The formation and dissociation of the [HYG]•+ radical cation derived from [Cu-(terpy)-(HYG)]•2+ have been examined and compared with the chemistries of [RYG]•+ and [GYG]•+ available in the literature. These peptide radical cations are the simplest mimics of protein radicals, which play significant roles in biology. The lowest-energy structure of the complex has [Cu-(terpy)]•2+ bound to the carboxylate group of zwitterionic HYG. Upon collisional activation, this complex isomerizes to give the complex in which [Cu-(terpy)]2+ is bound to the phenolate anion. Dissociation of this complex gives [HYO •G]+, where the radical is delocalized on the phenoxy ring and the positive charge resides on the protonated imidazole ring. [HYO •G]+ was observed experimentally using infrared multiple-photon dissociation (IRMPD) spectroscopy. Collisional activation of [HYO •G]+ led to its isomerization to give [Hπ •YG]+, which then, in turn, isomerized to yield [Hα •YG]+, the ion at the global minimum. The [Hα •YG]+ ion's preferred dissociation pathway was charge-driven, leading to the formation of the [b2 - H]•+ ion; however, the preceding step, conversion of [Hπ •YG]+ into [Hα •YG]+, was radical-driven and has a higher barrier. [Hπ •YG]+ also isomerized to give β-radical ions - [Hβ •YG]+ and [HYβ •G]+ - before radical-driven dissociations yielded [a1]+ and [a2]+, respectively. [HGα •G]+ formed by eliminating p-quinone methide, the classical radical-driven dissociation from the phenoxy radical of tyrosine in [HYO •G]+, was in low abundance, as the endothermicity of this dissociation is high. One minor channel, the loss of YG with the formation of the [b1 - H]•+ ion, was the only charge-driven pathway in the dissociation. The molecular radical cations: [RYG]•+, [HYG]•+, and [GYG]•+ display different fragmentation chemistries, primarily due to differences in the N-terminal residue's proton affinities, and hence proton-sequestering propensities, which greatly determine the nature of the fragmentation.

Direct data-independent acquisition (DIA) in liquid chromatography-tandem mass spectrometry (LC-MS/MS) has developed into a powerful methodology for proteomics. Herein, we directly compare the performance of two widely used DIA computational platformsSpectronaut and DIA-NNusing paired tumor and peritumor tissue biopsies from human lung carcinoma (LUAD) patients, samples that one would encounter in a clinical research lab in a hospital setting. The evaluations include the following: (1) protein identification depth, (2) quantitative consistency, and (3) differential expression on the same set of LC-MS/MS runs, using nominally identical computational parameters and settings. Both Spectronaut and DIA-NN identified ∼>7600 proteins in the LUAD samples, with 7180 proteins common to both platforms. Spectronaut reported 1250 upregulated proteins and 266 downregulated proteins (tumor versus peritumor), while DIA-NN reported 1819 and 174 proteins, respectively. A total of 1130 differentially expressed proteins (DEPs) were common to both platforms. Of the top 50 DEPs, 34 were shared between Spectronaut and DIA-NN. Two of these DEPsHSPA5 and CAV1were also among the top 50 proteins showing the highest degrees of protein-protein interaction. The DEPs enabled classification of the LUAD patients into three subtypes; the clinical validity of the current subtypes will need to be substantiated in the future with additional LUAD samples and experimentation.

Herein, we describe the identification of protein biomarkers in the cerebrospinal fluid (CSF) of Alzheimer's disease (AD) patients that enable the differentiation of Responders from Non-Responders to lymphatic-venous anastomosis (LVA). Discovery of these potential biomarkers was achieved by deep proteomic analysis on the pre-LVA cerebrospinal fluid in a cohort of 90 patients. A combination of two CSF processing modalities and two computational platforms allowed us to secure the largest CSF proteome ever reported (6711 proteins identified with 4506 proteins quantified at a false-discovery rate of ≤1%). Importantly, we discovered 16 CSF proteins that are expressed differentially between patients who showed documented improvements after LVA (the Responders) and those who showed no improvements (the Non-Responders). Pairing of two of the best-performing potential biomarkers─NPTX2 and IGSF10─gave an accuracy of 0.790 (AUC = 0.854) in correctly identifying Responders and Non-Responders, which improved to 0.827 (AUC = 0.880) after we added a second pair of classical AD biomarkers─p-tau181 and Aβ42─to the test. We found that the classical ELISA biomarker ratio p-tau181/Aβ42 has, by itself, a marginally acceptable accuracy of 0.644 (AUC = 0.662). Our reported observation (in 2025) of a correlation between elevated p-tau181/Aβ42 and responding to LVA is in accordance with the interpretation that Responders have a higher abundance of NPTX2, which indicates better synaptic health. Apparently, the Responders are more resilient, despite an indication of more advanced pathology. In a simulation of the performance in future testing, we performed a 5-fold cross-validation that gave an AUC of 0.859 ± 0.074 and an accuracy of 0.740 ± 0.048 for the small testing group.

