Loss Of Phosphoric Acid Research Articles

Sequencing of Phosphopeptides Using a Sequential Charge Inversion Ion/Ion Reaction and Electron Capture Dissociation Workflow.

Protein phosphorylation, a common post-translational modification (PTM), is fundamental in a plethora of biological processes, most importantly in modulating cell signaling pathways. Matrix-assisted laser desorption/ionization (MALDI) coupled to tandem mass spectrometry (MS/MS) is an attractive method for phosphopeptide characterization due to its high speed, low limit of detection, and surface sampling capabilities. However, MALDI analysis of phosphopeptides is constrained by relatively low abundances in biological samples and poor relative ionization efficiencies in positive ion mode. Additionally, MALDI tends to produce singly charged ions, generally limiting the accessible MS/MS techniques that can be used for peptide sequencing. For example, collision induced dissociation (CID) is readily amendable to the analysis of singly charged ions, but results in facile loss of phosphoric acid, precluding the localization of the PTM. Electron-based dissociation methods (e.g., electron capture dissociation, ECD) are well suited for PTM localization, but require multiply charged peptide cations to avoid neutralization during ECD. Conversely, phosphopeptides are readily ionized using MALDI in negative ion mode. If the precursor ions are first formed in negative ion mode, a gas-phase charge inversion ion/ion reaction could then be used to transform the phosphopeptide anions produced via MALDI into multiply charged cations that are well-suited for ECD. Herein we demonstrate a multistep workflow combining a charge inversion ion/ion reaction that first transforms MALDI-generated phosphopeptide monoanions into multiply charged cations, and then subjects these multiply charged phosphopeptide cations to ECD for sequence determination and phosphate bond localization.

Phosphate Migration versus the Loss of Phosphoric Acid in Protonated Phosphopeptides: A Computational Study.

Residue-specific phosphorylation is a protein post-translational modification that regulates cellular functions. Experimental determination of the exact sites of protein phosphorylation provides an understanding of the signaling and processes at work for a given cellular state. Any experimental artifact that involves migration of the phosphate group during measurement is a concern, as the outcome can lead to erroneous conclusions that may confound studies on cellular signal transduction. Herein, we examine computationally the mechanism by which a phosphate group migrates from one serine residue to another serine in monoprotonated pentapeptides [BA-pSer-Gly-Ser-BB + H]+ → [BA-Ser-Gly-pSer-BB + H]+ (where BA and BB are different combinations of the three basic amino acids, histidine, lysine, and arginine). In addition to moving the phosphate group, the overall mechanism involves transferring a proton from the N-terminal amino acid, BA, to the C-terminal amino acid, BB. This is not a synchronous process, and there is a key high-energy intermediate, structure C, that is zwitterionic with both the basic amino acids protonated and the phosphate group attached to both serine residues and carrying a negative charge. The barriers to moving the phosphate group are calculated to be in the range of 219-274 kJ mol-1 at the B3LYP/6-31G(d) level. These barriers are systematically slightly lower and in good agreement with single-point energy calculations at both M06-2X/6-311++G(d,p) and MP2/6-31++G(d,p) levels. The competitive reaction, loss of phosphoric acid from the protonated pentapeptides, has a barrier in the range of 176-202 kJ mol-1 at the B3LYP/6-31G(d) level. Extension of the theory to M06-2X/6-311++G(d,p)//B3LYP/6-31G(d) and MP2/6-31++G(d,p)// B3LYP/6-31G(d) gives higher values for the loss of phosphoric acid, falling in the range of 196-226 kJ mol-1; these are comparable to the barriers against phosphate migration at the same levels of theory. For larger peptides His-pSer-(Gly)n-Ser-His, where n has values from 2 to 5, the barriers against the loss of phosphoric acid are higher than those against the phosphate group migration. This difference is most pronounced and significant when n = 4 and 5 (the differences are approximately 80 kJ mol-1 under the single-point energy calculations at the M06-2X and MP2 levels). Energy differences using two more recent functionals, M08-HX and MN15, on His-pSer-(Gly)n-Ser-His, where n = 1 and 5, are in good agreement with the M06-2X and MP2 calculations. These results provide the mechanistic rationale for phosphate migration versus other competing reactions in the gas phase under tandem mass spectrometry conditions.

