Disulfide bonds are important structural moieties of proteins: they ensure proper folding, provide stability, and ensure proper function. With the increasing use of proteins for biotherapeutics, particularly monoclonal antibodies, which are highly disulfide bonded, it is now important to confirm the correct disulfide bond connectivity and to verify the presence, or absence, of disulfide bond variants in the protein therapeutics. These studies help to ensure safety and efficacy. Hence, disulfide bonds are among the critical quality attributes of proteins that have to be monitored closely during the development of biotherapeutics. However, disulfide bond analysis is challenging because of the complexity of the biomolecules. Mass spectrometry (MS) has been the go-to analytical tool for the characterization of such complex biomolecules, and several methods have been reported to meet the challenging task of mapping disulfide bonds in proteins. In this review, we describe the relevant, recent MS-based techniques and provide important considerations needed for efficient disulfide bond analysis in proteins. The review focuses on methods for proper sample preparation, fragmentation techniques for disulfide bond analysis, recent disulfide bond mapping methods based on the fragmentation techniques, and automated algorithms designed for rapid analysis of disulfide bonds from liquid chromatography-MS/MS data. Researchers involved in method development for protein characterization can use the information herein to facilitate development of new MS-based methods for protein disulfide bond analysis. In addition, individuals characterizing biotherapeutics, especially by disulfide bond mapping in antibodies, can use this review to choose the best strategies for disulfide bond assignment of their biologic products. Graphical Abstract This review, describing characterization methods for disulfide bonds in proteins, focuses on three critical components: sample preparation, mass spectrometry data, and software tools.

SynopsisDeuterated analogs of polyvinyl chloride (PVC) have been prepared and their infrared spectra analyzed as an aid to the more detailed assignment of bands in the spectrum of PVC. The following deuterated polymcrs were studied: PVC-adl, PVC-fldl, PVC-mod2, and PVCd,. These, as well as PVC, were polymerized in urea-complex, at -78"C., and a t +50°C. in order to determine which bands are of crystalline origin and whi-h of noncrystalline origin. Polarized spectra of oriented samples were obtained in each case to assist in the analysis. As a result of this study several of the previously uncertain assignments have been clarified. With the help of other studies on model chlorinecontaining compounds i t has been possible to identify the various conformations present in the polymer and to associate these with isotactic and syndiotactic pair configurations in the chain. A method is suggested for determining the proportion of each type of pair configuration in PVC. \ 2632 KRIMM, FOLT, SHIPMAN, BERENS r -

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HE important observation, made by Katz ( 6 ) in 1925 using T x-ray diffraction technique, showed that the mere stretching of crude or soft vulcanized Hevea rubber caused a t least a portion of the polymer to undergo crystallization. This and the subsequent experiments of Hock ( 4 ) emphasized the point that ~uccesaful attempts to synthesize a polymer resembling Hevea rubber mould have to take regularity into account. The situation is complicated in isoprene polymers by the variety of ways the monomer can enter the growing polymer chain.The four isomeric forms of polyisoprene are shown in Figure 1.Since head-to-tail orientations must be considered because of the asymmetry of the isoprene molecule, there are eight possible arrangements of the units that can occur in polymerized isoprene. Sodium, potassium, emulsion, and alfin polyisoprenes have been shown to contain all four forms of the repeating units (8). All of these uncured polymers have poor tack and their pure gum vulcanizates shorn poor physical properties. They do not crystallize when stretched or cooled. It has long been recognized that polymers of conjugated hydrocarbon dienes would have to be very regular in structure before their properties would approach those of Hevea rubber.Looking back over the last 15 to 20 years, there has been a elow development, almost iniperceptible at first, of catalyst systems m-hich produce polymers that in some degree shox-an oriented structure. This orientation could result if the monomer, during the propagation phase of the polymerization, is held in a definite position with respect to the growing chain, as by cybotactic forces where polymerization is initiated a t the melting point of the monomer or as by adsorption of the monomer onto a solid surface-perhaps to the surface of the catalyst itself.The woik reported here deals primarily mith the properties of a rubber made by directed polymerization. POLYMER STRUCTURE AND PROPERTIESCatalyst. Catalyst systems, based on polyolefin information purchased from Karl Ziegler, hare been applied to isoprene mononler systems. and modifications have been developed such that either cis-1,4-polyisoprene or trans-l,4-polyisoprene can now be prepared at n 111 Infrared Absorption Spectra. The effect of one of these catalyst modifications was first recognized when a modified olefin polymerization yielded an isolable unstabilized rubbery component n~liich showed an infrared absorption spectrum (2-to 25micron range) almost identical to that of Hevea rubber except for peaks due to oxidation at 2.8, 5.6, and 5.8 microns and, additionally, a slightly higher intensity in the peak at 11.25 microns. Subsequent samples of this rubber, a cis-1,4-poly-I The B. F. Goodrich Go., Tire and Equipment Division, Akron, Ohio.isoprene, now called Ameripol SN, when protected by age resistors did not show these oxidation peaks (Figure 2).Infrared absorption spectra indicate low ma,ximum concentrations of 1,2-and 3,4-addition products in Bmeripol SN. For these two isomeric units the discrimination in the presen...

