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Here we report the first example of alkyne trifunctionalization through simultaneous construction of C-C, C-O, and C-N bonds via gold catalysis. With the assistance of a γ-keto directing group, sequential gold-catalyzed alkyne hydration, vinyl-gold nucleophilic addition, and gold(III) reductive elimination were achieved in one pot. Diazonium salts were identified as both electrophiles (N source) and oxidants (C source). Vinyl-gold(III) intermediates were revealed as effective nucleophiles toward diazonium, facilitating nucleophilic addition and reductive elimination with high efficiency. The rather comprehensive reaction sequence was achieved with excellent yields (up to 95%) and broad scope (>50 examples) under mild conditions (room temperature or 40 °C).
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Alquinos/química , Oro/química , Compuestos de Vinilo/química , Catálisis , Compuestos de Diazonio/química , Oxidación-Reducción , Teoría Cuántica , TemperaturaRESUMEN
Recently, microdroplet reactions have aroused much interest because the microdroplet provides a unique medium where organic reactions could be accelerated by a factor of 103 or more. However, microdroplet reactions of proteins have been rarely studied. We report the occurrence of multiple-step reactions of a large protein, specifically, the digestion, reduction, and deglycosylation of an intact antibody, which can take place in microseconds with high reaction yields in aqueous microdroplets at room temperature. As a result, fast structural characterization of a monoclonal antibody, essential for assessing its quality as a therapeutic drug, can be enabled. We found that the IgG1 antibody can be digested completely by the IdeS protease in aqueous microdroplets in 250 microseconds, a 7.5 million-fold improvement in speed in comparison to traditional digestion in bulk solution (>30 min). Strikingly, inclusion of the reductant tris(2-carboxyethyl)phosphine in the spray solution caused simultaneous antibody digestion and disulfide bond reduction. Digested and reduced antibody fragments were either collected or analyzed online by mass spectrometry. Further addition of PNGase F glycosylase into the spray solution led to antibody deglycosylation, thereby producing reduced and deglycosylated fragments of analytical importance. In addition, glycated fragments of IgG1 derived from glucose modification were identified rapidly with this ultrafast digestion/reduction technique. We suggest that microdroplets can serve as powerful microreactors for both exploring large-molecule reactions and speeding their structural analyses.
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Anticuerpos Monoclonales , Inmunoglobulina G , Espectrometría de MasasRESUMEN
Accurate quantification is essential in the fields of proteomics, clinical assay, and biomarker discovery. Popular methods for absolute protein quantitation by mass spectrometry (MS) involve the digestion of target protein and employ isotope-labeled peptide internal standards to quantify chosen surrogate peptides. Although these methods have gained success, syntheses of isotope-labeled peptides are time-consuming and costly. To eliminate the need for using standards or calibration curves, herein we present a coulometric mass spectrometric (CMS) approach for absolute protein quantitation, based on the electrochemical oxidation of a surrogate peptide combined with mass spectrometric measurement of the oxidation yield. To demonstrate the utility of this method, several proteins were analyzed such as model proteins ß-casein, and apomyoglobin as well as circadian clock protein KaiB isolated from Escherichia coli. In our experiment, tyrosine-containing peptides were selected as surrogate peptides for quantitation, considering the oxidizable nature of tyrosine. Our data showed that the results for surrogate peptide quantity measured by our method and by traditional isotope dilution method are in excellent agreement, with the discrepancy of 0.3-3%, validating our CMS method for absolute quantitation. Furthermore, therapeutic monoclonal antibody (mAb) could be quantified by our method as well. Due to the high specificity and sensitivity of MS and no need to use isotope-labeled peptide standards, our CMS method would be of high value for the absolute proteomic quantification.
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Apoproteínas/análisis , Caseínas/análisis , Proteínas de Escherichia coli/análisis , Mioglobina/análisis , Proteínas Circadianas Period/análisis , Animales , Bovinos , Técnicas Electroquímicas , Escherichia coli/química , Caballos , Espectrometría de Masas , Oxidación-ReducciónRESUMEN
Gold-catalyzed alkyne and allene diselenations were developed. Excellent regioselectivity (trans) and good to excellent yields were achieved (up to 98 % with 2 % catalyst loading) with a wide range of substrates. Mechanistic investigation revealed the formation of a vinyl gold(I) intermediate followed by an intermolecular selenium cation migration, suggesting that a gold(I/III) redox process was successfully implemented under mild conditions.
