Enhanced efficacy of glycoengineered rice cell-produced trastuzumab.

For several decades, a plant-based expression system has been proposed as an alternative platform for the production of biopharmaceuticals including therapeutic monoclonal antibodies (mAbs), but the immunogenicity concerns associated with plant-specific N-glycans attached in plant-based biopharmaceuticals has not been completely solved. To eliminate all plant-specific N-glycan structure, eight genes involved in plant-specific N-glycosylation were mutated in rice (Oryza sativa) using the CRISPR/Cas9 system. The glycoengineered cell lines, PhytoRice®, contained a predominant GnGn (G0) glycoform. The gene for codon-optimized trastuzumab (TMab) was then introduced into PhytoRice® through Agrobacterium co-cultivation. Selected cell lines were suspension cultured, and TMab secreted from cells was purified from the cultured media. The amino acid sequence of the TMab produced by PhytoRice® (P-TMab) was identical to that of TMab. The inhibitory effect of P-TMab on the proliferation of the BT-474 cancer cell line was significantly enhanced at concentrations above 1 µg/mL (****P < 0.0001). P-TMab bound to a FcγRIIIa variant, FcγRIIIa-F158, more than 2.7 times more effectively than TMab. The ADCC efficacy of P-TMab against Jurkat cells was 2.6 times higher than that of TMab in an in vitro ADCC assay. Furthermore, P-TMab demonstrated efficient tumour uptake with less liver uptake compared to TMab in a xenograft assay using the BT-474 mouse model. These results suggest that the glycoengineered PhytoRice® could be an alternative platform for mAb production compared to current CHO cells, and P-TMab has a novel and enhanced efficacy compared to TMab.

Thermodynamic Reversal and Structural Correlation of 24-Crown-8/Protonated Tryptophan and 24-Crown 8/Protonated Serine Noncovalent Complexes in the Gas Phase vs in Solution: Quantum Chemical Analysis.

We investigate the structures of 24-crown-8/H+/l-tryptophan (CR/TrpH+) and 24-crown-8/H+/l-serine (CR/SerH+) noncovalent host-guest complex both in the gas phase and in an aqueous solution by quantum chemical methods. The Gibbs free energies of the complex in the two phases are calculated to determine the thermodynamically most favorable conformer in each phase. Our predictions indicate that both the carboxyl and the ammonium in CR/TrpH+ and the ammonium in the CR/SerH+ complexes in the lowest Gibbs free energy configurations form hydrogen bonds (H-bonds) with the CR host in the gas phase, while the conformer with the "naked" (devoid of H-bond with the CR host) -CO2H (and/or -OH) is much less favorable (Gibbs free energy higher by >3.6 kcal/mol). In the solution phase, however, a "thermodynamic reversal" occurs, making the higher Gibbs free energy gas-phase CR/TrpH+ and CR/SerH+ conformers thermodynamically more favorable under the influence of solvent molecules. Consequently, the global minimum Gibbs free energy structure in solution is structurally correlated with the thermodynamically much less gas-phase conformer. Discussions are provided concerning the possibility of elucidating host-guest-solvent interactions in solution from the gas-phase host-guest configurations in molecular detail.

Linker-Preserved Iron Metal-Organic Framework-Based Lateral Flow Assay for Sensitive Transglutaminase 2 Detection in Urine Through Machine Learning-Assisted Colorimetric Analysis.

