Affinity‐Labeling‐Based Introduction of a Reactive Handle for Natural Protein Modification

A new chemical method to site‐specifically modify natural proteins without the need for genetic manipulation is described. Our strategy involves the affinity‐labeling‐based attachment of a unique reactive handle at the surface of the target protein, and the subsequent selective transformation of the reactive handle by a bioorthogonal reaction to introduce a variety of functional probes into the protein. To demonstrate this approach, we synthesized labeling reagents that contain: 1) a benzenesulfonamide ligand that directs specifically to bovine carbonic anhydrase II (bCA), 2) an electrophilic epoxide group for protein labeling, 3) an exchangeable hydrazone bond linking the ligand and the epoxide group, and 4) an iodophenyl or acetylene handle. By incubating the labeling reagent with bCA, the reactive handle was covalently attached at the surface of bCA through epoxide ring opening. Either after or before removing the ligand by a hydrazone/oxime‐exhange reaction, which restores the enzymatic activity, the reactive handle incorporated could be derivatized by Suzuki coupling or Huisgen cycloaddition reactions. This method is also applicable to the target‐specific multiple modification in a protein mixture. The availability of various (photo)affinity‐labeling reagents and bioorthogonal reactions should extend the flexibility of this strategy for the site‐selective incorporation of many functional molecules into proteins.     

A note on edge-graceful spectra of the square of paths

For a simple path P_r on r vertices, the square of P_r is the graph on the same set of vertices of P_r, and where every pair of vertices of distance two or less in P_r is connected by an edge. Given a (p,q)-graph G with p vertices and q edges, and a nonnegative integer k, G is said to be k-edge-graceful if the edges can be labeled bijectively by k,k+1,…,k+q−1, so that the induced vertex sums are pairwise distinct, where the vertex sum at a vertex is the sum of the labels of all edges incident to such a vertex, modulo the number of vertices p. We call the set of all such k the edge-graceful spectrum of G, and denote it by egI(G). In this article, the edge-graceful spectrum for the square of paths is completely determined for odd r.     

The profile of the Cartesian product of graphs

Given a graph G, a proper labelingf of G is a one-to-one function from V(G) onto {1,2,…,|V(G)|}. For a proper labeling f of G, the profile widthw_f(v) of a vertex v is the minimum value of f(v)−f(x), where x belongs to the closed neighborhood of v. The profile of a proper labelingfofG, denoted by P_f(G), is the sum of all the w_f(v), where v∈V(G). The profile ofG is the minimum value of P_f(G), where f runs over all proper labeling of G. In this paper, we show that if the vertices of a graph G can be ordered to satisfy a special neighborhood property, then so can the graph G×Q_n. This can be used to determine the profile of Q_n and K_m×Q_n.     

Sensitivity enhancement using paramagnetic relaxation in MAS solid-state NMR of perdeuterated proteins

Previously, Ishii et al., could show that chelated paramagnetic ions can be employed to significantly decrease the recycle delay of a MAS solid-state NMR experiment [N.P. Wickramasinghe, M. Kotecha, A. Samoson, J. Past, Y. Ishii, Sensitivity enhancement in C-13 solid-state NMR of protein microcrystals by use of paramagnetic metal ions for optimizing H-1 T-1 relaxation, J. Magn. Reson. 184 (2007) 350-356]. Application of the method is limited to very robust samples, for which sample stability is not compromised by RF induced heating. In addition, probe integrity might be perturbed in standard MAS PRE experiments due to the use of very short duty cycles. We show that these deleterious effects can be avoided if perdeuterated proteins are employed that have been re-crystallized from D(2)O:H(2)O=9:1 containing buffer solutions. The experiments are demonstrated using the SH3 domain of chicken alpha-spectrin as a model system. The labeling scheme allows to record proton detected (1)H, (15)N correlation spectra with very high resolution in the absence of heteronuclear dipolar decoupling. Cu-edta as a doping reagent yields a reduction of the recycle delay by up to a factor of 15. In particular, we find that the (1)H T(1) for the bulk H(N) magnetization is reduced from 4.4s to 0.3s if the Cu-edta concentration is increased from 0mM to 250 mM. Possible perturbations like chemical shift changes or line broadening due to the paramagnetic chelate complex are minimal. No degradation of our samples was observed in the course of the experiments.     

