprisingly, the family B DNA polymerase (Pol B) also increased PCNA loading, probably by stabilizing the clamp on primed DNA via an essential motif. In contrast, on an RNA-primed DNA template, the PCNA/Pol B complex was destabilized in the presence of dNTPs, allowing the family D DNA polymerase (Pol D) to perform RNA-primed DNA synthesis. Then, Pol D is displaced by Pol B to perform processive DNA synthesis, at least on the leading strand.     

Using chemical derivatization and mass spectrometric analysis to characterize the post-translationally modified Staphylococcus aureus surface protein G

The Staphylococcus aureus surface protein G (SasG) is an important mediator of biofilm formation in virulent S. aureus strains. A detailed analysis of its primary sequence has not been reported to date. SasG is highly abundant in the cell wall of the vancomycin-intermediate S. aureus strain HIP5827, and was purified and subjected to sequence analysis by MS. Data from MALDI-TOF and LC-MS/MS experiments confirmed the predicted N-terminal signal peptide cleavage site at residue A₅₁ and the C-terminal cell wall anchor site at residue T₁₀₈₆. The protein was also derivatized with N-succinimidyloxycarbonyl-methyl-tris(2,4,6-trimethoxyphenyl) phosphonium bromide (TMPP-Ac-OSu) to assess the presence of additional N-terminal sites of mature SasG. TMPP-derivatized SasG peptides featured m/z peaks with a 572 Da mass increase over the equivalent underivatized peptides. Multiple N-terminal peptides, all of which were observed in the 150 amino acid segment following the signal peptide cleavage at the residue A₅₁, were characterized from MS and MS/MS data, suggesting a series of successive N-terminal truncations of SasG. A strategy combining TMPP derivatization, multiple enzyme digestions to generate overlapping peptides and detailed MS analysis will be useful to determine and understand functional implications of PTMs in bacterial cell wall-anchored proteins, which are frequently involved in the modulation of virulence-associated bacterial surface properties.     

Effect of cortisol on the cellular proliferation and maturation in the cerebrum and the cerebellum of the rat: Importance of the age of the animals at the beginning of treatment]

Young rats were given either a single subcutaneous injection (1 mg at 0, 1, 4 or 8 days), or four consecutive daily injections (0.2 mg/day between 0 and 3 days; 0.4 mg/day between 4 and 7 days; 0.6 mg/day between 8 and 11 days) of cortisol acetate in order to test the influence of age on the action of corticosteroids on the biochemical maturation of the cerebrum and cerebellum in terms of their DNA, RNA, and protein contents. The results showed that: 1 The diminution of the DNA content at 35 days was greater in the cerebellum (- 16 to - 32%) than in the cerebrum (- 9 to 20%); the DNA content of the cerebrum was more affected by treatment at birth, whereas that of the cerebellum was more affected by the delayed treatments. Results were different when expressed in terms of reduction of the normal increase: the gain of DNA decreased more in the cerebrum (-70%) than in the cerebellum (-40%); but the most delayed treatment induced a greater effect in both organs. These abnormalities were not always accompanied by a significant decrease of the body weight. 2 Generally, the treatments led to an increase of the mean cell territory, expressed either in terms of decrease of the DNA concentration, or in terms of increase of the organ weight/DNA ratio. Moreover, the increase of the RNA/DNA and the protein/DNA ratios constituted an indication of an accelerated cellular maturation.     

Tomato SlIDA has a critical role in tomato fertilization by modifying reactive oxygen species homeostasis

