RESUMEN
Microdroplet reactions have aroused much interest due to significant reaction acceleration (e.g., ultrafast protein digestion in microdroplets could occur in less than 1 ms). This study integrated a microdroplet protein digestion technique with automated sample flow injection and online mass spectrometry (MS) analysis, to develop a rapid and robust method for structural characterization of monoclonal antibodies (mAbs) that is essential to assess the antibody drug's safety and quality. Automated sequential aspiration and mixing of an antibody and an enzyme (IdeS or IgdE) enabled rapid analysis with high reproducibility (total analysis time: 2 min per sample; reproducibility: â¼2% coefficient of variation). Spraying the sample in ammonium acetate buffer (pH 7) using a jet stream source allowed efficient digestion of antibodies and efficient ionization of resulting antibody subunits under native-pH conditions. Importantly, it also provided a platform to directly study specific binding of an antibody and an antigen (e.g., detecting the complexes mAb/RSFV antigen and F(ab')2/RSVF in this study). Furthermore, subsequent tandem MS analysis of a resulting subunit from microdroplet digestion enabled localizing post-translational modifications on particular domains of a mAb in a rapid fashion. In combination with IdeS digestion of an antibody, additional tris(2-carboxyethyl)phosphine (TCEP) reduction and N-glycosidase F (PNGase F) deglycosylation reactions that facilitate antibody analysis could be realized in "one-pot" spraying. Interestingly, increased deglycosylation yield in microdroplets was found, simply by raising the sample temperature. We expect that our method would have a high impact for rapid characterization of monoclonal antibodies.
Asunto(s)
Anticuerpos Monoclonales , Espectrometría de Masas en Tándem , Reproducibilidad de los Resultados , Anticuerpos Monoclonales/química , Procesamiento Proteico-Postraduccional , Concentración de Iones de HidrógenoRESUMEN
Alkyl isothiocyanates (R-NCS) have pharmacological applications and provide a synthetic handle to various functional groups including thioureas. There are however few methods to access alkyl isothiocyanates through the creation of the C-N bond. We have developed a simple approach for the conjugate isothiocyanation of enones by trimethylsilyl isothiocyanate (TMSNCS), which proceeds through the 1,4-addition of the weak isothiocyanate nucleophile to activated enones in the absence of external promoters. This method avoids the direct use of highly toxic acids and bases, produces ß-isothiocyanato carbonyl products in yields of 87-98% under mild conditions (less than 6 hours at 0 °C), and displays wide functional group tolerance. Density functional theory calculations highlighted competing cationic and anionic mechanisms, where the enone activation by the TMSNCS reagent is accelerated in protic solvents. The selective formation of the isothiocyanate vs. thiocyanate isomers is explained by the thermodynamically-controlled nature of the reaction in which only the conjugate isothiocyanation is exergonic.
RESUMEN
Proteomic absolute quantitation strategies mainly rely on the use of synthetic stable isotope-labeled peptides or proteins as internal standards, which are highly costly and time-consuming to synthesize. To circumvent this limitation, we recently developed a coulometric mass spectrometry (CMS) approach for absolute quantitation of proteins without the use of standards, based on the electrochemical oxidation of oxidizable surrogate peptides, followed by mass spectrometry measurement of the peptide oxidation yield. Previously, CMS was only applied for single-protein quantitation. In this study, first, we demonstrated absolute quantitation of multiple proteins in a mixture (e.g., ß-lactoglobulin B, α-lactalbumin, and carbonic anhydrase) by CMS in one run, without using any standards. The CMS quantitation result was validated with a traditional isotope dilution method. Second, CMS can be used for absolute quantitation of a low-level target protein in a mixture; for instance, 500 ppm of PLBL2, a problematic host cell protein (HCP), in the presence of a highly abundant monoclonal antibody (mAb) was successfully quantified by CMS with no use of standards. Third, taking one step further, this study demonstrated the unprecedented quantitative analysis of deamidated peptide products arising from the mAb heavy chain deamidation reaction. In particular, absolute quantitation of the deamidation succinimide intermediate which had not been performed before due to the lack of standard was conducted by CMS, for the first time. Overall, our data suggest that CMS has potential utilities for quantitative proteomics and biotherapeutic drug discovery.
Asunto(s)
Péptidos , Proteómica , Anticuerpos Monoclonales , Espectrometría de Masas/métodos , Péptidos/química , Proteómica/métodos , Técnica de Dilución de RadioisótoposRESUMEN
The capture of reactive intermediates is important for the elucidation of reaction mechanisms. We report the first observation of electrochemically generated, short-lived radical cations of carbazole (t1/2 ≈ 97 µs) and two N-substituted carbazole derivatives by mass spectrometry. In addition, online investigation of the reactivity of electrochemically generated carbazole radical cations supports that the carbazole dimerization mechanism involves the reaction of one radical cation with one neutral molecule rather than the previously proposed coupling of two radical cations.
