Chao-Cheng Wang

Labeling reactions applicable to chromatography and electrophoresis of minute amounts of proteins

Chromatography and electrophoresis have become extremely valuable and important methods for the separation, purification, detection and analysis of biopolymers and HPLC/HPCE may become the premier, preferable approaches for both qualitative and quantitative analyses of most proteins, especially from recombinant materials. This includes smaller peptides, polypeptides, proteins, antibodies and all types of protein or antibody-conjugates (antibody-enzyme, protein-fluorescent probe, antibody-drug and so forth). This entire Topical Issue of Journal of Chromatography emphasizes the application of chromatography and electrophoresis to protein analysis. This particular review deals with approaches to the selective tagging or labeling of proteins at trace (minute) levels, again using either chromatography or electrophoresis, with the emphasis on modern HPLC/HPCE methods and approaches. We discuss here both pre- and post-column labeling methods and reagents, techniques for realizing selective labeling of proteins or antibodies, applicable approaches to protein preconcentration in both HPLC and HPCE areas and in general, methods for improving (lowering) detection limits for proteins utilizing chemical or physical derivatization and/or preconcentration techniques. There are really two major goals or emphases in that which follows: (1) methods for selective labeling of proteins prior to or after HPLC/HPCE and (2) labeling of proteins at trace levels for improved separation-detection and lowered detection limits. We discuss here a large number of specific references related to both pre- and post-column/capillary derivatizations for proteins, as well as methods for improved detectability in both HPLC and HPCE by, for example, analyte preconcentration on a solid-phase extractor or membrane support, capillary isotachophoresis and other methods. Selective reactions or derivatizations on proteins refers to the ability to tag the protein at specific (e.g. reactive amino sites) in a controlled manner, with the products having the same number of tags all at the very same site or sites. The products are all the same species, having the same number of tags at the same locations on the protein. Selective reactions can also refer to the idea of tagging all of the protein sample at only a single, same site or at all available sites, homogeneously.

Surfactant Sodium Lauryl Sulfate Enhances Skin Vaccination Molecular Characterization via a Novel Technique using Ultrafiltration Capillaries and Mass Spectrometric Proteomics

The skin is a highly accessible organ and thus provides an attractive immune environment for cost-effective, simple, and needle-free delivery of vaccines and immunomodulators. In this study, we pretreated mouse skin with an anionic surfactant, sodium lauryl sulfate (SLS), for a short period of time (10 min) followed by epicutaneous vaccination with hen egg lysozyme antigen. We demonstrated for the first time that pretreatment of skin with surfactant SLS significantly enhances the production of antibody to hen egg lysozyme. Short term pretreatment with SLS disorganized the stratum corneum, extracted partial lamellar lipids, induced the maturation of Langerhans cells, and did not result in epidermis thickening. To reveal the mechanism underlying these changes, particularly at the molecular level, we used a novel proteomic technique using ultrafiltration capillaries and mass spectrometry to identify in vivo proteins/peptides secreted in the SLS-pretreated skin. Two secretory proteins, named as calcium-binding protein S100A9 and thymosin β4, were identified by this novel technique. These two proteins thus may provide new insight into the enhancing effect of surfactants on skin vaccination.

HPLC-Mass Spectrometry of Isoflavonoids in Soy and the American Groundnut, Apios Americana

There is an ever growing interest in the study of the polyphenols. Their importance in the prevention of chronic disease is gradually being unfolded. Examples range from those such as the flavonoid quercitin and the stilbene resveratrol in red wine (Das et al., 1999) to the isoflavonoids daidzein, genistein and glycitein in soy (Barnes, 1998).