A groundbreaking demonstration of the utilization of the metal-organic framework MIL-101(Fe) as an exceptionally perceptive visual label in colorimetric lateral flow assays (LFA) is described. This pioneering approach enables the precise identification of transglutaminase 2 (TGM2), a recognized biomarker for chronic kidney disease (CKD), in urine specimens, which offers a remarkably sensitive naked-eye detection mechanism. The surface of MIL-101(Fe) was modified with oxalyl chloride, adipoyl chloride, and poly(acrylic) acid (PAA); these not only improved the labeling material stability in a complex matrix but also achieved a systematic control in the detection limit of the TGM2 concentration using our LFA platform. The advanced LFA with the MIL-101(Fe)-PAA label can detect TGM2 concentrations down to 0.012, 0.009, and 0.010 nM in Tris-HCl buffer, urine, and desalted urine, respectively, which are approximately 55-fold lower than those for a conventional AuNP-based LFAs. Aside from rapid TGM2 detection (i.e., within 20 min), the performance of the MIL-101(Fe)-PAA-based LFA on reproducibility [coefficients of variation (CV) < 2.9%] and recovery (95.9-103.2%) along with storage stability within 25 days of observation (CV < 6.0%) shows an acceptable parameter range for quantitative analysis. A sophisticated sensing method grounded in machine learning principles was also developed, specifically aimed at precisely deducing the TGM2 concentration by analyzing immunoreaction sites. More importantly, our developed LFA offers potential for clinical measurement of TGM2 concentration in normal human urine and CKD patients' samples.

Unveiling host-guest-solvent interactions in solution by identifying highly unstable host-guest configurations in thermal non-equilibrium gas phase.

We propose a novel scheme of examining the host-guest-solvent interactions in solution from their gas phase structures. By adopting the permethylated ß-cyclodextrin (perm ß-CD)-protonated L-Lysine non-covalent complex as a prototypical system, we present the infrared multiple photon dissociation (IRMPD) spectrum of the gas phase complex produced by electrospray ionization technique. In order to elucidate the structure of perm ß-CD)/LysH+ complex in the gas phase, we carry out quantum chemical calculations to assign the two strong peaks at 3,340 and 3,560 cm-1 in the IRMPD spectrum, finding that the carboxyl forms loose hydrogen bonding with the perm ß-CD, whereas the ammonium group of L-Lysine is away from the perm ß-CD unit. By simulating the structures of perm ß-CD/H+/L-Lysine complex in solution using the supramolecule/continuum model, we find that the extremely unstable gas phase structure corresponds to the most stable conformer in solution.

TEMPO-Assisted Free-Radical-Initiated Peptide Sequencing Mass Spectrometry for Ubiquitin Ions: An Insight on the Gas-Phase Conformations.

TEMPO ((2,2,6,6-tetramethylpiperidine-1-yl)oxyl)-assisted free-radical-initiated peptide sequencing mass spectrometry (FRIPS MS) is applied to the top-down tandem mass spectrometry of guanidinated ubiquitin (UB(Gu)) ions, i.e., p-TEMPO-Bn-Sc-guanidinated ubiquitin (UBT(Gu)), to shed a light on gas-phase ubiquitin conformations. Thermal activation of UBT(Gu) ions produced protein backbone fragments of radical character, i.e., a-/x- and c-/z-type fragments. It is in contrast to the collision-induced dissociation (CID) results for UB(Gu), which dominantly showed the specific charge-remote CID fragments of b-/y-type at the C-terminal side of glutamic acid (E) and aspartic acid (D). The transfer of a radical "through space" was mainly observed for the +5 and +6 UBT(Gu) ions. This provides the information about folding/unfolding and structural proximity between the positions of the incipient benzyl radical site and fragmented sites. The analysis of FRIPS MS results for the +5 charge state ubiquitin ions shows that the +5 charge state ubiquitin ions bear a conformational resemblance to the native ubiquitin (X-ray crystallography structure), particularly in the central sequence region, whereas some deviations were observed in the unstable second structure region (ß2) close to the N-terminus. The ion mobility spectrometry results also corroborate the FRIPS MS results in terms of their conformations (or structures). The experimental results obtained in this study clearly demonstrate a potential of the TEMPO-assisted FRIPS MS as one of the methods for the elucidation of the overall gas-phase protein structures.

Characteristics of the Molecular Weight of Polyhexamethylene Guanidine (PHMG) Used as a Household Humidifier Disinfectant.

