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Mass spectrometry affords rapid and sensitive analysis of peptides and proteins. Coupling spectroscopy with mass spectrometry allows for the development of new methods to enhance biomolecular structure determination. Herein, we demonstrate two new energy acceptors that can be utilized for action-excitation energy transfer experiments. In the first system, C-S bonds in methionine act as energy acceptors from native chromophores, including tyrosine, tryptophan, and phenylalanine. Comparison among chromophores reveals that tyrosine transfers energy most efficiently at 266 nm, but phenylalanine and tryptophan also transfer energy with comparable efficiencies. Overall, the C-S bond dissociation yields following energy transfer are low for methionine, which led to an investigation of selenomethionine, a common analog that is found in many naturally occurring proteins. Sulfur and selenium are chemically similar, but C-Se bonds are weaker than C-S bonds and have lower lying σ* anti-bonding orbitals. Excitation of peptides containing tyrosine and tryptophan results in efficient energy transfer to selenomethionine and abundant C-Se bond dissociation. A series of helical peptides were examined where the positions of the donor or acceptor were systematically scanned to explore the influence of distance and helix orientation on energy transfer. The distance was found to be the primary factor affecting energy transfer efficiency, suggesting that selenomethionine may be a useful acceptor for probing protein structure in the gas phase.

Proteinaceous aggregation is a well-known observable in Alzheimer's disease (AD), but failure and storage of lysosomal bodies within neurons is equally ubiquitous and actually precedes bulk accumulation of extracellular amyloid plaque. In fact, AD shares many similarities with certain lysosomal storage disorders though establishing a biochemical connection has proven difficult. Herein, we demonstrate that isomerization and epimerization, which are spontaneous chemical modifications that occur in long-lived proteins, prevent digestion by the proteases in the lysosome (namely, the cathepsins). For example, isomerization of aspartic acid into l-isoAsp prevents digestion of the N-terminal portion of Aβ by cathepsin L, one of the most aggressive lysosomal proteases. Similar results were obtained after examination of various target peptides with a full series of cathepsins, including endo-, amino-, and carboxy-peptidases. In all cases peptide fragments too long for transporter recognition or release from the lysosome persisted after treatment, providing a mechanism for eventual lysosomal storage and bridging the gap between AD and lysosomal storage disorders. Additional experiments with microglial cells confirmed that isomerization disrupts proteolysis in active lysosomes. These results are easily rationalized in terms of protease active sites, which are engineered to precisely orient the peptide backbone and cannot accommodate the backbone shift caused by isoaspartic acid or side chain dislocation resulting from epimerization. Although Aβ is known to be isomerized and epimerized in plaques present in AD brains, we further establish that the rates of modification for aspartic acid in positions 1 and 7 are fast and could accrue prior to plaque formation. Spontaneous chemistry can therefore provide modified substrates capable of inducing gradual lysosomal failure, which may play an important role in the cascade of events leading to the disrupted proteostasis, amyloid formation, and tauopathies associated with AD.

Photoinitiated radical chemistry has proven to be useful for breaking covalent bonds within many biomolecules in the gas phase. Herein, we demonstrate that radical chemistry is useful for bond synthesis in the gas phase. Single peptides containing two cysteine residues capped with propylmercaptan (PM) often form disulfide bonds following ultraviolet excitation at 266 nm and loss of both PM groups. Similarly, noncovalently bound peptide pairs where each peptide contains a single cysteine residue can be induced to form disulfide bonds. Comparison with disulfide bound species sampled directly from solution yields identical collisional activation spectra, suggesting that native disulfide bonds have been recapitulated in the gas phase syntheses. Another approach utilizing radical chemistry for covalent bond synthesis involves creation of a reactive diradical that can first abstract hydrogen from a target peptide, creating a new radical site, and then recombine the second radical with the new radical to form a covalent bond. This chemistry is illustrated with 2-(hydroxymethyl-3,5-diiodobenzoate)-18-crown-6 ether, which attaches noncovalently to protonated primary amines in peptides and proteins. Following photoactivation and crosslinking, the site of noncovalent adduct attachment can frequently be determined. The ramifications of these observations on peptide structure and noncovalent attachment of 18-crown-6-based molecules is discussed.