Liquid Chromatography: Mass Spectrometry of Isoflavones

High-performance liquid chromatography (HPLC) or capillary electrophoresis (CE) is particularly suited to the separation and analysis of a wide range of biological compounds. When investigating such compounds in complex matrices (e.g., blood, tissue, urine, foods), specificity of the detection method becomes an important issue. This can be overcome by extensive purification procedures before HPLC or CE analysis, but at the cost of a lot of effort and often unknown losses during the extraction and purification procedures. Such losses can be overcome by the addition of isotopically labeled internal standards, subject to their availability. The coupling of HPLC or CE with mass spectrometry provides a very specific method of detection. Compounds are transferred as ions to the gas phase by two types of spraying technologies, electrospray ionization (ESI) and heated nebulizer-atmospheric pressure chemical ionization. Although the mass-to-charge ratios of the molecular ions can be used to provisionally identify a compound, full confirmation of structure requires collision-induced dissociation and analysis of the resulting fragment ions. Combination of specific parent ion-daughter ion pairs allows for the quantitative measurement of isoflavones in the 1-5 pmol range with coefficients of variation for duplicate samples in the range of 5-9%. CE places greater demands on the mass spectrometer than does HPLC, because it generates narrow peak widths. A mass spectrometer with a scanning quadrupole analyzer does not enable full exploitation of the power of CE; in this context, an instrument with an ESI interface and a time-of-flight analyzer is ideal.

Proteomic characterization of skin and epidermis in response to environmental agents

The skin and its outer epidermis layer in particular, prevent access of various environmental agents including potential allergens, irritants, carcinogens, ultraviolet radiation and microbes. Cells in the epidermis make a significant contribution to innate as well as adaptive immune reactions in skin. The skin immunity thus provides a biologic defense in response to hazardous environmental agents. Although proteomics has been utilized to establish skin proteomes and investigate skin responses to some environmental agents, it has not been extensively used to address the complexity of skin responses to various environments. This review summarizes cutaneous genes and proteins that have been characterized as related to skin exposure to environmental agents. In parallel, this review emphasizes functional proteomics and systems biology, which are believed to be an important future direction toward characterizing the skin proteome–environmental interaction and developing successful therapeutic strategies for skin diseases caused by environmental insults.

Preparation of linear polyacrylamide gel step gradients for capillary electrophoresis

Abstract A means for casting step-gradients in linear polyacrylamide gel concentration for capillary electrophoresis is presented. A UV-Vis whole-column detector is used to profile the gel gradients cast in the capillary, while detection of fluorescein-labeled proteins is accomplished with an epi-illumination laser-induced fluorescence whole-column detection system. Fluorescence images of the capillary during the separation indicate a sharpening of the zones as they traverse the interface between gel concentrations. The potential of gel gradients in capillary electrophoresis for the analysis of wide molecular mass range protein or peptide samples is demonstrated. A mixture of proteins that range in molecular mass from 6·103 to 97.4·103 is separated in less than 15 min with baseline resolution of closely sized proteins that were not be resolved in a single concentration gel. Finally, analysis of several capillary images acquired during solute migration through the gel gradient permits the generation of a Ferguson plot from a single electrophoretic run.

Genistein and genistein-containing dietary supplements accelerate the early stages of cataractogenesis in the male ICR/f rat.

Cataract-related loss of vision affects large numbers of people in todays aging populations and presents a healthcare burden to many nations. The role of dietary supplements within the lens is largely unknown, although benefits from dietary anti-oxidants are expected. In this study, the effects of genistein as its aglycone, a genistein-containing dietary supplement (Novasoy(®)200), and a genistein-containing food (soy protein isolate, PRO-FAM 932) on the development of lens opacity were examined in the hereditary cataractous ICR/f rat. These studies were carried out in a background diet of semi-purified, isoflavone-free AIN-76A with casein as its protein source. The amount of genistein for the experimental diets was standardized to its concentration (as genistein aglycone as well as simple and complex β-glucoside conjugates) in the soy protein isolate supplement. Also tested was a high-dose genistein diet containing an 11-fold higher amount of genistein aglycone. The composition of each diet was verified by reverse-phase HPLC and blood plasma isoflavone concentrations were determined by LC-tandem mass spectrometry. The development of opacity in each lens was monitored and digitally recorded using slit-lamp examination over the course of the study. Each of the genistein-containing diets caused a significantly more rapid development of fibrous opacification in the anterior cortical region and development of apparent water clefts or vacuoles in the posterior subcapsular region than the AIN-76A control diet; however, the establishment of dense lens opacification was not significantly different between each of the diets. There was also no significant difference observed between the low-dose and high-dose genistein aglycone groups. These data suggest that genistein-containing dietary supplements accelerate the early stages of cataractogenesis in the male ICR/f rat, with no dose-dependent effects.