(1) Background: Household humidifier disinfectant (HD) brands containing polyhexamethylene guanidine (PHMG) have been found to cause the most HD-associated lung injuries (HDLIs) in the Republic of Korea. Nevertheless, no study has attempted to characterize the potential association of the health effects, including HDLI, with the physicochemical properties of PHMG dissolved in different HD brands. This study aimed to characterize the molecular weight (MW) distribution, the number-average molecular weight (Mn), the weight-average molecular weight (Mw), and the structural types of PHMG used in HD products. (2) Methods: Quantitative measurements were made using matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS). The Mn, Mw, and MW distributions were compared among various HD products. (3) Results: The mean Mn and Mw were 542.4 g/mol (range: 403.0-692.2 g/mol) and 560.7 g/mol (range: 424.0-714.70 g/mol), respectively. The degree of PHMG oligomerization ranged from 3 to 7. The MW distribution of PHMG indicated oligomeric compounds regardless of the HD brands. (4) Conclusions: Based on the molecular weight distribution, the average molecular weight of PHMG, and the degree of polymerization, the PHMG collected from HDLI victims could be regarded as an oligomer. PHMG, as used in household humidifiers, should not be exempted from toxic chemical registration as a polymer. Further study is necessary to examine the association of PHMG oligomeric compounds and respiratory health effects, including HDLI.

Characteristics of Exposure to Chloromethylisothiazolinone (CMIT) and Methylisothiazolinone (MIT) among Humidifier Disinfectant-Associated Lung Injury (HDLI) Patients in South Korea.

This study aimed to quantify both chloromethylisothiazolinone (CMIT) and methylisothiazolinone (MIT) dissolved in different product brands and to characterize the exposure to these chemicals among humidifier disinfectant-associated lung injury (HDLI) patients. Both CMIT and MIT dissolved in different humidifier disinfectant (HD) products were quantified using gas chromatography-mass spectrometry. The inhalation level of CMIT and MIT was estimated based on HD-associated factors as reported by HDLI patients. A total of eleven HD products marketed until the end of 2011 were found to contain CMIT and/or MIT. The level of combined CMIT and/or MIT dissolved in these HD products ranged from 12 to 353 ppm. The level varied among HD products and the year of manufacture. The average inhalation levels were estimated to be 7.5, 4.1, and 3.2 µg/m3 for the definite, probable, and possible groups, respectively. If probable and possible groups were collapsed together, the inhalation level of the collapsed group was significantly different from that of the definite group (p < 0.001). All HDLI patients responded as having used HD not only while sleeping, but also as having a humidifier treated with HD within close proximity every day in insufficiently ventilated spaces. These HD use characteristics of patients may be directly/indirectly linked to the HDLI development.

Noncovalent Complexes of Cyclodextrin with Small Organic Molecules: Applications and Insights into Host-Guest Interactions in the Gas Phase and Condensed Phase.

Cyclodextrins (CDs) have drawn a lot of attention from the scientific communities as a model system for host-guest chemistry and also due to its variety of applications in the pharmaceutical, cosmetic, food, textile, separation science, and essential oil industries. The formation of the inclusion complexes enables these applications in the condensed phases, which have been confirmed by nuclear magnetic resonance (NMR) spectroscopy, X-ray crystallography, and other methodologies. The advent of soft ionization techniques that can transfer the solution-phase noncovalent complexes to the gas phase has allowed for extensive examination of these complexes and provides valuable insight into the principles governing the formation of gaseous noncovalent complexes. As for the CDs' host-guest chemistry in the gas phase, there has been a controversial issue as to whether noncovalent complexes are inclusion conformers reflecting the solution-phase structure of the complex or not. In this review, the basic principles governing CD's host-guest complex formation will be described. Applications and structures of CDs in the condensed phases will also be presented. More importantly, the experimental and theoretical evidence supporting the two opposing views for the CD-guest structures in the gas phase will be intensively reviewed. These include data obtained via mass spectrometry, ion mobility measurements, infrared multiphoton dissociation (IRMPD) spectroscopy, and density functional theory (DFT) calculations.

Properties of Polyhexamethylene Guanidine (PHMG) Associated with Fatal Lung Injury in Korea.

