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Predicting the fragmentation patterns of proteins would be beneficial for the reliable identification of intact proteins by mass spectrometry. However, the ability to accurately make such predictions remains elusive. An approach to predict the specific cleavage sites in whole proteins resulting from collision-induced dissociation by use of an improved electrostatic model for calculating the proton configurations of highly-charged protein ions is reported. Using ubiquitin, cytochrome c, lysozyme and β-lactoglobulin as prototypical proteins, this approach can be used to predict the fragmentation patterns of intact proteins. For sufficiently highly charged proteins, specific cleavages occur near the first low-basicity amino acid residues that are protonated with increasing charge state. Hybrid QM/QM' (QM=quantum mechanics) and molecular dynamics (MD) simulations and energy-resolved collision-induced dissociation measurements indicated that the barrier to the specific dissociation of the protonated amide backbone bond is significantly lower than competitive charge remote fragmentation. Unlike highly charged peptides, the protons at low-basicity sites in highly charged protein ions can be confined to a limited sequence of low-basicity amino acid residues by electrostatic repulsion, which results in highly specific fragmentation near the site of protonation. This research suggests that the optimal charge states to form specific sequence ions of intact proteins in higher abundances than the use of less specific ion dissociation methods can be predicted a priori.

Because of the large abundance of sodium (Na) as a source material and the easy fabrication of Na-containing compounds, the sodium (Na) battery is a more environmentally friendly and sustainable technology than the lithium-ion battery (LIB). Na-metal batteries (SMBs) are considered promising to realize a high energy density to overtake the cost effectiveness of LIBs, which is critically important in large-scale applications such as grid energy storage. However, the cycling stability of the Na-metal anode faces significant challenges particularly under high cycling capacities, due to the complex failure models caused by the formation of Na dendrites. Here, a universal surface strategy, based on a self-regulating alloy interface of the current collector, to inhibit the formation of Na dendrites is reported. High-capacity (10 mAh cm-2 ) Na-metal anodes can achieve stable cycling for over 1000 h with a low overpotential of 35 mV. When paired with a high-capacity Na3 V2 (PO4 )2 F3 cathode (7 mAh cm-2 ), the SMB delivers an unprecedented energy density (calculated based on all the cell components) over 200 Wh kg-1 with flooded electrolyte, or over 230 Wh kg-1 with lean electrolyte. The dendrite-free SMB also shows high cycling stability with a capacity retention per cycle over 99.9% and an ultrahigh energy efficiency of 95.8%.

Rationale Under certain conditions some amino acid (AA) residues undergo special reactions in the gas phase, generating characteristic neutral losses and product ions. Taking these special fragments into account and understanding the effect of AA residues on peptide cleavages will consummate database search algorithms and manual data interpretation in peptide sequencing by mass spectrometry (MS). In this study, the details of the characteristic NH3 and CO losses of glutamine (Gln) residues located at the C-terminus of peptides are presented. Methods A number of selected peptides were fragmented under collision-induced dissociation (CID) in electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-QTOF-MS). Density functional theory (DFT) quantum mechanical calculations at the B3LYP/6-31+G(d,p) level were carried out to optimize the geometry of peptide ions and provide energy barriers of ions in each step during fragmentations. Results Two characteristic peaks appear near the precursor ions of sodiated Gln C-terminated peptides, suggesting the loss of neutral NH3 and CO via a two-step process. The proposed mechanism of their formation is as follows: after losing NH3 , a non-classical bn * ion is formed with a glutaric anhydride structure that further dissociates to lose CO. The sodiated peptides show more intensive peaks corresponding to the loss of neutral molecules than the protonated ones. This type of neutral loss can also occur at the Gln residue that is rearranged to the C-terminus of sodiated peptides. Conclusions The experiments and calculations suggest that the two-step characteristic NH3 and CO loss of sodiated peptides is energetically favored, and can be applied to identify C-terminated Gln residues. This study provides a mechanistic insight into the role of sodium ion during peptide fragmentation. Copyright © 2017 John Wiley & Sons, Ltd.