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Due to the high oxidation potential between AuI and AuIII , gold redox catalysis requires at least stoichiometric amounts of a strong oxidant. We herein report the first example of an electrochemical approach in promoting gold-catalyzed oxidative coupling of terminal alkynes. Oxidation of AuI to AuIII was successfully achieved through anode oxidation, which enabled facile access to either symmetrical or unsymmetrical conjugated diynes through homo-coupling or cross-coupling. This report extends the reaction scope of this transformation to substrates that are not compatible with strong chemical oxidants and potentiates the versatility of gold redox chemistry through the utilization of electrochemical oxidative conditions.
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Gold-catalyzed alkyne hydration was studied by using in situ reacting mass spectrometry (MS) technology. By monitoring the reaction process in solution under different conditions (regular and very diluted catalyst concentrations, different pH values) and examining the reaction occurrence in the early reaction stage (1-2â ms after mixing) with MS, we collected a series of experimental evidence to support that the bis-gold complex is a potential key reaction intermediate. Furthermore, both experimental and computational studies confirmed that the σ,π-bis-gold complexes are not active intermediates toward nucleophilic addition. Instead, formation of geminally diaurated complex C is crucial for this catalytic process.
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P-glycoprotein (Pgp) is a prototypical ATP-binding cassette (ABC) transporter of great biological and clinical significance.Pgp confers cancer multidrug resistance and mediates the bioavailability and pharmacokinetics of many drugs (Juliano and Ling, 1976; Ueda et al., 1986; Sharom, 2011). Decades of structural and biochemical studies have provided insights into how Pgp binds diverse compounds (Loo and Clarke, 2000; Loo et al., 2009; Aller et al., 2009; Alam et al., 2019; Nosol et al., 2020; Chufan et al., 2015), but how they are translocated through the membrane has remained elusive. Here, we covalently attached a cyclic substrate to discrete sites of Pgp and determined multiple complex structures in inward- and outward-facing states by cryoEM. In conjunction with molecular dynamics simulations, our structures trace the substrate passage across the membrane and identify conformational changes in transmembrane helix 1 (TM1) as regulators of substrate transport. In mid-transport conformations, TM1 breaks at glycine 72. Mutation of this residue significantly impairs drug transport of Pgp in vivo, corroborating the importance of its regulatory role. Importantly, our data suggest that the cyclic substrate can exit Pgp without the requirement of a wide-open outward-facing conformation, diverting from the common efflux model for Pgp and other ABC exporters. The substrate transport mechanism of Pgp revealed here pinpoints critical targets for future drug discovery studies of this medically relevant system.
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Miembro 1 de la Subfamilia B de Casetes de Unión a ATP , Translocación Genética , Humanos , Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP , MutaciónRESUMEN
Isotope-labeled internal standards are routinely used for mass spectrometry (MS)-based absolute quantitation. However, syntheses of isotope-labeled peptides are time-consuming and costly. To tackle this issue, we recently developed a coulometric mass spectrometric (CMS) approach for absolute quantitation without the use of standards, based on the electrochemical oxidation of cysteine or tyrosine-containing peptides followed by mass spectrometric measurement of the oxidation yield. To further expand the utility of this method, herein we present the CMS method for absolute quantitation of peptides based on tryptophan electrochemical oxidation. Several tryptophan-containing peptides, such as WGG, WQPPRARI, WAGGDASGE, RTRPLWVRME, and KVPRNQDWL, were successfully quantified with a quantification error ranging from -4.5 to +4.3%. Furthermore, this quantitation approach is also applicable to protein, in which protein can be digested and a surrogate peptide can be selected for quantification to reflect the amount of the parent protein, as exemplified by CMS analysis of peptide GITWK from cytochrome c. The CMS result agreed well with the traditional isotope dilution method, with only a small difference of 3.5%. In addition, CMS was used to successfully quantify amyloid beta (Aß) peptide fragments (up to 28 amino acid residues) based on tyrosine oxidation. The validity of the CMS method for peptide and protein absolute quantitation without using isotope-labeled peptide standards would greatly facilitate proteomics research.