The use of humidifier disinfectant (HD) has been determined to be associated with lung injuries (HDLI) in Korea. Although HD brands containing polyhexamethylene guanidine (PHMG) oligomers have been found to cause more HDLI compared to brands containing other disinfectants, the physicochemical properties of PHMG have been poorly defined. We aimed to quantify the PHMG dissolved in HD brands, characterize the number-average (Mn) and weight-average (Mw) molecular masses, and identify the polymerization degree of PHMG. Analysis of the PHMG oligomers was performed using a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (MALDI-TOF MS) operated in positive-ion reflectron mode. Eight brands of HD containing PHMG were identified. The PHMG concentrations in these brands ranged from 160 to 37,200 ppm (mean = 3100.9 ppm). Concentration was a significant variable among and within HD brands. The degree of PHMG oligomerization fell within the range of two to four. The averages of Mn and Mw were 517.2 g/mol (range: 422-613 g/mol) and 537.3 g/mol (range: 441.0-678.0 g/mol), respectively. Based on the average molecular weight and the degree of polymerization, the PHMG examined here could be regarded as oligomers, which may be associated with the highest proportion of HDLI being caused by PHMG.

Reliable screening and classification of phosphodiesterase type 5 inhibitors in dietary supplements using gas chromatography / mass spectrometry combined with specific common ions.

Illegal dietary supplements adulterated with phosphodiesterase type 5 inhibitors (PDE-5i) are increasingly widely distributed through internet markets and underground routes. For this reason, it demands development of reliable screening methods to determine a wide range of PDE-5i drugs in various types of dietary supplements. Herein, we developed a screening method using gas chromatography-mass spectrometry (GC-MS) for simultaneous detection of 53 PDE-5i drugs in supplements. Common formulations (such as capsule, powder, pill, and tablet) of supplements with complicated matrices were treated by simple liquid-liquid extraction and trimethylsilyl (TMS) derivatization. With the aid of TMS derivatization, 53 PDE-5i drugs could be successfully separated and detected within 15 min, using a short microbore GC column (15 m). Moreover, owing to enhanced detection sensitivity and selectivity of PDE-5i TMS derivatives, 0.5 mg of sample was sufficient to screen and confirm targeted PDE-5i drugs. In this study, specific common ions according to structural characteristics of PDE-5i drugs were found under the electron ionization (EI) of their TMS derivatives. These specific common fragments could reflect the common pharmacophores for 4 classes of PDE-5i drugs (sildenafil, other sildenafil, vardenafil, and tadalafil analogues). Based on characteristic EI fragment ions, extracted common ion chromatograms (ECICs) and discriminant analysis (DA) were effectively used for reliable screening and classification of various types of PDE-5i drugs. Specific ECICs and DA using characteristic EI fragments here will aid in identification of newly emerging PDE-5i counterfeits in supplements. This study will be helpful to supervise illegal adulteration of PDE-5i drugs in dietary supplements to protect public health and consumer safety.

Graph theory-based reaction pathway searches and DFT calculations for the mechanism studies of free radical-initiated peptide sequencing mass spectrometry (FRIPS MS): a model gas-phase reaction of GGR tri-peptide.

Graph theory-based reaction pathway searches (ACE-Reaction program) and density functional theory calculations were performed to shed light on the mechanisms for the production of [an + H]+, xn+, yn+, zn+, and [yn + 2H]+ fragments formed in free radical-initiated peptide sequencing (FRIPS) mass spectrometry measurements of a small model system of glycine-glycine-arginine (GGR). In particular, the graph theory-based searches, which are rarely applied to gas-phase reaction studies, allowed us to investigate reaction mechanisms in an exhaustive manner without resorting to chemical intuition. As expected, radical-driven reaction pathways were favorable over charge-driven reaction pathways in terms of kinetics and thermodynamics. Charge- and radical-driven pathways for the formation of [yn + 2H]+ fragments were carefully compared, and it was revealed that the [yn + 2H]+ fragments observed in our FRIPS MS spectra originated from the radical-driven pathway, which is in contrast to the general expectation. The acquired understanding of the FRIPS fragmentation mechanism is expected to aid in the interpretation of FRIPS MS spectra. It should be emphasized that graph theory-based searches are powerful and effective methods for studying reaction mechanisms, including gas-phase reactions in mass spectrometry.