基于液质联用技术的蛋白质-蛋白质相互作用研究进展※

蛋白质-蛋白质相互作用调控着细胞内众多生物过程, 蛋白质间相互作用网络的绘制对解析复杂的生物过程至关重要. 面对生物体中复杂的蛋白质-蛋白质相互作用, 液质联用技术不仅具有灵敏度高的鉴定优势, 还可以对数以千计的蛋白质进行定量分析. 因此, 对目标蛋白质进行富集、标记或共分级的处理后, 结合液质联用技术对蛋白质准确而灵敏的鉴定, 这类技术已被广泛应用于复杂样本中蛋白质-蛋白质相互作用网络的解析. 目前基于液质联用技术的几种常用的方式, 包括亲和纯化质谱方法(AP-MS)、近程标记质谱方法(PDB-MS)、化学交联质谱方法(XL-MS)和共分级偶联质谱方法(CF-MS)等. 本综述讨论了这些方法的基本原理、优点和在细胞内解析蛋白质间相互作用的应用.     

A study of reproducibility of guanidination-dimethylation labeling and liquid chromatography matrix-assisted laser desorption ionization mass spectrometry for relative proteome quantification

The combination of dimethylation after guanidination (2MEGA) isotope labeling with microbore liquid chromatography (LC)-matrix-assisted laser desorption ionization (MALDI) MS and MS/MS [C. Ji, N. Guo, L. Li, J. Proteome Res. 4 (2005) 2099] has been reported as a promising strategy for abundance ratio-dependent quantitative proteome analysis. A critical step in using this integrated strategy is to set up the abundance ratio threshold of peptide pairs, above which the peptide pairs are used for quantifying and identifying the protein that is considered to be differentially expressed between two different samples. The threshold is determined by technical variation (i.e., the overall abundance ratio variation caused by the experimental process including sample workup, MS analysis and data processing) as well as biological variation (i.e., the abundance ratio variation caused by the biological process including cell growth), which can be defined and assessed by a coefficient of variation (CV). We have designed experiments and measured three different levels of variations, starting with the same membrane protein preparation, the same batch of cells and three batches of cells from the same cell line grown under the same conditions, respectively. It is shown that technical variation from the experimental processes involved in 2MEGA labeling LC-MALDI MS has a CV of <15%. In addition, the measured biological variation from cell growth was much smaller than the measured technical variation. From the studies of the occurrence rate of outliers in the distribution of the abundance ratio data within a comparative dataset of peptide pairs, it is concluded that, to compare the proteome changes between two sets of cultured cells without the use of replicate experiments, a relative abundance ratio of greater than 2X or less than 0.5X (X is the average abundance ratio of the dataset) on peptide pairs can be used as a stringent threshold to quantify and identify differentially expressed proteins with high confidence.     

Carbohydrate pyrolysis mechanisms from isotopic labeling: Part 2. The pyrolysis of d-glucose: General disconnective analysis and the formation of C1 and C2 carbonyl compounds by electrocyclic fragmentation mechanisms

The flash pyrolysis of d-glucose was investigated by the use of isotopic labeling with ¹³C, in conjunction with GC/MS. Co-pyrolysis of uniformly labeled and unlabeled d-glucose established the extent of unimolecular formation of each of the pyrolysis products. A complete set of singly labeled d-glucose isotopologs was used to determine the origin of specific carbons within each of the pyrolysis products. The Cyclic Grob 1,3-diol fragmentation and the tandem alkaline pinacol rearrangement/retro-aldol fragmentation (TAPRRAF) discovered from the pyrolysis of glycerin were applied to the analysis of pyrolytic fragmentation pathways for d-glucose. These mechanisms provide means of initial carbon–carbon bond breakage, and are consistent with the high proportion of carbon-unimolecularity observed for many of the volatile low-molecular weight products of the reaction. These and other reactions, including the retro-aldol fragmentation, carbonyl migration, dehydration, ene-reaction, retro-Claisen cleavage, hydrolysis, or alcoholysis were applied conceptually to the initial fragments resulting from either mechanism to ascertain the ultimate fate of the carbons of d-glucose. The “predicted” results were then compared with labeling patterns observed by experiment. The most promising rationalizations provided by this exercise are presented herein, for the formation of five C₁ and C₂ carbonyl-containing pyrolysis products: formaldehyde, formic acid, acetaldehyde, glycolaldehyde and acetic acid.     

Facile and convenient method of deuterium gas generation using a Pd/C-catalyzed H2-D2 exchange reaction and its application to synthesis of deuterium-labeled compounds

The Pd/C-catalyzed H(2)-D(2) exchange reaction using a H(2)-D(2)O combination provided a general, efficient and environmentally friendly route for the preparation of deuterium gas (D(2)). H(2) sealed in a reaction flask was converted into nearly pure D(2), which could be used for the Pd/C-catalyzed one-pot reductive deuteration of various reducible functionalities and the chemoselective one-pot deuterogenation of olefin and acetylene. Additionally, we established the capturing method of the generated D(2) in a balloon, which was successfully applied to the Pd/C-catalyzed reductive mono-N-alkylation of a primary amine using nitrile as the alkylating reagent.