Optimization on Composition and Structure of Catalyst Layer for High-Temperature Polymer Electrolyte Membrane Fuel Cells

Abstract High-temperature polymer membrane fuel cells (HT-PEMFCs) are considered the trend of PEMFC future development due to their accelerated electrochemical reaction kinetics, simplified water/thermal management, and improved tolerance to impurities (CO). As the core part of the membrane electrode assembly in HT-PEMFCs, the catalyst layer significantly affects the cost, performance, and lifetime of HT-PEMFCs. However, platinum (Pt) catalyst degradation and carbon corrosion are apparently accelerated because of the high-temperature and acid environment in HT-PEMFC. Moreover, the loss of phosphoric acid (PA) that serves as the proton conductor is observed after long-term operation. In addition, the adsorption of phosphate on the Pt surface leads to poor Pt utilization. Thus, high cost and fast performance decay must be addressed to achieve better commercialization of HT-PEMFC. Optimizing the composition and structure of the catalyst layer is demonstrated as an effective strategy to resolve these problems. In this review, we first summarize the latest progress in the optimization of the catalyst layer composition for HT-PEMFC, including catalysts, binders, electrolytes (PAs), and additives. Thereafter, the structural characteristics of the catalyst layer are introduced, and the optimization strategies are reviewed. Finally, the current challenges and research perspectives of the catalyst layer in HT-PEMFC are discussed.

Numerical Study on Thermal Stress of High Temperature Proton Exchange Membrane Fuel Cells during Start-Up Process

High-temperature proton-exchange membrane fuel cells (HT-PEMFCs) with phosphoric-doped polybenzimidazole (PBI) membranes have a higher operating temperature compared to the PEMFCs operating below 373.15 K. The fuel cell is first heated from room temperature to the minimum operating temperature to avoid the generation of liquid water. The existence of liquid water can result in the loss of phosphoric acid and then affect the cell performance. In this study, the start-up process of HT-PEMFCs is numerically studied by establishing a three-dimensional non-isothermal mathematical model. Preheated gas is supplied into gas flow channels to heat the fuel cell, and then voltage load is applied to accelerate the start-up process. Effects of voltage (0.9 V, 0.7 V and 0.5 V) and flow arrangement (co-flow and counter flow) on temperature, current density, proton conductivity and stress distributions of fuel cells are examined. It is found that the maximum stress is increased when a lower voltage is adopted, and the counter-flow arrangement provides a more uniform stress distribution than that of co-flow arrangement.

A Structured Hybrid Catalyst to Improve ORR Activity and Phosphoric Acid in the High-Temperature Exchange Membrane Fuel Cell