Ionization of Gas-Phase Polycyclic Aromatic Hydrocarbons in Electrospray Ionization Coupled with Gas Chromatography.

Herein, gas-phase polycyclic aromatic hydrocarbons (PAHs) as nonpolar compounds were ionized to protonated molecular ions [M + H]+ without radical cations and simultaneously analyzed using gas chromatography (GC)/electrospray ionization (ESI)-tandem mass spectrometry (MS/MS). The ionization profile, dissociation, and sensitivity were first investigated to understand the significant behavior of gas-phase PAHs under ESI. The formation of protonated molecular ions of PAHs was distinguished according to the analyte phase and ESI spray solvents. The protonated PAHs exhibited characteristic dissociations, such as H-loss, H2-loss, and acetylene-loss, via competition of internal energy. In addition, GC/ESI-MS/MS resulted in relatively lower concentration levels (better sensitivity) for the limits-of-detection (LODs) of PAHs than liquid chromatography (LC)/ESI-MS/MS, and it seems to result from the characteristic ionization mechanism of the gas-phase analyte under ESI. Furthermore, the LODs of gas-phase PAHs depended on molecular weight and proton affinity (PA). Consequently, we demonstrated the relationship among the analyte phases, sensitivities, and structural characteristics (molecular weight and PA) under ESI. The gas-phase PAHs provided enhanced protonation efficiency and sensitivity using GC/ESI-MS/MS, as their molecular weight and PA increased. Based on these results, we offered important information regarding the behavior of gas-phase analytes under ESI. Therefore, the present GC/ESI-MS/MS method has potential as an alternative method for simultaneous analysis of PAHs.

Microbial production of uracil by an isolated Methylobacterium sp. WJ4 using methanol.

In this study, we report the production of uracil from methanol by an isolated methylotrophic bacterium, Methylobacterium sp. WJ4. The use of methanol as alternative carbon feedstock is attractive option in biotechnology. As a feedstock of biotechnological processes, methanol has distinct advantages over methane. This is not only due to physical and chemical considerations, but also to the properties of the pertinent organisms. Besides, with a wide array of biological activities and synthetic accessibility, uracil is considered as privileged structures in drug discovery. Uracil analogues have been applied to treatments of patients with cancer or viral infections. In this respect, it is meaningful to produce uracil using methanol. The effect of process parameters and methanol concentration for uracil production were investigated and optimized. Uracil production was remarkably increased to 5.76mgg cell dry weight-1 in optimized condition. The results were significant for further understanding of methylotrophic bacteria on uracil production.

Online Simultaneous Hydrogen/Deuterium Exchange of Multitarget Gas-Phase Molecules by Electrospray Ionization Mass Spectrometry Coupled with Gas Chromatography.

In this study, a hydrogen/deuterium (H/D) exchange method using gas chromatography-electrospray ionization/mass spectrometry (GC-ESI/MS) was first investigated as a novel tool for online H/D exchange of multitarget analytes. The GC and ESI source were combined with a homemade heated column transfer line. GC-ESI/MS-based H/D exchange occurs in an atmospheric pressure ion source as a result of reacting the gas-phase analyte eluted from GC with charged droplets of deuterium oxide infused as the ESI spray solvent. The consumption of the deuterated solvent at a flow rate of 2 µL min-1 was more economical than that in online H/D exchange methods reported to date. In-ESI-source H/D exchange by GC-ESI/MS was applied to 11 stimulants with secondary amino or hydroxyl groups. After H/D exchange, the spectra of the stimulants showed unexchanged, partially exchanged, and fully exchanged ions showing various degrees of exchange. The relative abundances corrected for naturally occurring isotopes of the fully exchanged ions of stimulants, except for etamivan, were in the range 24.3-85.5%. Methylephedrine and cyclazodone showed low H/D exchange efficiency under acidic, neutral, and basic spray solvent conditions and nonexchange for etamivan with an acidic phenolic OH group. The in-ESI-source H/D exchange efficiency by GC-ESI/MS was sufficient to determine the number of hydrogen by elucidation of fragmentation from the spectrum. Therefore, this online H/D exchange technique using GC-ESI/MS has potential as an alternative method for simultaneous H/D exchange of multitarget analytes.