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Péptidos beta-Amiloides/análisis , Péptidos beta-Amiloides/química , Espectrometría de Masas/métodos , Proteómica/métodos , Triptófano/análisis , Oxidación-Reducción , Fragmentos de Péptidos/análisis , Fragmentos de Péptidos/químicaRESUMEN
Conventional mass spectrometry (MS)-based analytical methods for small carbohydrate fragments (oligosaccharides, degree of polymerization 2-12) are time-consuming due to the need for an offline sample pretreatment such as desalting. Herein, we report a new paper spray ionization method, named desalting paper spray (DPS), which employs a piece of triangular filter paper for both sample desalting and ionization. Unlike regular paper spray ionization (PSI) and nanoelectrospray ionization (nanoESI), DPS-MS allows fast and sensitive detection of oligosaccharides in biological samples having complex matrices (e.g., Tris, PBS, HEPES buffers, or urine). When an oligosaccharide sample is loaded onto the filter paper substrate (10 × 5 mm, height × base) made mostly of cellulose, oligosaccharides are adsorbed on the paper via hydrophilic interactions with cellulose. Salts and buffers can be washed away using an ACN/H2O (90/10 v/v) solution, while oligosaccharides can be eluted from the paper using a solution of ACN/H2O/formic acid (FA) (10/90/1 v/v/v) and directly spray-ionized from the tip of the paper. Various saccharides at trace levels (e.g., 50 fmol) in nonvolatile buffer can be quickly analyzed by DPS-MS (<5 min per sample). DPS-MS is also applicable for direct detection of oligosaccharides from glycosyltransferase (GT) reactions, a challenging task that typically requires a radioactive assay. Quantitative analysis of acceptor and product oligosaccharides shows increased product with increased GT enzymes used for the reaction, a result in line with the radioactivity assay. This work suggests that DPS-MS has potential for rapid oligosaccharide analysis from biological samples.
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Espectrometría de Masas/instrumentación , Oligosacáridos/análisis , Papel , Límite de Detección , Espectrometría de Masas/economía , Sales (Química)/aislamiento & purificación , Factores de TiempoRESUMEN
A synergistic gold/iron catalytic system was developed for sequential alkyne hydration and vinyl gold addition to aldehydes or ketones. Fe(acac)3 was identified as an essential co-catalyst in preventing vinyl gold protodeauration and facilitating nucleophilic additions. Effective C-C bond formation was achieved under mild conditions (r.t.) with excellent regioselectivity and high efficiency (1% [Au], up to 95% yields). Extending reaction scope to intramolecular fashion achieved successful macrocyclization (16-31 ring sizes) with excellent yields (up to 90%, gram-scale) without extended dilution (0.2 M), which highlighted the great potential of this new crossed-aldol strategy in challenging target molecule synthesis.
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Quantitation methods for peptides using mass spectrometry have advanced rapidly. These methods rely on using standard and/or isotope-labeled peptides, which might be difficult or expensive to synthesize. To tackle this challenge, we present a new approach for absolute quantitation without the use of standards or calibration curves based on coulometry combined with mass spectrometry (MS). In this approach, which we call coulometric mass spectrometry (CMS), the mass spectrum of a target peptide containing one or more tyrosine residues is recorded before and after undergoing electrochemical oxidation. We record the total integrated oxidation current from the electrochemical measurement, which according to the Faraday's Law of coulometry, provides the number of moles of oxidized peptide. The ion intensity ratio of the target peptide before and after oxidation provides an excellent estimate of the fraction of the peptide that has been oxidized, from which the total amount of peptide is calculated. The striking strength of CMS is that it needs no standard peptide, but CMS does require the peptide to contain a known number of oxidizable groups. To illustrate the power of this method, we analyzed various tyrosine-containing peptides such as GGYR, DRVY, oxytocin, [Arg8]-vasotocin and angiotensinogen 1-14 with a quantification error ranging from - 7.5 to + 2.4%. This approach is also applicable to quantifying phosphopeptides and could be useful in proteomics research.