Developing electronic doping of colloidal quantum dots is important for basic science and technology. In this article, the doping of colloidal CdSe quantum dots with gallium atoms is reported. Gallium doping of CdSe quantum dots produces important changes in electronic and optical properties of the material. The gallium doping shows a significant impact on the growth kinetics of quantum dots, which reveals important clues about the mechanism of the gallium dopant incorporation into the CdSe. The results show that the gallium doping significantly impacts the conductivity of CdSe thin film made of the quantum dots as well as the photoluminescence and chemical reactivity of the quantum dots, in agreement with the expected ntype character.

Recombinantly expressed proteins are susceptible to oxidation during expression, purification, storage, and analysis; the residue most susceptible to oxidation is methionine. Methionine oxidation can be overestimated using current quantitative analysis methods because oxidation can occur during sample preparation, and researchers often do not use methods that account for this possibility. An experimental strategy had been developed previously to solve this problem through the use of an O-labeled hydrogen peroxide reagent. However, the method did not address the analysis of peptides that contained multiple methionine residues. Herein, we develop and validate a new analysis method that uses theoretical isotope distributions and experimental spectra to quantify methionine oxidation that is present prior to sample preparation. The newly described approach is more rapid than the previously described method, and it needs only half the amount of protein for analysis. This method was validated using model proteins; then, it was applied to the analysis of recombinant HIV-1 Env, the key protein in HIV vaccine candidates. While Met oxidation of this protein could not be analyzed using previous methods, the approach described herein was useful for determining the oxidation state of HIV-Env. Graphical Abstract ᅟ.

Epithelial-adipose interaction is an integral step in breast cancer cell invasion and progression towards lethal metastatic disease. Understanding the physiological contribution of obesity, a major contributor to breast cancer risk and negative prognosis in post-menopausal patients, on cancer cell invasion requires detailed co-culture constructs that reflect mammary microarchitecture. Using laser direct-write, a laser-based CAD/CAM bioprinting technique, we have demonstrated the ability to construct breast cancer cell-laden hydrogel microbeads into spatially defined patterns in hydrogel matrices containing differentiated adipocytes. Z-stack imaging confirmed the three-dimensional nature of the constructs, as well as incorporation of cancer cell-laden microbeads into the adipose matrix. Preliminary data was gathered to support the construct as a potential model for breast cancer cell invasion into adipose tissue. MCF-7 and MDA-MB-231 breast cancer cell invasion was tracked over 2 weeks in an optically transparent hydrogel scaffold in the presence of differentiated adipocytes obtained from normal weight or obese patient tissue. Our model successfully integrates adipocytes and gives us the potential to study cellular and tissue-level interactions towards the early detection of cancer cell invasion into adipose tissue.

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