A characteristic neutral loss of 44 Da is observed in the MS/MS spectra of Thr-containing sodiated peptides. A combination of tandem mass spectrometry and quantum chemical calculations calculated at the B3LYP/6-311G (d, p) level of ab initio theory is used to elucidate this fragmentation pathway. The high resolution mass spectrometry data indicate this neutral loss is acetaldehyde lost from the side chain of Thr rather than CO2. The intensity of this neutral loss can be enhanced when Thr residue is far from the C-terminus and when the C-terminus is esterified as well. The mechanism of the acetaldehyde loss is proposed to adopt a McLafferty-type rearrangement reaction, which involves a proton transfer from the hydroxyl of Thr side chain to its C-terminal neighboring carbonyl oxygen inducing the cleavage of the Ca–Cβ bond. This mechanism is further supported by examining the fragmentation of a [GT(tBu)G + Na]+ peptide derivative and by comparing the product ion spectra of [M + Na-44]+ of [GTGA + Na]+ with [M + Na]+ of [GGGA + Na]+. A similar neutral loss of HCHO can also be detected in Ser-containing peptides. Our computational results reveal that the most stable [GTG + Na]+ ion is present as a tridentate charge-solvated structure and the dissociation leading to the 44 loss is dynamically and energetically favorable. Copyright © 2015 John Wiley & Sons, Ltd.

A good understanding of gas-phase fragmentation chemistry of peptides is important for accurate protein identification. Additional product ions obtained by sodiated peptides can provide useful sequence information supplementary to protonated peptides and improve protein identification. In this work, we first demonstrate that the sodiated a3 ions are abundant in the tandem mass spectra of sodium-cationized peptides although observations of a3 ions have rarely been reported in protonated peptides. Quantum chemical calculations combined with tandem mass spectrometry are used to investigate this phenomenon by using a model tetrapeptide GGAG. Our results reveal that the most stable [a3 + Na − H]+ ion is present as a bidentate linear structure in which the sodium cation coordinates to the two backbone carbonyl oxygen atoms. Due to structural inflexibility, further fragmentation of the [a3 + Na − H]+ ion needs to overcome several relatively high energetic barriers to form [b2 + Na − H]+ ion with a diketopiperazine structure. As a result, low abundance of [b2 + Na − H]+ ion is detected at relatively high collision energy. In addition, our computational data also indicate that the common oxazolone pathway to generate [b2 + Na − H]+ from the [a3 + Na − H]+ ion is unlikely. The present work provides a mechanistic insight into how a sodium ion affects the fragmentation behaviors of peptides. Copyright © 2015 John Wiley & Sons, Ltd.

Tumor-associated proteases (TAPs), such as legumain, are actively involved in cancer progression; they have been used as biomarkers for diagnosis, prognosis, and drug targeting. As a result, in-vivo detection and trafficking of TAPs have attracted a great deal of attention. TAP-specific probes for in-vivo imaging, however, remain rare. A TAP-responsive hybrid nanoprobe system based on quantum dots (QD) and the fluorescence resonance energy transfer (FRET) effect is presented for the detection of legumain (asparaginyl endopeptidase), which is overexpressed in many tumors. A novel hybrid construction method is developed for fabricating the nanoprobe, by which the strong heparin–protamine affinity is used for conjugation. The hybrid comprises two components: 1) low-molecular-weight heparin (LH)-modified QD, and 2) low-molecular-weight protamine (LMWP)-conjugated fluorescence quencher QSY21, through a legumain-cleavable linker. The hybrid nanoprobe (i.e., a FRET system) is self-assembled via the LH–LMWP affinity. The linker between LMWP and QSY21 is selectively cleaved by legumain, leading to QSY21 detachment and fluorescence recovery in the tumor. In-vivo imaging is successfully achieved in the colon tumor mouse model. Importantly, such a hybrid nanoprobe system is adaptable for the detection of other TAPs (e.g., matrix metalloproteinase -2) by using an established, corresponding substrate–peptide linker, thereby offering a universal platform for TAP detection and tumor imaging.