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Técnicas Electroquímicas/métodos , Espectrometría de Masas/métodos , Péptidos/análisis , Péptidos/química , Oxidación-Reducción , Tirosina/análisis , Tirosina/químicaRESUMEN
Mass spectrometry-based quantification method has advanced rapidly. In general, the methods for accurate quantification rely on the use of authentic target compounds or isotope-labeled compounds as standards, which might be not available or difficult to synthesize. To tackle this grand challenge, this paper presents a novel approach, based on electrochemistry (EC) combined with mass spectrometry (MS). In this approach, a target compound is allowed to undergo electrochemical oxidation and then subject to MS analysis. The oxidation current recorded from electrochemistry (EC) measurement provides information about the amount of the oxidized analyte, based on the Faraday's Law. On the other hand, the oxidation reaction yield can be determined from the analyte MS signal changes upon electrolysis. Therefore, the total amount of analyte can be determined. In combination with liquid chromatography (LC), the method can be applicable to mixture analysis. The striking strength of such a method for quantitation is that neither standard compound nor calibration curve is required. Various analyte molecules such as dopamine, norepinephrine, and rutin as well as peptide glutathione in low quantity were successfully quantified using our method with the quantification error ranging from - 2.6 to + 4.6%. Analyte in a complicated matrix (e.g., uric acid in urine) was also accurately measured. Graphical Abstract.
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Elucidation of ion dissociation patterns is particularly important to structural analysis by mass spectrometry (MS). However, typically, only the charged fragments from an ion dissociation event are detected in tandem MS experiments; neutrals are not identified. In recent years, we have developed an atmospheric pressure thermal dissociation (APTD) technique that can be applied to dissociate ions at atmosphere pressure and thus provide one way to characterize neutral fragments. In this paper, we focus on the detection of neutral CO resulting from amino acid and peptide ion dissociation. In the first set of experiments, several protonated amino acids (e.g., + 1 ion of phenylalanine) were found to undergo loss of a neutral (s) of total mass 46 Da, a process leading to iminium ion formation. We successfully detected the neutral species CO by using a CO sensor, UV-Vis and MS analysis following selective CO trapping with a rhodium complex. The capture of CO from dissociation of protonated amino acids supports the assignment of the loss of 46 Da to neutral losses of CO and H2O, rather than loss of formaldehyde or dihydroxycarbene, other possible fragmentation pathways that have been subject of debate for a long time. In a second experiment, we used the APTD method in combination with the CO detection technique, to demonstrate the formation of CO in the conversion of b ions to a ions during peptide ion dissociations. These results showed the potential of APTD in the elucidation of ion dissociation mechanisms, using simple home-built apparatus. Graphical Abstract á .
RESUMEN
Ion dissociation is the usual basis for tandem MS analysis but a significant limitation is that only charged fragments from ion dissociation events are detected while neutral fragments are simply lost. This study reports our continued effort to solve this problem by developing atmospheric pressure neutral reionization mass spectrometry (APNR). In APNR, analyte ions are thermally dissociated (atmospheric pressure thermal dissociation, APTD) followed by soft reionization using electrosonic spray ionization (ESSI). Our results show that APNR is a powerful method for structural analysis of various biomolecules such as peptides, saccharides and nucleotides, as well as for elucidating unimolecular ion dissociation mechanisms. It was found that APNR provides extensive fragment ions including a series of y ions in peptides, which benefit sequencing and provide complementary information to collision induced dissociation (CID). In particular, direct cleavage of disulfide bonds of peptides occurs during APTD, facilitating peptide sequencing and disulfide bond mapping. In addition, many cross-ring cleavage fragments are detected during APNR analysis of oligosaccharides, indicating that the APTD dissociation process is energetic and potentially useful for identifying glycan linkage sites. Fragmentation patterns of oligosaccharide isomers can be used for their differentiation. Furthermore, in the cases of dissociation of nucleotides and synthetic naphthoylindole drugs, the putative neutral, phosphorylated riboses and indoles, were successfully detected using APNR, providing strong evidence to confirm previously proposed unimolecular ion dissociation mechanisms. We believe this APNR technique along with APTD should be of high value in structure determination of biomolecules.