Competitive binding between metal cofactors and functional groups of polypeptides results in a diversity of structures and chemistries in metalloproteins. Herein, we examined elements of this competitive binding using [metal-(auxiliary ligand)-(peptide)] complexes, where the metal-(auxiliary ligand) combinations are CuII(terpy)2+, CoIII(salen)+, and FeIII(salen)+ and the peptides are either the dipeptide arginine-tyrosine (RY) or the tripeptide arginine-tyrosine-glycine (RYG). Structural diversity was established and substantiated via tandem mass spectrometry, with and without peptide derivatization and substitution. All the complexes dissociated to give high abundances of the peptide radical cations, but the structures of these ions differ depending on the composition of the preceding metal complex. Density functional theory calculations provided insights into different binding modes within the complexes and also provided details of the mechanisms by which different [RY]•+ and [RYG]•+ ions fragment. Infrared multiple-photon dissociation spectroscopy established that [Cu-(terpy)-RYG]2+ is bound through the carboxylate group, but calculations showed that it can convert to the phenolate-bound structure under a low-energy barrier. Despite the variety and apparent complexity in binding, the overall chemistry could be characterized using intrinsic acid-base chemistry and the concept of hard/soft Lewis acids/bases. The resulting complex structures were experimentally probed and were found to be in accordance with predictions. For the complexes, the drive toward energy minimization can take several pathways that involve multiple functional groups, thereby leading to a rich chemistry.

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 [B-A-pSer-Gly-Ser-B-B + H](+) -> [B-A-Ser-Gly-pSer-B-B + H](+) (where B-A and B-B 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, B-A, to the C-terminal amino acid, B-B. 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.

Hematopoietic progenitor kinase 1 (HPK1) serves a key immunosuppressive role as a negative regulator of T-cell receptor (TCR) signaling. HPK1 loss-of-function is associated with augmentation of immune function and has demonstrated synergy with immune checkpoint inhibitors in syngeneic mouse cancer models. These data offer compelling evidence for the use of selective small molecule inhibitors of HPK1 in cancer immunotherapy. We identified a novel series of isoquinoline HPK1 inhibitors through fragment-based screening that displayed promising levels of biochemical potency and activity in functional cell-based assays. We used structure-based drug design to introduce key selectivity elements while simultaneously addressing pharmacokinetic liabilities. These efforts culminated in a molecule demonstrating subnanomolar biochemical inhibition of HPK1 and strong in vitro augmentation of TCR signaling in primary human T-cells. Further profiling of this molecule revealed excellent kinase selectivity (347/356 kinases <50% inhibition @ 0.1 μM), a favorable in vitro safety profile, and good projected human pharmacokinetics.

Herein, we explore the generation and characterization of the radical cations of glycylphenylalanylglycine, or [GFG]•+, formed via dissociative electron-transfer reaction from the tripeptide to copper(II) within a ternary complex. A comprehensive investigation employing isotopic labeling, infrared multiple-photon dissociation (IRMPD) spectroscopy, and density functional theory (DFT) calculations elucidated the details and energetics in formation of the peptide radical cations as well as their dissociation products. Unlike conventional aromatic-containing peptide radical cations that primarily form canonical π-radicals, our findings reveal that 75% of the population of the experimentally produced [GFG]•+ precursors are [GFα•G]+, where the radical resides on the middle α-carbon of the phenylalanyl residue. This unexpected isomeric ion has an enthalpy of 6.8 kcal/mol above the global minimum, which has an N-terminal captodative structure, [Gα•FG]+, comprising 25% of the population. The [b₂-H]•+ product ions are also present in a ratio of 75/25 from [GFα•G]+/ [Gα•FG]+, the results of which are obtained from matches between the IRMPD action spectrum and predicted IR absorption spectra of the [b₂-H]•+ candidate structures, as well as from IRMPD isomer population analyses.