TEMPO-Assisted Free Radical-Initiated Peptide Sequencing Mass Spectrometry (FRIPS MS) in Q-TOF and Orbitrap Mass Spectrometers: Single-Step Peptide Backbone Dissociations in Positive Ion Mode.

The present study demonstrates that one-step peptide backbone fragmentations can be achieved using the TEMPO [2-(2,2,6,6-tetramethyl piperidine-1-oxyl)]-assisted free radical-initiated peptide sequencing (FRIPS) mass spectrometry in a hybrid quadrupole time-of-flight (Q-TOF) mass spectrometer and a Q-Exactive Orbitrap instrument in positive ion mode, in contrast to two-step peptide fragmentation in an ion-trap mass spectrometer (reference Anal. Chem. 85, 7044-7051 (30)). In the hybrid Q-TOF and Q-Exactive instruments, higher collisional energies can be applied to the target peptides, compared with the low collisional energies applied by the ion-trap instrument. The higher energy deposition and the additional multiple collisions in the collision cell in both instruments appear to result in one-step peptide backbone dissociations in positive ion mode. This new finding clearly demonstrates that the TEMPO-assisted FRIPS approach is a very useful tool in peptide mass spectrometry research. Graphical Abstract ᅟ.

Photodissociation of TEMPO-modified peptides: new approaches to radical-directed dissociation of biomolecules.

Radical-directed dissociation of gas phase ions is emerging as a powerful and complementary alternative to traditional tandem mass spectrometric techniques for biomolecular structural analysis. Previous studies have identified that coupling of 2-[(2,2,6,6-tetramethylpiperidin-1-oxyl)methyl]benzoic acid (TEMPO-Bz) to the N-terminus of a peptide introduces a labile oxygen-carbon bond that can be selectively activated upon collisional activation to produce a radical ion. Here we demonstrate that structurally-defined peptide radical ions can also be generated upon UV laser photodissociation of the same TEMPO-Bz derivatives in a linear ion-trap mass spectrometer. When subjected to further mass spectrometric analyses, the radical ions formed by a single laser pulse undergo identical dissociations as those formed by collisional activation of the same precursor ion, and can thus be used to derive molecular structure. Mapping the initial radical formation process as a function of photon energy by photodissociation action spectroscopy reveals that photoproduct formation is selective but occurs only in modest yield across the wavelength range (300-220 nm), with the photoproduct yield maximised between 235 and 225 nm. Based on the analysis of a set of model compounds, structural modifications to the TEMPO-Bz derivative are suggested to optimise radical photoproduct yield. Future development of such probes offers the advantage of increased sensitivity and selectivity for radical-directed dissociation.

One-step peptide backbone dissociations in negative-ion free radical initiated peptide sequencing mass spectrometry.

