Enhancement of phosphoprotein analysis using a fluorescent affinity tag and mass spectrometry

A fluorescent affinity tag (FAT) was synthesized and was utilized to selectively modify phosphorylated serine and threonine residues via beta-elimination and Michael addition chemistries in a 'one-step' reaction. This labeling technique was used for covalent modification of both phosphoproteins and phosphopeptides, allowing identification of these molecular species by fluorescence imaging after solution- or gel-based separation methods. In addition to the strong fluorescence of the rhodamine tag, a commercially available antibody can be used to enrich low-abundance post-labeled phosphopeptides present in complex mixtures. Application of this methodology to phosphorylation-site mapping has been evaluated for a phosphoprotein standard, bovine beta-casein. Initial results demonstrated low femtomole detection limits after fluorescence image analysis of FAT-labeled proteins or peptides.     

Synthesis and Absorption Properties of New Na Specific Adsorbent Li（1＋x）AlxTi（2-x）（PO4）3

A new kind of adsorbent Li（1＋x）AlxTi（2-x）（PO4）3 was synthesized by solid state reaction method. The influence of the content of doping aluminum on the adsorbent Li（1＋x）AlxTi（2-x）（PO4）3 was investigated by XRD, while the morphology of powders was observed by SEM. The investigation of the adsorption properties showed that the adsorbent can selectively adsorb sodium with the adsorption capacity of 11.76 mg/g. The optimum conditions of adsorption are at pH 10.0-11.0 in LiCl solution.     

Association between the photodynamic loss of Bcl-2 and the sensitivity to apoptosis caused by phthalocyanine photodynamic therapy

We have reported that photodynamic therapy (PDT) using the photosensitizer phthalocyanine (Pc) 4 and red light damages the antiapoptotic protein Bcl-2. Recently, using transient transfection of Bcl-2 deletion mutants, we identified the membrane anchorage domains of Bcl-2 as necessary to form the photosensitive target. However, it is not clear how Bcl-2 photodamage sensitizes cells to Pc 4-PDT-induced apoptosis, whether overall cell killing is also sensitized or how up-regulation of Bcl-2 in tumors might make them more or less responsive to Pc 4-PDT. In this study we report on MCF-7c3 cells (human breast cancer cells expressing stably transfected procaspase-3) overexpressing wild-type Bcl-2 or certain deletion mutants in either a transient or a stable mode. By flow cytometric analysis of transiently transfected cells, we found that wild-type Bcl-2, Bcl-2delta33-54 and Bcl-2delta37-63 (each of which can be photodamaged) protected cells from apoptosis caused by Pc 4-PDT. In contrast, Bcl-2delta210-239, which lacks the C-terminal transmembrane domain and cannot be photodamaged, afforded no protection. We then evaluated the PDT sensitivity of transfected cell lines stably overexpressing high levels of wild-type Bcl-2 or one of the Bcl-2 mutants. Overexpression of wild-type Bcl-2, Bcl-2delta33-54 or Bcl-2delta37-63 resulted in relative resistance of cells to Pc 4-PDT, as assessed by morphological apoptosis or loss of clonogenicity. Furthermore, overexpression of Bcl-2 also inhibited the activation-associated conformational change of the proapoptotic protein Bax, and higher doses of Pc 4 and light were required to activate Bax in cells expressing high levels of Bcl-2. Many advanced cancer cells have elevated amounts of Bcl-2. Our results show that increasing the dose of Pc 4-PDT can overcome the resistance afforded by either Bcl-2 or the two mutants. PDT regimens that photodamage Bcl-2 lead to activation of Bax, induction of apoptosis and elimination of the otherwise resistant tumor cells.     

Secondary deuterium kinetic isotope effects in irreversible additions of allyl reagents to benzaldehyde

The competitive kinetics of additions of allyl to benzaldehyde-h and -d from allyltributyl tin, from diisopropyltartrylallyl boronate, and from allyl bromide and zinc dust in aqueous tetrahydrofuran have inverse secondary deuterium kinetic isotope effects, SDKIEs. These inverse SDKIEs are in contrast to the normal SDKIEs that were obtained with allyl lithium and allyl Grignard, suggesting rate-determining single-electron transfer in these cases. By various MO calculations the transition state for addition of allyl boronate occurs with substantial B-O bond formation and little C-C bond formation. The magnitudes of the SDKIEs with the other two allylating reagents, when compared with reasonable equilibrium isotope effects for the addition, suggest transition states with substantial C-C bond formation.     

Automated multi-attribute method sample preparation using high-throughput buffer exchange tips

Rationale

The multi-attribute method (MAM) has become a valuable mass spectrometry (MS)-based tool that can identify and quantify the site-specific product attributes and purity information for biotherapeutics such as monoclonal antibodies (mAbs) and fusion molecules in recent years. As we expand the use of the MAM at various stages of drug development, it is critical to enhance the sample preparation throughput without additional chemical modifications and variability.

Methods

In this study, a fully automated MAM sample preparation protocol is presented, where rapid desalting in less than 15 minutes is achieved using miniaturized size-exclusion chromatography columns in pipette tips on an automated liquid handler. The peptide samples were analyzed using an electrospray ionization (ESI) orbitrap mass spectrometer coupled to an ultra-high-performance liquid chromatography (UHPLC) system with a dual column switching system.