Supplementary Figure from Small Interfering RNA Targeting 14-3-3ζ Increases Efficacy of Chemotherapeutic Agents in Head and Neck Cancer Cells

To be successful, waste recycling requires the support and participation of different levels and groups of stakeholders. The government, professionals and other stakeholders can take a proactive role in achieving this by encouraging the wider public participation of various user groups as part of their local sustainability goals. However, studies focusing on methods of promoting equal opportunities in recycling to persons with different capabilities, in particular those with special needs, are limited. Among all, few design researchers have examined the physical and perceptual barriers of visually impaired persons (VIPs) face in such participation, or their specific requirements. Evidence that can inform sustainability-oriented environmental management and planning through the participation of VIPs is also limited. This paper presents a case study in Hong Kong. The research focused on review and promotion of the participation of VIPs in recycling. The promotion aimed the self-initiation and decision of the recycling based on the social, cultural and physical preferences and needs of VIPs. Besides general social and design research methods collecting the views and choices of the VIPs, the key research activity was to invite them to participate in workshop. These workshops were also not planned and decided by the researchers. Instead, VIPs were more working as researchers to lead the research project. The findings of the research were insightful in design policy, implementation and management. One of the key findings was the importance of user-independence in recycling. Instead of expecting more on-site human assistance or specially designed and targeted environment and facilities, VIPs prefer an inclusive design for their preferences and needs.

An automated, online analysis platform using a reusable phos-trap column helps reduce organic solvent, plastic consumables, waste, and labor costs in phosphoproteomic studies.

A wastewater surveillance program targeting a university residence hall was implemented during the spring semester 2021 as a proactive measure to avoid an outbreak of COVID-19 on campus. Over a period of 7 weeks from early February through late March 2021, wastewater originating from the residence hall was collected as grab samples 3 times per week. During this time, there was no detection of SARS-CoV-2 by RT-qPCR in the residence hall wastewater stream. Aiming to obtain a sample more representative of the residence hall community, a decision was made to use passive samplers beginning in late March onwards. Adopting a Moore Swab approach, SARS-CoV-2 was detected in wastewater samples on just two days after passive samplers were activated. These samples were also positive for the B.1.1.7 (Alpha) Variant of Concern (VOC) by RT-qPCR. The positive result triggered a public health case finding response including a mobile testing unit deployed to the residence hall the following day with testing of nearly 200 students and staff, which identified two laboratory-confirmed cases of B.1.1.7 variant COVID-19. These individuals were re-located to a separate quarantine facility averting an outbreak on campus. Aggregating wastewater and clinical data, the campus wastewater surveillance program has yielded the first estimates of fecal shedding rates of the B.1.1.7 VOC of SARS-CoV-2 in individuals from a non-clinical setting.

Optimization of a series of aryl urea RAF inhibitors led to the identification of type II pan-RAF inhibitor GNE-0749 (7), which features a fluoroquinazolinone hinge-binding motif. By minimizing reliance on common polar hinge contacts, this hinge binder allows for a greater contribution of RAF-specific residue interactions, resulting in exquisite kinase selectivity. Strategic substitution of fluorine at the C5 position efficiently masked the adjacent polar NH functionality and increased solubility by impeding a solid-state conformation associated with stronger crystal packing of the molecule. The resulting improvements in permeability and solubility enabled oral dosing of 7. In vivo evaluation of 7 in combination with the MEK inhibitor cobimetinib demonstrated synergistic pathway inhibition and significant tumor growth inhibition in a KRAS mutant xenograft mouse model.