The high-temperature proton exchange membrane fuel cell (HT-PEMFC) based on a phosphoric acid (PA)-doped polybenzimidazole (PBI) membrane electrolyte has been attracted attention because of its higher kinetics of oxygen reduction reaction, no flooding issues and excellent carbon monoxide tolerance [1, 2]. However, PA leaching from PBI membrane to electrode is one of the biggest problems as the loss of phosphoric acid causing worse proton conductivity of PBI membrane and preventing gas transfer in the electrode [3]. Furthermore, HT-PEMFC requires higher amount of platinum (Pt) (normally between 0.7 and 1.2 mg cm-2) to provide high electrochemical surface area, which accounts for a higher proportion in the cost of a total fuel cell stack [4]. Therefore, the reduction of phosphoric acid permeation and catalyst dosage are two challenges in HT-PEMFC.Many studies and technologies have been conducted to prevent the permeation of PA. Currently, the modification of PBI membrane structure is the main method by introducing materials with more -NH2 functional groups to form stable chemical bonds with phosphoric acid molecules and prevent their movement [5-7]. However, the composite membrane materials are not uniform and the intercalation of materials (SiO2, Graphene et al) will cause crossover issues to influence the durability of HT-PEMFC [7].Based on our previous research, platinum supported on nitrogen doped reduced electrochemically exfoliated graphene oxide/carbon black hybrid catalysts (Pt/NrEGOx-CBy) have higher oxygen reduction reaction (ORR) activity. This work aims to optimize the ratio of NrEGO and CB in HT-PEMFC and explore the effect of NrEGO flakes on PA permeation to improve the durability of HT-PEMFC. The preliminary results showed that Pt/NrEGO2-CB3 catalyst in a membrane electrode assembly (MEA) had the mass power density of 1.652 W mgPt -1 with a decay rate of 0.01 mV h-1 within 100 hours, which is much lower than that of 1.125 mV h-1 in the commercial Pt/C as shown in Figure 1.Reference[1] J. Lobato, H. Zamora, J. Plaza, P. Cañizares, M.A. Rodrigo. Enhancement of high temperature PEMFC stability using catalysts based on Pt supported on SiC based materials. Applied Catalysis B: Environmental. 2016. 198: 516-524.[2] Y. Hu, Y. Jiang, J.O. Jensen, L.N. Cleemann, Q. Li. Catalyst Evaluation for Oxygen Reduction Reaction in Concentrated Phosphoric Acid at Elevated Temperatures. Journal of Power Sources. 2018. 375: 77-78.[3] G. Liu, H. Zhang, J. Hu, Y. Zhai, D. Xu, Z.-g. Shao. Studies of performance degradation of a high temperature PEMFC based on H3PO4-doped PBI. Journal of Power Sources. 2006. 162 (1): 547-552.[4] H. Su, T.-C. Jao, O. Barron, B.G. Pollet, S. Pasupathi. Low platinum loading for high temperature proton exchange membrane fuel cell developed by ultrasonic spray coating technique. Journal of Power Sources. 2014. 267: 155-159.[5] F. Liu, S. Wang, J. Li, X. Tian, X. Wang, H. Chen, Z. Wang. Polybenzimidazole/ionic-liquid-functional silica composite membranes with improved proton conductivity for high temperature proton exchange membrane fuel cells. Journal of Membrane Science. 2017. 541: 492-499.[6] K.-J. Peng, J.-Y. Lai, Y.-L. Liu. Nanohybrids of graphene oxide chemically-bonded with Nafion: Preparation and application for proton exchange membrane fuel cells, Journal of Membrane Science. 2016. 514: 86-94.[7] C.-L. Lu, C.-P. Chang, Y.-H. Guo, T.-K. Yeh, Y.-C. Su, P.-C. Wang, K.-L. Hsueh, F.-G. Tseng. High-performance and low-leakage phosphoric acid fuel cell with synergic composite membrane stacking of micro glass microfiber and nano PTFE. Renewable Energy. 2019. 134: 982-988. Figure 1

Proteomics with Enhanced In-Source Fragmentation/Annotation: Applying XCMS-EISA Informatics and Q-MRM High-Sensitivity Quantification.

Enhanced in-source fragmentation/annotation (EISA) has recently been shown to produce fragment ions that match tandem mass spectrometry data across a wide range of small molecules. EISA has been developed to facilitate data-dependent acquisition (DDA), data-independent acquisiton (DIA), and multiple-reaction monitoring (MRM), enabling molecular identifications in untargeted metabolomics and targeted quantitative single-quadrupole MRM (Q-MRM) analyses. Here, EISA has been applied to peptide-based proteomic analysis using optimized in-source fragmentation to generate fragmentation patterns for a mixture of 38 peptides, which were comparable to the b- and y-type fragment ions typically observed in tandem MS experiments. The optimal in-source fragmentation conditions at which high-abundance peptide fragments and precursor ions coexist were compared with automated data-dependent acquisition (DDA) in the same quadrupole time-of-flight (QTOF-MS) mass spectrometer, generating a significantly higher fragment percentage of peptides from both singly and doubly charged b- and y-type fragment (b+, y+, b2+, and y2+) ions. Higher fragment percentages were also observed for these fragment ion series over linear ion trap instrumentation. An XCMS-EISA annotation/deconvolution program was developed, making use of the retention time and peak shape continuity between precursor fragment ions, to perform automated proteomic data analysis on the enhanced in-source fragments. Post-translational modification (PTM) characterization on peptides was demonstrated with EISA, producing fragment ions corresponding to a neutral loss of phosphoric acid with greater intensity than observed with DDA on a QTOF-MS. Moreover, Q-MRM demonstrated the ability to use EISA for peptide quantification. The availability of more sophisticated in-source fragmentation informatics, beyond XCMS-EISA, will further enable EISA for sensitive autonomous identification and Q-MRM quantitative analyses in proteomics.