Photoinitiated radical chemistry has proven to be useful for breaking covalent bonds within many biomolecules in the gas phase. Herein, we demonstrate that radical chemistry is useful for bond synthesis in the gas phase. Single peptides containing two cysteine residues capped with propylmercaptan (PM) often form disulfide bonds following ultraviolet excitation at 266 nm and loss of both PM groups. Similarly, noncovalently bound peptide pairs where each peptide contains a single cysteine residue can be induced to form disulfide bonds. Comparison with disulfide bound species sampled directly from solution yields identical collisional activation spectra, suggesting that native disulfide bonds have been recapitulated in the gas phase syntheses. Another approach utilizing radical chemistry for covalent bond synthesis involves creation of a reactive diradical that can first abstract hydrogen from a target peptide, creating a new radical site, and then recombine the second radical with the new radical to form a covalent bond. This chemistry is illustrated with 2-(hydroxymethyl-3,5-diiodobenzoate)-18-crown-6 ether, which attaches noncovalently to protonated primary amines in peptides and proteins. Following photoactivation and crosslinking, the site of noncovalent adduct attachment can frequently be determined. The ramifications of these observations on peptide structure and noncovalent attachment of 18-crown-6-based molecules is discussed.

The sudden transition from in-person to virtual learning during the spring 2020 semester posed challenges for students and faculty alike. This paper explores the techniques used by faculty to transform the chemistry and biochemistry curriculum at a Hispanic-serving institution with a significant proportion of first-generation students and the response of students to these changes. Faculty utilized an assortment of techniques, including synchronous lectures by video conference, asynchronous prerecorded lectures, online examinations with varying timing policies, and virtual office hours. A survey was completed by 332 students across 26 different courses taught by 11 faculty regarding their preferences for course delivery format and the academic and nonacademic challenges they faced. A paired samples "t"-test indicated a statistically significant difference in satisfaction with face-to-face instruction (M = 4.13, SD = 0.765) and satisfaction with virtual instruction (M = 3.57, SD = 0.961), t(440) = 13.30, p < 0.001, Cohen's "d" of 0.63, and Cronbach's [alpha] of 0.767. The results indicated a preference for face-to-face instruction. Open-ended survey questions identify that the primary academic challenges faced by students involved technology, understanding material, and difficulty obtaining help, while the primary nonacademic challenges were work issues, focus/motivation, and family issues. These results may inform future practices for virtual instruction.

Science educators have developed a variety of assessment techniques to help students connect their scientific knowledge and bridge conceptual gaps. In chemistry, concept maps and creative exercises are the two notable assessments that have been implemented into multiple chemistry courses and indicated promising effects on students' conceptual learning and connection-making between chemistry concepts. These two assessment techniques were usually implemented individually in research studies. Herein, we employed a quasi-experimental, mixed-methods approach to explore whether combining concept maps and creative exercises would reveal any synergistic effects for student learning of chemical equilibrium and acid-base chemistry in a college general chemistry course. In this study, student perceptions of the use of the two assessments were examined by open-ended surveys. Interestingly, students perceived creative exercises as an assessment technique while concept maps were viewed as a learning tool for studying or reviewing exams. Additionally, Students believed that concept maps assisted them in answering creative exercises, but not vice versa. The four study groups (control group, concept maps only, creative exercises only, and both concept maps and creative exercises) were compared through concept inventory pre and post-test questions. The results of an ANCOVA indicated that participation in the experimental groups did not significantly impact conceptual learning gains, as measured by the concept inventory post-test scores. However, focus group interviews indicated students from the experimental group that used both concept maps and creative exercises were able to provide more sophisticated scientific explanations for conceptual questions related to the topics of chemical equilibrium and acid-base chemistry. Implications of these research results, best practices for implementation of the two assessments, and future research are discussed.

