Ultra acidic strong cation exchange enabling the efficient enrichment of basic phosphopeptides

We present a straightforward method to enrich phosphopeptides with multiple basic residues, an under-represented class in common enrichment strategies. Our method is based on a two-dimensional strong cation exchange (SCX) strategy, operating at two different acidic pHs, enabling both separation and enrichment of different classes of phosphopeptides. The principle of enrichment is based on the change of net charge of phosphorylated peptides under strong acidic conditions in the second SCX, whereas the net charge of regular peptides remains unchanged, thus enabling separation based on net charge. Application of our tandem SCX approach to a modest amount of human cells allowed the identification of over 10,000 unique "basic" phosphopeptides of which many represent putative targets of basophilic kinases.     

Enrichment of carbonylated peptides using Girard P reagent and strong cation exchange chromatography

It has been shown that oxidatively modified forms of proteins accumulate during oxidative stress, aging, and in some age-related diseases. One of the unique features of protein oxidation by a wide variety of routes is the generation of carbonyl groups. Of major interest in the study of oxidative stress diseases is which proteins in a proteome are being oxidized and the site(s) of oxidation. Based on the fact that proteins are generally characterized through tryptic peptide fragments, this paper reports a method for the isolation of oxidized peptides, which involves (1) derivatization of oxidized proteins with Girard P reagent (GRP; 1-(2-hydrazino-2-oxoethyl)pyridinium chloride), (2) following proteolysis enrichment of the derivatized peptide using strong cation exchange (SCX) chromatography, and (3) identification of oxidation sites using tandem mass spectrometry. Derivatization of aldehydes and ketones in oxidized proteins was accomplished by reacting protein carbonyls with the hydrazide of GRP. The resulting hydrazone bond was reduced by sodium cyanoborohydride to further stabilize the labeling. Derivatization time and concentrations of the derivatizing agent were optimized with model peptides. Oxidized transferrin was used as model protein to study derivatization efficiency at the protein level. Following metal-catalyzed oxidation of transferrin, the protein was derivatized with GRP and trypsin digested. Positively charged peptides were then selected from the digest with SCX chromatography at pH 6.0. Seven GRP-derivatized peptides were found to be selected from transferrin by MALDI-TOF-TOF analysis. Fourteen underivatized native peptides were also captured by the SCX column at pH 6.0. Mapping of the derivatized peptides onto the primary structure of transferrin indicated that the oxidation sites were all on solvent-accessible regions at the protein surface. Efficiency of the method was further demonstrated in the identification of oxidized proteins from yeast.     

间隔基长度对基于ATRP的偶氮苯三嵌段共聚物液晶性影响

研究了间隔基长度对基于ATRP的ABA型嵌段共聚物液晶性影响。利用含不同长度间隔基（亚甲基数目分别为0，2，6）的，n-[4-4-乙氧基苯基偶氮）酚氧基]烷基甲基丙烯酸甲酯为单体，通过原子转移自由基聚合合成系列含偶氮苯的两亲性三嵌段共聚物PAnC-PEG-PAnC。示差扫描量热法（Dsc）测试和偏光显微镜（POM）观察表明：随着n增加，共聚物的玻璃化转变温度下降，PAOC-PEG-PAOC无液晶性，而PA2C-PEG-PA2C与PA6C-PEG-PA6C则为向列型液晶。其不同源于聚合物主链与侧链之间的间隔基，说明此类共聚物的液晶性强烈依赖于间隔基的长度。     

pH对阳离子交换膜分离去除Cu~(2+)效果的影响

基于Donnan dialysis原理,在无外加电压作用下采用阳离子交换膜分离去除原水中的Cu2+,研究pH对阳离子交换膜分离去除Cu2+效果的影响.研究结果表明:原水及补偿离子溶液pH≥4时,H+浓度较低,其对阳离子交换膜分离去除Cu2+无明显影响,去除率均在85%左右;原水pH=3时,阳离子交换膜分离去除Cu2+的能力降低,去除率为60%～62%左右;原水pH=6,补偿离子溶液pH=3时,H+与补偿离子K+具有累加作用,但累加作用不明显,Cu2+去除率只有少量增加.     