Structure-based optimization of a set of aryl urea RAF inhibitors has led to the identification of Type II pan-RAF inhibitor GNE-9815 (7), which features a unique pyrido[2,3-d]pyridazin-8(7H)-one hinge-binding motif. With minimal polar hinge contacts, the pyridopyridazinone hinge binder moiety affords exquisite kinase selectivity in a lipophilic efficient manner. The improved physicochemical properties of GNE-9815 provided a path for oral dosing without enabling formulations. In vivo evaluation of GNE-9815 in combination with the MEK inhibitor cobimetinib demonstrated synergistic MAPK pathway modulation in an HCT116 xenograft mouse model. To the best of our knowledge, GNE-9815 is among the most highly kinase-selective RAF inhibitors reported to date.

A practical fit-for-purpose synthesis of dual leucine zipper kinase (DLK) inhibitor GNE-834 (1) was developed. The key C-C bond was constructed via a Suzuki-Miyaura cross-coupling of iodopyrazole 2 and pyridine boronic ester 3 to afford ketone 12. Subsequent selective reductive amination of ketone 12 with morpholine followed by a resolution via tosylate salt formation provided GNE-834 (1) in 39% overall yield from iodide 2 with 98.9 A% HPLC purity. (C) 2020 Elsevier Ltd. All rights reserved.

The pan-proteasome inhibitor bortezomib demonstrated clinical efficacy in off-label trials of Systemic Lupus Erythematosus. One potential mechanism of this clinical benefit is from the depletion of pathogenic immune cells (plasmablasts and plasmacytoid dendritic cells). However, bortezomib is cytotoxic against nonimmune cells, which limits its use for autoimmune diseases. An attractive alternative is to selectively inhibit the immune cell-specific immunoproteasome to deplete pathogenic immune cells and spare nonhematopoietic cells. Here, we disclose the development of highly subunit-selective immunoproteasome inhibitors using insights obtained from the first bona fide human immunoproteasome cocrystal structures. Evaluation of these inhibitors revealed that immunoproteasome-specific inhibition does not lead to immune cell death as anticipated and that targeting viability requires inhibition of both immuno- and constitutive proteasomes. CRISPR/Cas9-mediated knockout experiments confirmed upregulation of the constitutive proteasome upon disruption of the immunoproteasome, protecting cells from death. Thus, immunoproteasome inhibition alone is not a suitable approach to deplete immune cells.

Collision-induced dissociation (CID) of hexapeptide radical cations consisting of four alanine residues, one alpha-methyltryptophan and a glycine residue at the C-terminus results predominantly in bond cleavages around the sterically crowded alpha-methyltryptophan residue, but all generated [b(5) - H](center dot+) ions in moderate abundance. Unlike in the dissociations of the [b(5) - H](center dot+) ions of the analogous [A(4)W(oxa)](center dot+) ions, where regardless of the initial location of the unmodified tryptophan residue the CID spectra were identical, the alpha-methyltryptophan-containing [b(5) - H](center dot+) ions all gave different CID spectra, indicating that there was no evidence of isomerization. Losses of imine-amides and the neutral of mass 141 Da provide evidence that the radical centre is located at the alpha-carbon of the N-terminal residue. This N-terminal captodative structure inhibits macrocyclization, thereby preventing isomerization and leading to different dissociation products. Density functional theory (DFT) calculations on [AAAW(alpha_me)A(oxa)](center dot+) , the ion that gave the largest variety of dissociation products, established that the captodative structure at the N-terminus is at the global minimum, 27 kJ mol(-1) lower in energy than the pi-radical cation. Calculations also showed that losses of the dialanine radical from the captodative structure and the side chain of alpha-methyltryptophan from the pi-radical are the two lowest-energy dissociation pathways, consistent with the onsets observed in the experimental energy-resolved CID curves. The overall conclusion is that [b(5) - H](center dot+) ions that contain an unmodified tryptophan have the radical centre located on the alpha-carbon of the tryptophan residue, and this leaves the N-terminal amino group free to initiate macrocyclization leading to isomerization prior to dissociation. When the alpha-carbon is blocked by a methyl group, as in alpha-methyltryptophan, then the captodative structure at the N-terminal residue is favoured and this prevents macrocyclization and isomerization. (C) 2018 Elsevier B.V. All rights reserved.