Peptide dissociation behavior in TEMPO (2,2,6,6-tetramethylpiperidine-1-oxyl)-based FRIPS (free radical initiated peptide sequencing) mass spectrometry was analyzed in both positive- and negative-ion modes for a number of peptides including angiotensin II, kinetensin, glycoprotein IIb fragment (296-306), des-Pro(2)-bradykinin, and ubiquitin tryptic fragment (43-48). In the positive mode, the ·Bz-C(O)-peptide radical species was produced exclusively at the initial collisional activation of o-TEMPO-Bz-C(O)-peptides, and two consecutive applications of collisional activation were needed to observe peptide backbone fragments. In contrast, in the negative-ion mode, a single application of collisional activation to o-TEMPO-Bz-C(O)-peptides produced extensive peptide backbone fragmentations as well as ·Bz-C(O)-peptide radical species. This result indicates that the duty cycle in the TEMPO-based FRIPS mass spectrometry can be reduced by one-half in the negative-ion mode. In addition, the fragment ions observed in the negative-ion experiments were mainly of the a-, c-, x-, and z-types, indicating that radical-driven tandem mass spectrometry was mainly responsible for the TEMPO-based FRIPS even with a single application of collisional activation. Furthermore, the survival fraction analysis of o-TEMPO-Bz-C(O)-peptides was made as a function of the applied normalized collision energy (NCE). This helped us to better understand the differences in FRIPS behavior between the positive- and negative-ion modes in terms of dissociation energetics. The duty-cycle improvement made in the present study provides a cornerstone for future research aiming to achieve a single-step FRIPS in the positive-ion mode.

Host-guest chemistry in the gas phase: complex formation with 18-crown-6 enhances helicity of alanine-based peptides.

The gas-phase helix propensities of alanine-based polypeptides are studied with different locations of a Lys residue and host-guest interactions with 18-Crown-6 (18C6). A series of model peptides Ac-Ala(9-n)-LysH(+)-Ala(n) (n = 0, 1, 3, 5, 7, and 9) is examined alone and with 18C6 using traveling wave ion mobility mass spectrometry combined with molecular dynamics (MD) simulations. The gas-phase helices are observed from the peptides whose Lys residue is located close to the C-terminus so that the Lys exerts its capping effect on the C-terminal carbonyl groups. The peptides, which interact with 18C6 in the gas phase, show enhanced helical propensities. The enhanced helicity of the peptide in the complex is attributed by isolation of the Lys butylammonium group from the helix backbone and the interaction of methylene groups of 18C6, which possess localized positive partial charges, with C-terminal carbonyl groups serving as a cap to stabilize the helix.

Investigation of the MALDI process used to characterize self-assembled monolayers of alkanethiolates on gold.

In this work, we investigated the surface processes involved in matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS), which produce intact characteristic ions, typically in disulfide form, from self-assembled monolayers (SAMs) of alkanethiolates on gold. For the study, SAMs decorated with peptides and a THAP matrix were employed. Using two-laser MS, it was previously found that irradiation with a UV laser gave rise to the direct desorption of SAM molecules from alkanethiol SAMs on gold, producing disulfide species in vacuum. However, a closer examination of this study suggests that the MALDI process in which the matrix is used may not be the case. Instead, the results indicate that the treatment of the matrix solution is responsible for the characteristic ion formation in MALDI MS. We propose that the matrix solution dissolves alkanethiolate molecules from SAMs, leading to the generation of characteristic disulfide species in the solution. The disulfides are then cocrystallized with matrix molecules and subsequently detected by MALDI MS. Because MALDI MS is a powerful tool for biopolymers with high molecular weights, it has been successfully applied to SAMs presenting large biomolecules. This understanding of the MALDI process in the surface MS of alkanethiol SAMs on gold may allow advances in the biomolecular application of SAMs in combination with mass spectrometric analysis.

Gas-phase peptide sequencing by TEMPO-mediated radical generation.

Collisional activation of 2-[(2,2,6,6-tetramethylpiperidin-1-yloxy)methyl]benzoic acid (TEMPO-Bz)-conjugated peptide cations, prepared by attaching a TEMPO-derived precursor 1 to an N-terminal amino group or a lysine side chain, resulted in the formation of radical species. The subsequent tandem mass spectrometry on the radical cations exhibited odd-electron peptide backbone dissociations in the same manner as that observed by electron capture dissociation (ECD) or electron transfer dissociation (ETD). For example, a-, x-, or z-types of ions were major fragments and the disulfide bond was readily cleaved. The TEMPO-FRIPS (free radical initiated peptide sequencing) was also applicable to characterizing even singly protonated peptides, in contrast to ECD or ETD in which only doubly or highly protonated cations are responsive. The TEMPO-FRIPS approach also has universality in that it can be used in any type of a tandem mass spectrometer.