Effect of a quaternary surfactant upon the kinetics of H+-Mg2+ exchange on sulfonic acid cation exchangers

The dynamic and static effect of a quaternay surfactant on the rate of H⁺-Mg²⁺ exchange on sulfonic acid cation exchange resins of various porosity (gelular and macroporous) in the ranges of film diffusion control and particle diffusion control has been investigated.     

Hydrous oxides of titanium: cation exchange properties and kinetics of exchange

The ion exchange properties of hydrous titania gels of different particle sizes, precipitated from titanous chloride through the agency of ammonium carbonate and hydroxide have been studied. Such studies were carried out under acidic and alkaline conditions with respect to Cu²⁺, Ni²⁺, Co²⁺ and Cr³⁺ ions.In the case of gels precipitated by ammonium carbonate, oxygen gas was used as the oxidizing agent whereas with ammonium hydroxide as precipitant, oxidation was performed with hydrogen peroxide.Ion exchange capacities were determined by visible spectrophotometry. Increasing the pH of preparation lead to an increase in exchange capacities of the hydroxide precipitated gels that are characterized to be mesoporous. Such an increase is not observed in the case of carbonate precipitated microporous gels. It is shown that in the latter case the NH ₄ ⁺ ions generated by the initial interaction of (NH₄)₂CO₃ with the acidic titanous chloride lead to the formation of titania exchangers that are predominantly in the ammonium form. The textural characteristics of the exchanger resulting from different conditions of preparation is a significant contributing parameter to the resulting data.Ageing of the microporous titania samples markedly reduces the exchanger capacity of the smaller Ni²⁺ ions but increases that of the bulkier Cr³⁺ as a result of the presence of some wide pores that appear upon agglomeration. The presence of Cr³⁺ ions in the hydroxo form in solution seems to inhibit its exchange with the appropriate surface species.Studies on the kinetics of exchange with respect to the Ni²⁺ ions seem to indicate that a particle diffusion mechanism is partly or completely responsible for the rate of exchange.     

Ultra-high-efficiency strong cation exchange LC/RPLC/MS/MS for high dynamic range characterization of the human plasma proteome

High-efficiency nanoscale reversed-phase liquid chromatography (chromatographic peak capacities of approximately 1000: Shen, Y.; Zhao, R.; Berger, S. J.; Anderson, G. A.; Rodriguez, N.; Smith, R. D. Anal. Chem. 2002, 74, 4235. Shen, Y.; Moore, R. J.; Zhao, R.; Blonder, J.; Auberry, D. L.; Masselon, C.; Pasa-Tolic, L.; Hixson, K. K.; Auberry, K. J.; Smith, R. D. Anal. Chem. 2003, 75, 3596.) and strong cation exchange LC was used to obtain ultra-high-efficiency separations (combined chromatographic peak capacities of >10(4)) in conjunction with tandem mass spectrometry (MS/MS) for characterization of the human plasma proteome. Using conservative SEQUEST peptide identification criteria (i.e., without considering chymotryptic or elastic peptides) and peptide LC normalized elution time constraints, the separation quality enabled the identification of proteins over a dynamic range of greater than 8 orders of magnitude in relative abundance using ion trap MS/MS instrumentation. Between 800 and 1682 human proteins were identified, depending on the criteria used for identification, from a total of 365 microg of human plasma. The analyses identified relatively low-level (approximately pg/mL) proteins (e.g., cytokines) coexisting with high-abundance proteins (e.g., mg/mL-level serum albumin).     

Enrichment and identification of cysteine-containing peptides from tryptic digests of performic oxidized proteins by strong cation exchange LC and MALDI-TOF/TOF MS

The extreme complexity of sample and uninformative fragmentation of peptides in MS/MS experiments are two of several real challenges faced by proteomics. In this work, a strategy aimed at tackling these two problems is presented. Briefly, proteins were first oxidized by performic acid to cleave the disulfide bonds and simultaneously convert cysteine residue into its sulfonic form. Then the resultant sulfonic peptides were enriched by SCX chromatography, exploiting the negative solution charge of sulfonic group. The sulfonic peptide could be easily detected by MALDI-MS in negative mode and showed both enhanced fragmentation efficiency and a simplified spectrum in MALDI-MS/MS experiment in positive mode. The strength of the strategy was demonstrated by applying it to bovine serum albumin. Potential use of the strategy in proteomics was also discussed.     