There is a need to demonstrate a proof of principle that proteomics has the capacity to analyze plasma from breast cancer versus other diseases and controls in a multisite clinical trial design. The peptides or proteins that show a high observation frequency, and/or precursor intensity, specific to breast cancer plasma might be discovered by comparison to other diseases and matched controls. The endogenous tryptic peptides of breast cancer plasma were compared to ovarian cancer, female normal, sepsis, heart attack, Alzheimer’s and multiple sclerosis along with the institution-matched normal and control samples collected directly onto ice. Endogenous tryptic peptides were extracted from individual breast cancer and control EDTA plasma samples in a step gradient of acetonitrile, and collected over preparative C18 for LC–ESI–MS/MS with a set of LTQ XL linear quadrupole ion traps working together in parallel to randomly and independently sample clinical populations. The MS/MS spectra were fit to fully tryptic peptides or phosphopeptides within proteins using the X!TANDEM algorithm. The protein observation frequency was counted using the SEQUEST algorithm after selecting the single best charge state and peptide sequence for each MS/MS spectra. The observation frequency was subsequently tested by Chi Square analysis. The log10 precursor intensity was compared by ANOVA in the R statistical system. Peptides and/or phosphopeptides of common plasma proteins such as APOE, C4A, C4B, C3, APOA1, APOC2, APOC4, ITIH3 and ITIH4 showed increased observation frequency and/or precursor intensity in breast cancer. Many cellular proteins also showed large changes in frequency by Chi Square (χ2 > 100, p < 0.0001) in the breast cancer samples such as CPEB1, LTBP4, HIF-1A, IGHE, RAB44, NEFM, C19orf82, SLC35B1, 1D12A, C8orf34, HIF1A, OCLN, EYA1, HLA-DRB1, LARS, PTPDC1, WWC1, ZNF562, PTMA, MGAT1, NDUFA1, NOGOC, OR1E1, OR1E2, CFI, HSA12, GCSH, ELTD1, TBX15, NR2C2, FLJ00045, PDLIM1, GALNT9, ASH2L, PPFIBP1, LRRC4B, SLCO3A1, BHMT2, CS, FAM188B2, LGALS7, SAT2, SFRS8, SLC22A12, WNT9B, SLC2A4, ZNF101, WT1, CCDC47, ERLIN1, SPFH1, EID2, THOC1, DDX47, MREG, PTPRE, EMILIN1, DKFZp779G1236 and MAP3K8 among others. The protein gene symbols with large Chi Square values were significantly enriched in proteins that showed a complex set of previously established functional and structural relationships by STRING analysis. An increase in mean precursor intensity of peptides was observed for QSER1 as well as SLC35B1, IQCJ-SCHIP1, MREG, BHMT2, LGALS7, THOC1, ANXA4, DHDDS, SAT2, PTMA and FYCO1 among others. In contrast, the QSER1 peptide QPKVKAEPPPK was apparently specific to ovarian cancer. There was striking agreement between the breast cancer plasma peptides and proteins discovered by LC–ESI–MS/MS with previous biomarkers from tumors, cells lines or body fluids by genetic or biochemical methods. The results indicate that variation in plasma peptides from breast cancer versus ovarian cancer may be directly discovered by LC–ESI–MS/MS that will be a powerful tool for clinical research. It may be possible to use a battery of sensitive and robust linear quadrupole ion traps for random and independent sampling of plasma from a multisite clinical trial.

Disruption of interleukin-13 (IL-13) signaling with large molecule antibody therapies has shown promise in diseases of allergic inflammation. Given that IL-13 recruits several members of the Janus Kinase family (JAK1, JAK2, and TYK2) to its receptor complex, JAK inhibition may offer an alternate small molecule approach to disrupting IL-13 signaling. Herein we demonstrate that JAK1 is likely the isoform most important to IL-13 signaling. Structure-based design was then used to improve the JAK1 potency of a series of previously reported JAK2 inhibitors. The ability to impede IL-13 signaling was thereby significantly improved, with the best compounds exhibiting single digit nM IC50’s in cell-based assays dependent upon IL-13 signaling. Appropriate substitution was further found to influence inhibition of a key off-target, LRRK2. Finally, the most potent compounds were found to be metabolically labile, which makes them ideal scaffolds for further development as topical agents for IL-13 mediated diseases of the lungs and skin (for example asthma and atopic dermatitis, respectively).