The tetra-peptide AVPI, derived from the Smac/DIABLO N-terminal epitope, is able to trigger caspase activation and apoptotic process. However, its clinical value is greatly hampered by the nature of membrane-impermeability. Herein, the cell-penetrating chimeric apoptotic peptide of AVPIR8 is synthesized, of which the apoptosis-induced AVPI is strategically blended with the cell-penetrating sequence of octaarginine (R8). The dual-functionalized AVPIR8 is not only potent in inducing apoptosis in tumor cells due to the cell penetration ability, but also is able to work as gene carrier for transfering the tumor suppressor p53 DNA into cells, thus constructing a co-delivery drug system (AVPIR8/p53). Such system efficiently promotes apoptosis in cancer cells while sparing normal cells, and its antitumor activity is further significantly enhanced in combination with doxorubicin as cocktail therapy. More importantly, the anticancer efficacy of the cocktail is demonstrated to be able to arrest tumor growth in two animal tumor models (melanoma and cervical cancers), respectively. The chemotherapeutic dose in the AVPIR8/p53-based cocktail is significantly reduced by 80%, compared to the monotherapy of doxorubicin. The present results show the promise of the co-delivered AVPIR8/p53 as adjuvant therapy for boosting the conventional chemotherapeutics, with a unique benefit of enhanced productive treatment outcomes yet greatly reduced adverse toxicity.

Peptide fragments such as b and y sequence ions generated upon low-energy collision-induced dissociation have been routinely used for tandem mass spectrometry (MS/MS)-based peptide/protein identification. The underlying formation mechanisms have been studied extensively and described within the literature. As a result, the 'mobile proton model' and 'pathways in competition model' have been built to interpret a majority of peptide fragmentation behavior. However, unusual peptide fragments which involve unfamiliar fragmentation pathways or various rearrangement reactions occasionally appear in MS/MS spectra, resulting in confused MS/MS interpretations. In this work, a series of unfamiliar c ions are detected in MS/MS spectra of the model peptides having an N-terminal Arg or deuterohemin group upon low-energy collision-induced dissociation process. Both the protonated Arg and deuterohemin group play an important role in retention of a positive charge at the N-terminus that is remote from the cleavage sites. According to previous reports and our studies involving amino acid substitutions and hydrogen-deuterium exchange, we propose a McLafferty-type rearrangement via charge-remote fragmentation as the potential mechanism to explain the formation of c ions from precursor peptide ions or unconventional b ions. Density functional theory calculations are also employed in order to elucidate the proposed fragmentation mechanisms. Copyright © 2016 John Wiley & Sons, Ltd.

To isolate genes with different expression levels in human esophageal SCC, SSH and reverse Northern were performed between cancer tissue and its normal counterpart. Among the differentially expressed genes identified, we report here cDNA corresponding to a 0.88 kb mRNA (NMES1), whose expression was observed in all 36 adjacent normal esophageal mucosae, while 31 (86%) matched cancer tissues showed a marked reduction or complete lack of its expression. Sequence analysis of its full-length cDNA revealed a gene encoding a predicted polypeptide of 9 kDa. Northern blot showed that NMES1 was distributed mainly in the alimentary tract. The gene was mapped to 15q21.1 by screening the GenBridge 4 RH panel. Immunohistochemistry confirmed the downregulation of NMES1 in esophageal SCC at the protein level and showed that it is a nuclear protein. In situ hybridization revealed its different expression during mouse embryonic development, especially in bone, brain, stomach and intestine. The negative correlation of NMES1 expression with esophageal oncogenesis suggests its suppressive role in tumorigenesis of the esophagus, while the precise function of NMES1 still needs further investigation. © 2002 Wiley-Liss, Inc.