In an effort to improve student conceptual understanding and help students better connect pre-existing knowledge to new ideas, a concept map assignment was implemented in a first-year college level general chemistry course. This implementation included a quasi-experiment that was carried out in discussion group recitation sections within a third-quarter general chemistry course. Students enrolled in a single section of the course were divided into two groups in which a concept map treatment was compared to a control group that completed short journal entries. Comparison of a concept inventory post-test using an independent samples t-test indicates students in the concept map treatment appear to perform better than the students in the journal control group (t = 2.34, mean difference = 0.844, p < 0.05). However, a multi-variable regression analysis in which the concept inventory post-test scores were compared between the treatment and control groups, while traits related to incoming academic preparation were held constant, suggests there was no significant difference in performance (unstandardized b = 0.222, p = 0.540). The quality of the students' concept maps was also evaluated and correlated to student performance on the concept inventory, and it appears students who were better at concept mapping made greater gains in conceptual understanding (Pearson's r = 0.295, p < 0.05). When the relationship between the quality of concept mapping and concept inventory post-test was determined while holding constant covariates related to incoming academic preparation, the unstandardized B coefficient was positive, but was not significant at the p = 0.05 level (unstandardized b = 0.215, p = 0.134) This study does not provide unequivocal evidence that a concept map treatment leads to greater gains in conceptual understanding compared to a control population, or that students with better concept mapping skills performed better on the concept inventory instrument. Nevertheless, a template for implementing a concept map assignment in a large enrollment course is provided, and the results presented herein might prompt chemistry instructors to consider including concept map assignments in their instructional toolbox.

Disulfide heterogeneity and other non-native crosslinks introduced during therapeutic antibody production and storage could have considerable negative effects on clinical efficacy, but tracking these modifications remains challenging. Analysis must also be carried out cautiously to avoid introduction of disulfide scrambling or reduction, necessitating the use of low pH digestion with less specific proteases. Herein we demonstrate that 213 nm ultraviolet photodissociation streamlines disulfide elucidation through bond-selective dissociation of sulfur-sulfur and carbon-sulfur bonds in combination with less specific backbone dissociation. Importantly, both types of fragmentation can be initiated in a single MS/MS activation stage. In addition to disulfide mapping, it is also shown that thioethers and trisulfides can be identified by characteristic fragmentation patterns. The photochemistry resulting from 213 nm excitation facilitates a simplified, two-tiered data processing approach that allows observation of all native disulfide bonds, scrambled disulfide bonds, and non-native sulfur-based linkages in a pepsin digest of Rituximab. Native disulfides represented the majority of bonds according to ion count, but the highly solvent-exposed heavy/light interchain disulfides were found to be most prone to modification. Production and storage methods that facilitate non-native links are discussed. Due to the importance of heavy and light chain connectivity for antibody structure and function, this region likely requires particular attention in terms of its influence on maintaining structural fidelity.

Ultraviolet photodissociation or UVPD is an increasingly popular option for tandem-mass spectrometry experiments. UVPD can be carried out at many wavelengths, and it is important to understand how the results will be impacted by this choice. Here, we explore the utility of 213 nm photons for initiating bond-selective fragmentation. It is found that bonds previously determined to be labile at 266 nm, including carbon-iodine and sulfur-sulfur bonds, can also be cleaved with high selectivity at 213 nm. In addition, many carbon-sulfur bonds that are not subject to direct dissociation at 266 nm can be selectively fragmented at 213 nm. This capability can be used to site-specifically create alaninyl radicals that direct backbone dissociation at the radical site, creating diagnostic d-ions. Furthermore, the additional carbon-sulfur bond fragmentation capability leads to signature triplets for fragmentation of disulfide bonds. Absorption of amide bonds can enhance dissociation of nearby labile carbon-sulfur bonds and can be used for stochastic backbone fragmentation typical of UVPD experiments at shorter wavelengths. Several potential applications of the bond-selective fragmentation chemistry observed at 213 nm are discussed. Graphical Abstract ᅟ.