Construction of Stable Wide-Temperature-Range Proton Exchange Membranes by Incorporating a Carbonized Metal-Organic Frame into Polybenzimidazoles and Polyacrylamide Hydrogels.

Proton exchange membrane fuel cells (PEMFCs) are promising devices for clean power generation in fuel cell electric vehicles applications. The further request of high-efficiency and cost competitive technology make high-temperature proton exchange membranes utilizing phosphoric acid-doped polybenzimidazole be favored because they can work well up to 180°C without extra humidifier. However, they face quick loss of phosphoric acid below 120°C and resulting in the limits of commercialization. Herein UiO-66 derived carbon (porous carbon-ZrO2 ), comprising branched poly(4,4'-diphenylether-5,5'-bibenzimidazole) and polyacrylamide hydrogels self-assembly (BHC1-4) membranes for wide-temperature-range operation (80-160°C) is presented. These two-phase membranes contained the hygroscopicity of polyacrylamide hydrogels improve the low-temperature proton conductivity, relatively enable the membrane to function at 80°C. An excellent cell performance of BHC2 membrane with high peak power density of 265 and 656mWcm-2 at both 80 and 160°C can be achieved. Furthermore, this membrane exhibits high stability of frequency cold start-ups (from room temperature to 80°C) and long-term cell test at 160°C. The improvement of cell performance and stability of BHC2 membrane indicate a progress of breaking operated temperature limit in existing PEMFCs systems.

Continuous durability study of a high temperature polymer electrolyte membrane fuel cell stack

High Temperature Polymer Electrolyte Membrane (HT-PEM) fuel cells are a promising choice for renewable power generation. We present an experimental study that investigates the durability of a full-size fuel cell stack, where membrane electrode assemblies were continuously extracted during operation of the stack, and analysed in detail with electron microscopy, energy dispersive X-ray spectroscopy and X-ray diffraction. The evolution of membrane thinning, the loss of phosphoric acid and the changes in the catalyst layer of the electrodes have been investigated as a function of operation time. A correlation between the acid loss and the decrease of cell voltage has been determined to provide detailed insight into durability data of the HT-PEM fuel cell on a commercial sized stack level.

Comparison of Carbon Based Bipolar Plate Materials for Polymer Electrolyte Membrane Fuel Cells

Polymer electrolyte membrane (PEM) fuel cells can be separated into two types, the high-temperature (HT) PEM and low-temperature (LT) PEM fuel cell. As main advantages operating at higher temperatures improved tolerances to impurities like carbon monoxide, easier heat rejection and simpler water management can be mentioned. In comparison to LT-PEM fuel cells where sulfuric acid is applied polybenzimidazole (PBI) membranes doped with phosphoric acid (PA) were used in HT-PEM fuel cells. Nevertheless, degradation and long term stability are still major challenges. Here, it is necessary to understand the degradation mechanisms regarding membrane and catalyst degradation, carbon corrosion as well as loss of phosphoric acid and degradation of bipolar plates (BPP). Titration investigations of carbon based bipolar plates after operation in PEM fuel cells showed that phosphoric acid can be found within the composite matrix [1, 2] which can lead to corrosion of the graphite material. In this work, a variety of graphite-polymer composite bipolar plate materials for PEM fuel cells were tested for their stability and compared with different methods. The electrochemical stability was investigated by means of corrosion measurements within a three-electrode cell setup. The bipolar plate material was applied as working electrode and a platinum wire with an activated felt was served as counter electrode. Potentials are shown with respect to the standard hydrogen electrode (SHE). Two different methods of aging were used: chemical storage and potentiostatic treatment. For determination of stability and corrosion rates OCP, electrochemical impedance spectroscopy (EIS), cyclic voltammetry (CV) and linear sweep voltammetry (LSV) were evaluated. Additionally, the bipolar plate materials were compared by electrical conductivity measurements (in-plane and through-plane), contact angle measurements as well as structural changes of the surface area. Here different imaging methods like scanning electron microscopy (SEM), micro-computed tomography (µ-CT), atomic force microscopy (AFM) and confocal microscopy were applied. First results of two graphite composite bipolar plate materials with different types and amount of polymer binder show different electrochemical behavior after chemical aging for 3 and 7 days in 0.5 M H2SO4. The bipolar plate material with higher polymer content (BPP-01) reveals lower double layer capacitance and a lower amount of oxygen functional groups in the potential range between 0.5-0.7 V vs. SHE (Figure 1) in comparison to the bipolar plate with higher polymer content (BPP-02). The reason for this behavior could be the lower amount of electrochemical active graphite at the BPP/electrolyte interface from BPP-01 in comparison to BPP-02. In addition, conductivity measurements show that BPP01 has a higher sheet resistance (53.6 +/- 5.6 mOhm) compared to BPP-02 (46.2 +- 4.5 mOhm) in good agreement to the content of polymer and graphite in the composite materials. The various methods can be used to identify the difference of graphite compound materials and correlate it to degradation mechanism. Further results will be given. [1] C. Hartnig and T. J. Schmidt, Electrochim. Acta, 56 (2011), 4237-4242.[2] N. Pilinski, M. Rastedt and P. Wagner, ECS Trans., 69 (2015), 323-335. Figure 1