Development of adjustable stiffness actuator by varying lever arm length

Abstract

For a robotic actuator to be able to work safely with humans and/or to adapt natural dynamics of locomotion, it should be able to change its stiffness. The research presented herein, in this context, discusses a new actuation concept of variable stiffness for robotic joints for the purposes of energy efficiency, safety, and enhanced speed. With the aid of two motors, the actuator developed by authors can control its angular position and link-stiffness, independently. A small motor regulates the output stiffness for intended application whereas the large motor can control the angular position of the link. Stiffness variation is accomplished using an energy-efficient way of changing the lever arm ratio using a ball screw mechanism, without inducing or removing the potential energy stored in the springs. Experiments successfully exhibit that the greater the lever arm ratio, the stiffer will be the link. Also, to observe the effect of changing the springs, a set of springs with different spring constants are tested and their results indicate a direct relation between spring and actuator stiffness.     

Integrated sample pretreatment system for N-linked glycosylation site profiling with combination of hydrophilic interaction chromatography and PNGase F immobilized enzymatic reactor via a strong cation exchange precolumn

An integrated sample pretreatment system, composed of a click maltose hydrophilic interaction chromatography (HILIC) column, a strong cation exchange (SCX) precolumn, and a PNGase F immobilized enzymatic reactor (IMER), was established for the simultaneous glycopeptide enrichment, sample buffer exchange, and online deglycosylation, by which the sample pretreatment for glycoproteome could be performed online automatically, beneficial to improve the efficiency and sensitivity of the N-linked glycosylation site identification. With such a system, the deglycosylated glycopeptide from the digests of avidin with the coexistence of 50 times (mass ratio) BSA could be selectively detected, and the detection limit as low as 5 fmol was achieved. Moreover, the sample pretreatment time was significantly shortened to ~1 h. Such a system was further successfully applied for analyzing the digest of the soluble fraction extracted from rat brain. A total of 120 unique glycoprotein groups and 196 N-linked glycosylation sites were identified by nanoreversed phase liquid chromatography-electrospray ionization-tandem mass spectrometry (nanoRPLC-ESI-MS/MS), with the injected digests amount as 6 μg. All these results demonstrate that the integrated system is of great promise for N-linked glycosylation site profiling and could be further online coupled with nanoHPLC-ESI-MS/MS to achieve high-throughput glycoproteome analysis.     

Quaternized trimethylaminated polystyrene-coated zirconia as a strong anion exchange material for HPLC

The synthesis and characterization of a new, base-stable, strong anion exchange phase by amination of polystyrene-coated zirconia (PS-ZrO2) are described. Even though the ion exchange capacity of the quaternized trimethylaminated PSZrO2 (QTMA-PS-ZrO2) is only 0.07 mequiv/g, it is able to separate various inorganic anions, benzoic acid derivatives, and nucleotides in their deprotonated states. The effects of ionic strength, eluent pH, and counterion type are discussed. In the presence of both phosphate and fluoride ions in the eluent, band broadening caused by Lewis acid/base interactions between zirconia and analytes is greatly suppressed. The mixed retention modes (ion exchange, hydrophobic interaction, and Lewis acid/base interactions) on QTMA-PS-ZrO2 offer a different selectivity toward various anionic analytes than do other zirconia- and nonzirconia-based ion exchangers.     

带间隔臂的荷负电超滤膜对水中天然有机物的去除

通过特定的化学反应对不同截留分子量的再生纤维素(RC)超滤膜进行改性,得到一系列间隔臂长度不同的荷负电超滤膜.选用腐殖酸(HA)作为天然有机物的代表物质,比较不同间隔臂长度和不同截留分子量的RC膜对腐殖酸去除效果和膜污染情况,分析膜阻力.实验结果表明,不同截留分子量的改性膜对腐殖酸的去除效果都比未改性膜好,同时膜通量的衰减变小,即膜污染现象减轻.间隔臂长度较大的膜对腐殖酸的去除更有效,同时膜污染更轻,这主要是由于间隔臂长度较大的改性荷负电膜上的Zeta电位较大,与带同种电荷的腐殖酸之间的静电排斥作用更大.     