RESUMEN
In this study, we developed a novel simple fluorescence resonance-energy transfer (FRET) system between two-color CdTe quantum dots (QDs) assisted by cetyltrimethylammonium bromide (CTAB). Mercaptopropionic (MPA)-capped CdTe QDs serving as both donors and acceptors were successfully synthesized by changing the refluxing time in aqueous solution. CTAB micelles formed in water and minimized the distance between the donors and acceptors significantly by electrostatic interactions, improving FRET efficiency. Several factors that affected the fluorescence spectra of the FRET system were investigated. The prepared FRET system was feasible as an effective fluorescent probe to detect Hg(II) in aqueous solution. At pH 7.0, a linear relationship between the quenched fluorescence intensity of orange-emitting acceptors (QDs(A) ) and Hg(II) concentration was acquired in the range 5-250 nmol/L with a detection limit of 1.95 nmol/L. The developed method showed excellent analytical performance for Hg(II) with high sensitivity and acceptable selectivity, reproducibility and stability. This finding indicated that the method has a promising potential application for environmental monitoring. This study demonstrated the great promise of QDs for expedient, low-cost and high-sensitivity detection of Hg(II).
Asunto(s)
Compuestos de Cadmio/química , Cetrimonio/química , Color , Transferencia Resonante de Energía de Fluorescencia , Mercurio/análisis , Puntos Cuánticos/química , Telurio/químicaRESUMEN
Currently, glycopeptide quantitation is mainly based on relative quantitation due to absolute quantitation requiring isotope-labeled or standard glycopeptides which may not be commercially available or are very costly and time consuming to synthesize. To address this grand challenge, coulometric mass spectrometry (CMS), based on the combination of electrochemistry (EC) and mass spectrometry (MS), was utilized to quantify electrochemically active glycopeptides without the need of using standard materials. In this study, we studied tyrosine-containing glycopeptides, NYIVGQPSS(ß-GlcNAc)TGNL-OH and NYSVPSS(ß-GlcNAc)TGNL-OH, and successfully quantified them directly with CMS with a discrepancy of less than 5% between the CMS measured amount and the theoretical amount. Taking one step further, we applied this approach to quantify glycopeptides generated from the digestion of NIST mAb, a monoclonal antibody reference material. Through HILIC column separation, five N297 glycopeptides resulting from NIST mAb tryptic digestion were successfully separated and quantified by CMS for an absolute amount without the use of any standard materials. This study indicates the potential utility of CMS for quantitative proteomics research.
Asunto(s)
Glicopéptidos , Espectrometría de Masas , Oxidación-Reducción , Glicopéptidos/análisis , Glicopéptidos/química , Espectrometría de Masas/métodos , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/análisis , Proteómica/métodos , Proteómica/normas , Tirosina/análisis , Tirosina/química , Técnicas Electroquímicas/métodosRESUMEN
Photobase generators (PBGs) are compounds that utilize light-sensitive chemical-protecting groups to offer spatiotemporal control of releasing organic bases upon targeted light irradiation. PBGs can be implemented as an external control to initiate anionic polymerizations such as thiol-ene Michael addition reactions. However, there are limitations for common PBGs, including a short absorption wavelength and weak base release that lead to poor efficiency in photopolymerization. Therefore, there is a great need for visible-light-triggered PBGs that are capable of releasing strong bases efficiently. Here, we report two novel BODIPY-based visible-light-sensitive PBGs for light-induced activation of the thiol-ene Michael "click" reaction and polymerization. These PBGs were designed by connecting the BODIPY-based light-sensitive protecting group with tetramethylguanidine (TMG), a strong base. Moreover, we exploited the heavy atom effect to increase the efficiency of releasing TMG and the polymerization rate. These BODIPY-based PBGs exhibit extraordinary activity toward thiol-ene Michael addition-based polymerization, and they can be used in surface coating and polymer network formation of different thiol and vinyl monomers.
RESUMEN
Recent studies have shown that ultrafast enzymatic digestion of proteins can be achieved in microdroplet within 250 µs. Further investigation of peptides resulting from microdroplet digestion (MD) would be necessary to evaluate it as an alternative to the conventional bulk digestion for bottom-up and biotherapeutic protein characterization. Herein we examined and compared protein tryptic digestion in both MD and bulk solution. In the case of MD of ß-lactoglobulin B, the preservation of long peptides was observed due to the short digestion time. In addition, MD is applicable to digest both high- and low-abundance proteins in mixture. In the case of digesting NIST 8671 mAb antibody containing a low level of commonly encountered host cell protein (HCP) PLBL2 (mAb:PLBL2 = 100:1 by weight), MD produced lower levels of digestion-induced chemical modifications of asparagine/glutamine deamidation, compared with overnight digestion. No significant difference between MD and bulk digestion was observed in terms of trypsin digestion specificity based on examination of semi- and unspecific-cleaved peptides. Our study suggests that MD, a fast digestion approach, could be adopted for bottom-up proteomics research and for peptide mapping of mAbs to characterize site-specific deamidation and glycosylation, for the purpose of development of biopharmaceuticals.
Asunto(s)
Anticuerpos Monoclonales , Péptidos , Secuencia de Aminoácidos , Anticuerpos Monoclonales/química , Cromatografía Liquida/métodos , Péptidos/química , Proteolisis , Tripsina/químicaRESUMEN
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.