A New Class of Fuel Cells Based on Ion Pair-Coordinated Proton Exchange Membranes

Low temperature proton exchange membrane (PEM) fuel cells that use perfluorinated sulfonic acid are at present being commercialized in fuel cell vehicles, but these cells can operate only at relatively low temperatures, ca. < 100°C, and high hydration levels. High temperature PEM fuel cells that use phosphoric acid-doped polybenzimidazole can operate effectively up to 180°C; however, these are difficult to operate below 140°C without suffering loss of phosphoric acid when exposed to water.1 In this presentation, we discuss a new class of fuel cells based on ion pair-coordinated proton exchange membranes.2 In the ion pair-coordinated proton exchange membranes, quaternary ammonium functionalized membranes, also known as anion exchange membranes, are doped with phosphoric acid. Due to the high basicity of the quaternary ammonium hydroxide, the acidic proton of phosphoric acid is completely deprotonated to form a strong ionic interaction between quaternary ammonium and biphosphate. Our density functional theory calculation indicated that the ion-pair interaction is 8.5 times greater than the conventional acid base interaction in the phosphoric acid doped polybenzimidazole. The strong interaction between the ion pair enables fuel cell operation at lower temperatures with substantially higher water partial pressure than are possible for high-temperature phosphoric acid doped polybenimidazole fuel cells. In this presentation, the chemical structural difference between this new system and the conventional phosphoric acid-doped polybenzimidazole system will be compared. The benefits and challenges of the new system in fuel cell operations will be further discussed. Acknowledgment This work was supported by the US Department of energy, Energy Efficiency and Renewable Energy, Fuel Cell Technology Office (Program Manager: Dimitrios Papageorgopoulos). References G. Bendlamudi Systematic Characterization of HT PEMFCs Containing PBI/H3PO4 Systems: Thermodynamic analysis and Experimental Investigations 104 (Logos Verlag Berlin GmbH, 2011).Kwan-Soo Lee, Jacob S. Spendelow, Yoong-Kee Choe, Cy Fujimoto, and Yu Seung Kim Nature Energy, 1, 16120 (2016).

Structural and catalytic alteration of sarcosine oxidase through reconstruction with coenzyme-like ligands