Anther development and pollen tube elongation are key steps for pollination and fertilization. The timing and spatial distribution of reactive oxygen species (ROS) and programmed cell death are central to these processes, but the regulatory mechanism of ROS production is not well understood. Inflorescence deficient in abscission (IDA) is implicated in many plant development and responses to environmental stimuli. However, their role in reproductive development is still unknown. We generated tomato knockout lines (CR‐slida) of an IDA homolog (SlIDA), which is expressed in the tapetum, septum and pollen tube, and observed a severe defect in male gametes. Further analysis indicated that there was a programmed cell death defect in the tapetum and septum and a failure of anther dehiscence in the CR‐slida lines, likely related to insufficient ROS signal. Liquid chromatography‐tandem mass spectrometry identified mature SlIDA as a 14‐mer EPIP peptide, which was shown to be secreted, and a complementation experiment showed that application of a synthetic 14‐mer EPIP peptide rescued the CR‐slida defect and enhanced the ROS signal. Moreover, the application of the ROS scavengers diphenyleneiodonium or Mn‐TMPP suppressed peptide function. Collectively, our results revealed that SlIDA plays an essential role in pollen development and pollen tube elongation by modulating ROS homeostasis.     

A compact form of DNA in solution. III. Influence of the ion composition of the solution on the compactization process of double-stranded DNA in the presence of peg]

The data showing the features of the DNA compactization process in PEG-containing solutions of chlorides of different alkaline metals (LiCl, KCl, RbCl and CsCl) and an ammonium salt (CH3-(CH2)17-N-(CH3)3Br) are presented. The data indicate that the formation of a compact form of the double-stranded DNA in PEG-containing water-salt solutions depends not only on the PEG concentration and ionic strength but on tha cation nature as well. The compactization occurs most easily in the presence of Na+-ions. This indicates a specific character of interaction between Na+-ions and DNA phosphate groups which may be due to an optimum structural fit between the hydrated Na+-ions and orientation of the phosphate groups in the DNA molecule. The nature of forces involved in the processes of the intramolecular compactization and intermolecular aggregation of double-stranded DNA molecules in water-salt solution is discussed. The difference between the effect of Na+ and that of K+-ions on the compactization process at the ionic strengths close to physiological values makes it possible to suggest that the changes of the tertiary structure of double-stranded DNA which accompany its function in vivo may take place under conditions of a decreased water activity at the expense of relatively slight changes in ion composition of the water surrounding DNA.     

Comparison of the cleavage of pyrimidine dimers by the bacteriophage T4 and Micrococcus luteus UV-specific endonucleases

A comparison was made of the activity of the UV-specific endonucleases of bacteriophage T4 (T4 endonuclease V) and of Micrococcus luteus on ultravilet light-irradiated DNA substrates of defined sequence. The two enzymes cleave DNA at the site of pyrimidine dimers with the same frequency. The products of the cleavage reaction are the same, suggesting that the scission of DNA by T4 endonuclease V occurs via the combined actin of a pyrimidine dimer specific DNA glycosylase and an apyrimidinic-apurinic (AP) endonuclease as was recently shown for the M. luteus enzyme. The pyrimidine dimer DNA-glycosylase activity of both enzymes is more active on double-stranded DNA than it is on single-stranded DNA.     

Comparative study of DNA enzymes and ribozymes against the same full-length messenger RNA of the vanilloid receptor subtype I.

The efficiencies of 32 antisense oligodeoxynucleotides, 35 DNA enzymes and 6 ribozymes to bind and cleave the full-length messenger RNA of the vanilloid receptor subtype I were analyzed. Systematic screening of the mRNA revealed that good accessibility of a putative cleavage site for antisense oligodeoxynucleotides is a necessary but not a sufficient prerequisite for efficient DNA enzymes. Comparison of DNA enzymes and ribozymes against the same target sites revealed: 1) DNA enzymes were more active with longer recognition arms (9 nucleotides on either side), whereas ribozymes revealed higher activities with shorter recognition arms (7 nucleotides on either side). 2) It does not only depend on the target site but also on the enzyme sequence, whether a DNA enzyme or a ribozyme is more active. 3) The most efficient DNA enzyme found in this study had an approximately 15-fold higher reaction rate, k(react), and a 100-fold higher k(react)/K(m) under single turnover conditions compared with the fastest ribozyme. DNA enzymes as well as ribozymes showed significant activity under multiple turnover conditions, the DNA enzymes again being more active. We therefore conclude that DNA enzymes are an inexpensive, very stable and active alternative to ribozymes for the specific cleavage of long RNA molecules.