Results

No significant change was observed in product attributes and their quantities compared with manual, low-artifact sample preparation. The sample recovery using the buffer exchange tips was greatly enhanced over the manual spin cartridges while still demonstrating excellent reproducibility for a wide variety of starting sample concentrations. Unlike a plate desalting system, the individual columns provide flexibility in the number of samples prepared at a time and sample locations within plates.

Conclusions

This automated protocol enables the preparation of up to 96 samples with less “at-bench” time than the manual preparation of a smaller batch of samples, thereby greatly facilitating support of process development and the use of the MAM in quality control.

     

Excess enthalpy and entropy of aqueous tetramethylguanidinium methanesulfonate and trifluoromethanesulfonate solutions

Relative apparent molal heat contents, φ_L, are reported for the tetramethylguanidinium salts of methanesulfonic (MS) and trifluoromethanesulfonic (TFMS) acids. Relative partial molal heat contents, L?₂, are calculated and combined with previously reported activity coefficient data, to yield excess entropies. It is found that the values of L?₂ make the principal contribution to the excess entropies of Me₄GuMS solutions while the excess free energies, as calculated from activity coefficients, make the greatest contribution to the excess entropies of Me₄GuTFMS solutions.     

Characterization of anthocyanin extracts from maize kernels

The aim of the present work is to characterize the pigments present in the kernel of four native maize varieties related to the races Arrocillo, Cónico, Peruano, and Purepecha to determine their possible use as natural dyes. Total anthocyanin content is determined by a conventional spectrophotometric method, and anthocyanin analysis is done by high-performance liquid chromatography. The stability of the pigment at pH is also evaluated. The four maize samples contained anthocyanin in both the pericarp and aleurone layer. Total anthocyanin content among samples ranged from 54 mg/100 g of sample to 115 mg/100 g of sample. Anthocyanin profiles are almost the same among the four samples. Differences are observed only in the relative percentage of each anthocyanin. The anthocyanins identified are cyanidin-3-glucoside, pelargonidin- 3-glucoside, peonidin-3-glucoside, cyanidin-3-(6" malonylglucoside), and cyanidin-3-(3",6"-dimalonylglucoside). Anthocyanin extracts showed similar behavior in solutions with different pH. From pH 1-6 lambdamax values are maintained almost constant; however, above this pH value, a marked increase is observed in the bathochromic shifts, but the bluish color did not continue to change above pH 8.     

Synthesis of New Chiral C2-Symmetrical Bis(oxazoline )Compounds

New chiral C2-symmetrical bis(oxazoline)compounds bearing one isobutyl-,secbutyl-benzyl- or isopropyl-substituent at the 4-position have been prepared and characterized.     

Sequestering of Eu(III) by a GAAA RNA tetraloop

The site-specific binding of metal ions maintains an important role in the structure, thermal stability, and function of folded RNA structures. RNA tetraloops of the "GNRA" family (where N = any base and R = any purine), which owe their unusual stability to base stacking and an extensive hydrogen bonding network, have been observed to bind metal ions having different chemical and geometric properties. We have used laser-induced lanthanide luminescence and isothermal titration calorimetry (ITC) to examine the metal-binding properties of an RNA stem loop of the GNRA family. Previous research has shown that a single Eu(III) ion binds the stem loop fragment in a highly dehydrated site with a K(d) of approximately 12 microM. Curve-fitting analysis of the broad luminescence excitation spectrum of Eu(III) upon complexation with the tetraloop fragment indicates the possibility of two microenvironments that do not differ in hydration number. Binding of Eu(III) to the loop was accompanied by positive enthalpic changes, consistent with energetic cost of removal of water molecules and suggesting that the binding is entropically driven. By comparison, binding of Mg(II) or Mn(II) to the RNA loop, or Eu(III) to the DNA analogue of the loop, was associated with exothermic changes, consistent with predominantly outer-sphere coordination. These results suggest specific binding, most probably involving ligands on the 5' side of the loop.     

Composition-controlled synthesis of bimetallic gold-silver nanoparticles

This paper reports findings of an investigation of the synthesis of monolayer-capped binary gold-silver (AuAg) bimetallic nanoparticles that is aimed at understanding the control factors governing the formation of the bimetallic compositions. The synthesis of alkanethiolate-capped AuAg nanoparticles was carried out using two related synthetic protocols using aqueous sodium borohydride as a reducing agent. One involves a two-phase reduction of AuCl(4)(-), which is dissolved in organic solution, and Ag(+), which is dissolved in aqueous solution. The other protocol involves a two-phase reduction of AuCl(4)(-) and AgBr(2)(-), both of which are dissolved in the same organic solution. AuAg nanoparticles of 2-3 nm core sizes with different compositions in the range of 0-100% Au have been synthesized. The two synthetic routes were compared in terms of bimetallic composition and size properties. Our new findings have allowed us to establish the correlation between synthetic feeding of metals and metal compositions in the bimetallic nanoparticles, which have important implications to the exploration of gold-based bimetallic nanoparticles for constructing sensing and catalytic nanomaterials.