A sarcosine oxidase (SOX) gene from Bacillus sp. (AY626822.2) was expressed in Escherichia coli BL21 (DE3) in the form of inclusion bodies. A 3D model of SOX was then built and refined, and molecular docking was used to investigate the interactions between SOX and natural or coenzyme-like ligands, including flavin adenine dinucleotide (FAD); flavin mononucleotide (FMN); riboflavin; isoalloxazine; 7-methyl-8-chloro-10-(1′-d-ribityl) isoalloxazine (7-M-8-C); 7-bromo-8-methyl-10-(1′-d-ribityl) isoalloxazine (7-B-8-M); 7-methyl-8-bromo-10-(1′-d-ribityl) isoalloxazine (7-M-8-B); 7-chloro-8-ethyl-10-(1′-d-ribityl) isoalloxazine (7-C-8-E); 7,8-diethyl-10-(1′-d-ribityl) isoalloxazine (7,8-D); and 3-methyl-7,8-dimethyl-10-(1′-d-ribityl) isoalloxazine (3-M-7,8-D). Unfolded SOX was extracted from inclusion bodies, and reconstructed with these ligands via a refolding process. The reconstructed enzymes were then subjected to structural and catalytic analysis. After structural simulation, refinement, and molecular docking, all ligands were able to recognize the coenzyme site of SOX. In addition, when the position 7- or 8-site of the compounds was modified, new pi-cation/sigma interactions were formed in the SOX-ligand complex. Fluorescent detection revealed that all the ligands could be successfully reconstructed with unfolded SOX. Circular dichroism (CD) spectra and nano differential scanning calorimetry (DSC) analysis indicated that the loss of phosphoric acid and adeninein natural coenzymes could significantly reduce the α-helix content, transition temperature (Tm), and calorimetric enthalpy (ΔH). In addition, although reconstruction with the position 7- or 8-site modified compounds led to variations in secondary structure, no significant shifts in Tm and ΔH were observed. Furthermore, in the evaluation of catalytic kinetic parameters, when SOX was reconstructed with ligands containing halogen atoms at the 7- or 8-sites, much higher relative specificities in the presence of organic solvents were noted.

Application of a Mass Spectrometric Approach to Detect the Presence of Fatty Acid Biosynthetic Phosphopeptides.

The details of plant lipid metabolism are relatively well known but the regulation of fatty acid production at the protein level is still not understood. Hence this study explores the importance of phosphorylation as a mechanism to control the activity of fatty acid biosynthetic enzymes using low and high oleic acid mesocarps of oil palm fruit (Elaeis guineensis variety of Tenera). Adaptation of neutral loss-triggered tandem mass spectrometry and selected reaction monitoring to detect the neutral loss of phosphoric acid successfully found several phosphoamino acid-containing peptides. These peptides corresponded to the peptides from acetyl-CoA carboxylase and 3-enoyl-acyl carrier protein reductase as identified by their precursor ion masses. These findings suggest that these enzymes were phosphorylated at 20th week after anthesis. Phosphorylation could have reduce their activities towards the end of fatty acid biosynthesis at ripening stage. Implication of phosphorylation in the regulation of fatty acid biosynthesis at protein level has never been reported.

Impact of Accelerated Stress Tests on High Temperature PEMFC Degradation

AbstractAccelerated stress tests (ASTs) have been used to provoke and investigate degradation mechanisms inside commercial high temperature proton exchange membrane electrode assemblies based on phosphoric acid doped polybenzimidazole (PBI). A novel load cycling test using high current densities of 0.6 and 1.0 A cm−2 has been developed and compared with a test utilizing lower current densities between 0.0 and 0.3 A cm−2. A high comparability is ensured by testing membrane electrode assemblies (MEAs) from the same supplier and batch under identical test conditions. Next to daily electrochemical characterization (polarization curves, electrochemical impedance spectroscopy, linear sweep and cyclic voltammetry), ex situ investigations including product water analysis by inductively coupled plasma mass spectrometry (ICP‐MS) and micro‐computed tomography (µ‐CT) as a powerful 3D imaging tool have been carried out. Clear demarcations of AST consequences for cell ageing have been identified. Especially, differences in platinum catalyst degradation and in loss of phosphoric acid as electrolyte over AST time have been revealed. Furthermore, influences of electrochemical characterization on MEA performance have been shown, which should be considered in future durability tests of fuel cells.

Complementary IMAC enrichment methods for HLA-associated phosphopeptide identification by mass spectrometry.