Electrochemical catalytic treatment of wastewater by metal ion supported on cation exchange resin

The electrochemical oxidation of phenol in synthetic wastewater and paper mill wastewater catalyzed by metal ion supported on cation exchange resin in suspended bed electrolytic reactor with graphite electrode has been investigated. The catalyst was characterized by SEM and XPS spectra and the effects of pH, the different metal ion and NaCl on the efficiency of the electrochemical oxidation phenol process were also studied. It was found that the catalyst containing Fe(3+) had the highest electrochemical catalytic activity for the electrochemical oxidation of phenol. When the initial concentration of phenol was 200 ppm, up to 90% chemical oxygen demand (COD) removal was obtained in 10 min. When the catalyst containing Fe(3+) was used to the paper mill wastewater, it still showed high efficiency. The COD removal could get to 75% in 60 min.     

Characteristic length and enhancement time of a runaway electron avalanche in strong electric fields

The dependences of the length of exponential enhancement of the runaway electron avalanche on the electric field strength and air and helium pressure were calculated using the Monte Carlo method. The calculation results allow one to arrive at the conclusion that the length and time of the exponential enhancement of the runaway avalanche electrons decrease with decreasing pressure and atomic number of the discharge gas, provided that the electric field strengths are equal.     

Continuous pH/salt gradient and peptide score for strong cation exchange chromatography in 2D-nano-LC/MS/MS peptide identification for proteomics

Tryptic digests of human serum albumin and human lung epithelial cell lysates were used as test samples in a novel proteomics study. Peptides were separated and analyzed using 2D-nano-LC/MS/MS with strong cation exchange (SCX) and reversed-phase chromatography and continuous gradient elution. The peptide elution conditions combined simultaneous pH gradient with ammonium acetate salt gradient elution modes. A novel empirical SCX peptide elution score was developed, which accounts for both the number of basic and acidic residues and, in part, their location within a sequence of a peptide. Average scores calculated for the fractionated peptide sequences correlated well with the pH of SCX elution fractions. Multiple peptides with identical amino acid sequences, but differing in cysteine tags possessing different positive charge and different SCX elution properties, were obtained by subjecting the samples to reduction and alkylation with different cysteine alkylating reagents: iodoacetamide, 4-vinylpyridine, and (3-acrylamidopropyl) trimethylammonium chloride. The structurally similar peptides were used as elution standards.     

Improving depth in phosphoproteomics by using a strong cation exchange-weak anion exchange-reversed phase multidimensional separation approach

Several enrichment and separation strategies are available that allow nearly pure phosphopeptide pools to be created. These phosphopeptide pools are too complex to be completely unraveled by RP-LC-MS analysis alone. Here, we implement weak anion exchange (WAX) chromatography as an additional, complementary dimension to strong cation exchange (SCX) and reversed phase (RP). Initially, we used SCX to fractionate a human lysate digest to generate a fraction highly enriched for phosphopeptides. Analysis of this single fraction by RP-LC-MS with a 140 min gradient method allowed the identification of 4045 unique phosphopeptides (false discovery rate (FDR) < 1%; Mascot score > 20) using an Orbitrap Velos. Triplicate analysis (420 min total gradient time) of the same sample increased the total to just over 5000 unique phosphopeptides. When we separated the same sample by WAX and analyzed 14 WAX fractions by 30 min gradient RP-LC-MS (420 min total gradient time) we were able to identify 7251 unique phosphopeptides, an approximate increase of 40%, while maintaining the same total gradient time. We performed a more comprehensive, albeit also more time-consuming, analysis of the same 14 WAX fractions by the use of 140 min gradient LC-MS analyses, which resulted in the detection of over 11?000 unique phosphopeptides. Our results clearly demonstrate that additional separation dimensions are still necessary for in-depth phosphoproteomics and that WAX is a suitable dimension to be combined and sandwiched between SCX and RP chromatography.