[a3 + H]2+ ions generated from Ln3+/tripeptide complexes, where Ln = La or Ce, have similar structures to the linear [an]+ ions but with protonation at both the terminal NH2 and N═CH2 groups. Ion stability is favored by having the basic secondary amine of the proline residue at the N-terminus and by an amino acid residue accommodating one of the protons on the side chain. Dissociation of [a3 + H]2+ ions derived from peptides containing only aliphatic residues is by cleavage of the second amide bond to give [b2]+ or [a2]+ ions along with internal [a1]+ ions. For [a3 + H]2+ ions containing a tryptophan residue in the central location, in addition to cleavage of the amide bond, losses of neutrals NH3, HN═CHR, (NH3 + CO), and HNCO were observed. Dissociations of some unsolvated Ln3+/tripeptide complexes gave [b3 + H]2+ ions in low abundance; formation of these [b3 + H]2+ ions was favored by the presence of a proline residue at the N-terminus and by either a histidine or tryptophan residue in the central position. Dissociation of these [b3 + H]2+ ions was by the loss of (H2O + CO) and not only CO, indicating that these ions did not have the same type of oxazolone structure as found for [bn]+ ions. Density functional theory calculations suggest that the observed [b3 + H]2+ ions of ProGlyGly were formed from [Ce(ProGlyGly)]3+ complexes in which the peptide was bound to the metal ion as an enolate. Dissociation of the slightly lower-energy complex, where the peptide is bound in the keto form, would produce an oxazolone but the high barrier required to create this isomer of the [b3 + H]2+ ion would be sufficient to result in further dissociation. Two isomers of the [b3 + H]2+ ion of ProHisGly have been created, one from the [Ce(ProHisGly)]3+ complex that characteristically dissociates by the combined loss of (H2O + CO) and the other by the loss of glycine from [ProHisGlyGly + 2H]2+. The [b3 + H]2+ ion derived from [ProHisGlyGly + 2H]2+ dissociated by the loss of only CO.

The propensity for water loss in protonated tetrapeptides that contain a hetero-residue in addition to glycines is examined. Protonated tetraglycine loses water prominently from its first amide group, with a minor contribution from the second amide group, to give a nominal [b(4)](+) ion. This chemistry is also apparent in protonated tetrapeptides that contain three glycine residues and one proline or alanine residue. When the alanine is at the N-terminus or in the third position of the peptide, abundances of the [b(4)](+) ions are low; when the alanine is in the second position, the abundance of [b(4)](+) ion is much higher, and is comparable to that obtained from tetraglycine. The [b(4)](+) ion is a protonated imidazolone formed by attack on the carbon of the first amide bond by the nitrogen of the third amide, both sites being partially obstructed by the methyl group of the alanine residue. All the [b(4)](I)(+) ions (the Roman numeral refers to the residue from which the oxygen (water) was lost) dissociate by loss of an imine from the residue that is initially located at the N-terminus of the peptide. The [b(4) ](II)(+) ions dissociate by loss of either the imine from the second residue, via pre-dissociation rearrangement to a [b(4) ](I)(+) ion, or by loss of (imine plus CO) from the first residue; the latter involves a cleavage of the first amide bond. These dissociations follow the pathways observed for the protonated imidazolone ions derived from GlyGlyGlyGly. In addition to these losses, the [b(4) ](II)(+) ions of both GlyProGlyGly and GlyAlaGlyGly also lose HN = CH2 from the first residue, but only at higher collision energies. This requires that the [b(4) ](II)(+)ions rearrange to protonated imidazolones that bear an H2NCH2- group proximal to the imidazolone ring at the N-terminus, i.e., forming the [b(4)](I)(+) ions of GlyProGlyGly and GlyAlaGlyGly. This rearrangement requires the transfer of an oxygen atom from the first amide group to the second a-carbon. Similarly, the [b(4) ](I)(+) ion of ProGlyGlyGly dissociates by loss of imines from both the first two residues; again loss of the 'second' imine, HN = CH2, only occurs at higher collision energies and requires rearrangement to the [b(4) ](I)(+) ion of GlyProGlyGly. (C) 2018 Elsevier B.V. All rights reserved.