Phosphorylation events within cancer cells often become dysregulated, leading to oncogenic signaling and abnormal cell growth. Phosphopeptides derived from aberrantly phosphorylated proteins that are presented on tumors and not on normal tissues by human leukocyte antigen (HLA) class I molecules are promising candidates for future cancer immunotherapies, because they are tumor specific and have been shown to elicit cytotoxic T cell responses. Robust phosphopeptide enrichments that are suitable for low input amounts must be developed to characterize HLA-associated phosphopeptides from clinical samples that are limited by material availability. We present two complementary mass spectrometry-compatible, iron(III)-immobilized metal affinity chromatography (IMAC) methods that use either nitrilotriacetic acid (NTA) or iminodiacetic acid (IDA) in-house-fabricated columns. We developed these protocols to enrich for subfemtomole-level phosphopeptides from cell line and human tissue samples containing picograms of starting material, which is an order of magnitude less material than what is commonly used. In addition, we added a peptide esterification step to increase phosphopeptide specificity from these low-input samples. To date, hundreds of phosphopeptides displayed on melanoma, ovarian cancer, leukemia and colorectal cancer have been identified using these highly selective phosphopeptide enrichment protocols in combination with a program called 'CAD Neutral Loss Finder' that identifies all spectra containing the characteristic neutral loss of phosphoric acid from phosphorylated serine and threonine residues. This methodology enables the identification of HLA-associated phosphopeptides presented by human tissue samples containing as little as nanograms of peptide material in 2 d.

Study of the gas-phase fragmentation behaviour of sulfonated peptides

A series of singly and doubly protonated peptides bearing sulfonated residue have been studied, using both experiment and molecular modelling, to elucidate fragmentation chemistry of sulfonated peptides. Collision-induced dissociation mass spectra indicate that the sulfo group loss (neutral loss of 80Da) is the dominant dissociation channel. Modelling results suggest the proton transfer mechanism, where upon vibrational excitation, the acidic side chain proton is transferred from the sulfo group hydroxyl to the ester oxygen resulting in SO bond cleavage and formation of the unmodified hydroxyl containing residue and SO3. Conformations associated with potential energy profile of the reaction imply the charge remote nature of the proposed mechanism. The proposed proton transfer mechanism was compared with the intramolecular nucleophilic substitution (SN2) mechanism, the main pathway suggested for neutral loss of phosphoric acid from phosphopeptides. Both pathways (proton transfer and SN2) are available for sulfonated and phosphorylated peptides; however, each posttranslational modification favours different mechanism. The change of the bond dissociation enthalpies and the ability of stabilising the transition state structures are demonstrated as main factors responsible for each posttranslational modification activating a different pathway.

Timeframe Dependent Fragment Ions Observed in In-Source Decay Experiments with β-Casein Using MALDI MS.

The fragment ions observed with time-of-flight (TOF) and quadrupole ion trap (QIT) TOF mass spectrometers (MS) combined with matrix-assisted laser desorption/ionization in-source decay (MALDI-ISD) experiments of phosphorylated analytes β-casein and its model peptide were compared from the standpoint of the residence timeframe of analyte and fragment ions in the MALDI ion source and QIT cell. The QIT-TOF MS gave fragment c-, z'-, z-ANL, y-, and b-ions, and further degraded fragments originating from the loss of neutrals such as H(2)O, NH(3), CH(2)O (from serine), C2H4O (from threonine), and H(3)PO(4), whereas the TOF MS merely showed MALDI source-generated fragment c-, z'-, z-ANL, y-, and w-ions. The fragment ions observed in the QIT-TOF MS could be explained by the injection of the source-generated ions into the QIT cell or a cooperative effect of a little internal energy deposition, a long residence timeframe (140 ms) in the QIT cell, and specific amino acid effects on low-energy CID, whereas the source-generated fragments (c-, z'-, z-ANL, y-, and w-ions) could be a result of prompt radical-initiated fragmentation of hydrogen-abundant radical ions [M + H + H](+) and [M + H - H](-) within the 53 ns timeframe, which corresponds to the delayed extraction time. The further degraded fragment b/y-ions produced in the QIT cell were confirmed by positive- and negative-ion low-energy CID experiments performed on the source-generated ions (c-, z'-, and y-ions). The loss of phosphoric acid (98 u) from analyte and fragment ions can be explained by a slow ergodic fragmentation independent of positive and negative charges.

Formation of Membrane Electrode Assembly for High Temperature Methanol Fuel Cells

In this work, a quaternized polysulfone/PTFE/H3PO4composite membrane was prepared and used to a high temperature sustainable proton exchange membrane (HTPEM). This HTPEM was prepared based on a porous PTFE membrane, which can sustainable for 200 °C. Pt/C nano-suspension was prepared and deposited layer-by-layer on the gas diffusion layer (GDL) using electrohydrodynamic atomization (EHDA) deposition technique for the formation of cathode and anode catalyst layers (CLs). The CLs presented well packed and porous features. This EHDA deposited cathode and anode CLs, GDL and HTPEM were assembled to a membrane electrode assembly (MEA) and high temperature methanol fuel cell (HTMFC). The results showed that low concentration and high flow rate of methanol aqueous solution led to the loss of phosphoric acid on HTPEM, which resulted in the decline of the HTPEM. When the concentration and the flow rate of the methanol aqueous solution was increased and reduced, respectively, the cell can work properly at a temperature of 170 °C.

Accelerated Degradation of High-Temperature Polymer Electrolyte Fuel Cells: Discussion and Empirical Modeling

Accelerated stress tests are commonly applied in order to obtain a prediction of fuel cell life time within a short testing period. The stress test in this work considers a fuel cell which is constantly under load with frequent periods of very high current. Four different cells were operated each with a specific load profile. As a result severe performance degradation was observed in the region of high current densities. A short overview over the phenomena which may contribute to this specific fuel cell degradation is compiled from literature. Based on this overview major degradation modes are identified and combined with a simple polarization curve model. The modeling results allow for two different interpretations. Carbon corrosion reactions can explain the observed effects if cell voltage is assumed as the driving force for degradation. A better fit was obtained by using the overall heat flux as degradation criterion. In this case an increase in local temperature could lead to redistribution and loss of phosphoric acid from the MEA. The results are supported by the fact that the model yields consistent time dependend results for four different and non-periodic load cycles.

Influence of thermal post-curing on the degradation of a cross-linked polybenzimidazole-based membrane for high temperature polymer electrolyte membrane fuel cells

The lifetime stability of membranes is one of the main requirements regarding reliability of high temperature polymer electrolyte membrane fuel cells. The present work has improved durability under cycled operation by thermal post-curing of cross-linked polybenzimidazole (PBI)-based membranes. The membranes were dried over 1, 2 and 3 h at 250 °C under air. Ex-situ experiments proved an increase in stability by post-curing. The liquid uptake and swelling in phosphoric acid increased with longer curing periods. The effect of thermal treatments on cycle stability, lifetime and begin-of-life performance of the membrane electrode assemblies (MEAs) was investigated. Longer post-curing periods of the membranes had no influence on the MEAs' begin-of-life performance and constant current behavior over 2300 h. However, the 3 h post-cured MEAs showed enhanced cycle stability. Post-mortem analysis was carried out to identify the occurring degradation mechanisms. While a significant loss of phosphoric acid and a reduction of electrochemical surface activity on the cathode were observed for both post-cured MEAs, the 3 h dried membrane sample had a significantly higher resistance against pinhole formation during the long term test. Altogether, this work presents thermal post-curing as a promising method for the reduction of degradation determining effects in fuel cell membranes.

Increased Diversity of the HLA-B40 Ligandome by the Presentation of Peptides Phosphorylated at Their Main Anchor Residue

Human leukocyte antigen (HLA) class I molecules bind peptides derived from the intracellular degradation of endogenous proteins and present them to cytotoxic T lymphocytes, allowing the immune system to detect transformed or virally infected cells. It is known that HLA class I-associated peptides may harbor posttranslational modifications. In particular, phosphorylated ligands have raised much interest as potential targets for cancer immunotherapy. By combining affinity purification with high-resolution mass spectrometry, we identified more than 2000 unique ligands bound to HLA-B40. Sequence analysis revealed two major anchor motifs: aspartic or glutamic acid at peptide position 2 (P2) and methionine, phenylalanine, or aliphatic residues at the C terminus. The use of immobilized metal ion and TiO2 affinity chromatography allowed the characterization of 85 phosphorylated ligands. We further confirmed every sequence belonging to this subset by comparing its experimental MS2 spectrum with that obtained upon fragmentation of the corresponding synthetic peptide. Remarkably, three phospholigands lacked a canonical anchor residue at P2, containing phosphoserine instead. Binding assays showed that these peptides bound to HLA-B40 with high affinity. Together, our data demonstrate that the peptidome of a given HLA allotype can be broadened by the presentation of peptides with posttranslational modifications at major anchor positions. We suggest that ligands with phosphorylated residues at P2 might be optimal targets for T-cell-based cancer immunotherapy.