Osteopontin facilitates ultraviolet B-induced squamous cell carcinoma development

BACKGROUND Osteopontin (OPN) is a matricellular glycoprotein that is markedly expressed in cutaneous squamous cell carcinomas (cSCCs) and in actinic keratoses implicating its role in photocarcinogenesis. OBJECTIVE To determine whether OPN facilitates the development of cSCC and its function. METHODS cSCCs development was compared between wild-type (WT) and OPN-null mice subjected to UVB irradiation for 43 weeks. UVB-induced OPN expression was determined by Western blot, immunoprecipitation, ELISA, and semi-quantitative RT-PCR. Epidermal layer and TUNEL analyses assessed if OPN mediates UVB-induced epidermal hyperplasia or suppresses UVB-induced apoptosis of basal keratinocytes, respectively. In vitro experiments determined whether OPN enhances cell survival of UVB-induced apoptosis and its potential mechanisms. Immunohistochemical analyses of epidermis assessed the expression of CD44 and focal adhesion kinase (FAK), molecules that mediate OPN survival function. RESULTS Compared to female WT mice, OPN-null mice did not develop cSCCs. UVB irradiation stimulated OPN protein expression in the dorsal skin by 11h and remains high at 24-48h. OPN did not mediate UVB-induced epidermal hyperplasia; instead, it protected basal keratinocytes from undergoing apoptosis upon UVB exposure. Likewise, the addition of OPN suppressed UVB-induced OPN-null cSCC cell apoptosis, the activation of caspase-9 activity, and increased phosphorylation of FAK at Y397. Furthermore, the expression of CD44 and FAK in WT mice epidermis was greater than that of OPN-null mice prior to and during early acute UVB exposure. CONCLUSION These data support the hypothesis that chronic UVB-induced OPN expression protects the survival of initiated basal keratinocytes and, consequently, facilitates cSCC develop.

Abstract P4-11-04: The Influence of Radiation on Survival in Patients with Triple Negative Stage II Breast Cancer

Background: Prospective trials of locally advanced breast cancer patients treated with adjuvant radiation (RT) have demonstrated a survival advantage; however, use of postmastectomy RT in stage II patients is controversial. This study explores the possibility that patients with aggressive variant molecular subtypes [triple receptor negative (TN)] treated with modern chemotherapy, may experience a survival benefit from adjuvant RT. Methods: Billing codes for all breast cancer patients treated with chemotherapy between 1/1998 and 5/2005 at the University of Alabama at Birmingham were reviewed to comprehensively capture all Stage II and III patients with intact data. Patient, tumor, and treatment related variables were recorded and patients were divided into 3 molecular subtypes based on receptor status: hormone receptor (HR) positive, Her2 negative; HR+/− , Her2+; and TN. Kaplan Meier curves to assess survival were performed by dividing the TN group into 2 groups: those who did or did not receive adjuvant RT. Results: 409 patients with stage II-III disease with were identified. Out of this group, 81 patients had TN breast cancer (60 Stage II and 21 Stage III). RT data was known in 79 of these patients. Median age was 49 years. Median follow-up was 72 months. Thirty-seven stage II and 18 stage III patients received adjuvant RT. Of the stage II patients who received radiation, 25 underwent lumpectomy and 11 underwent mastectomy. Stage II patients who received adjuvant RT had a statistically significant improvement in DFS (p=0.03), and had a trend towards improvement in OS (p=0.07) when compared with those who did not receive adjuvant RT. There was no significant difference in survival for the stage III patients with use of RT, however numbers in this group were small. Conclusion: Adjuvant RT was associated with an improvement in DFS and a trend towards improvement in OS in patients with Stage II, TN breast cancers treated with modern chemotherapy. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-11-04.

Abstract P5-10-19: The Influence of Time to Completion of Chemotherapy on Survival in Patients with Triple Negative Stage III Breast Cancer

Background: Neoadjuvant chemotherapy is increasingly delivered to facilitate breast conserving surgery through tumor downstaging. Prospective trials of neoadjuvant chemotherapy from the NSABP suggest no difference in survival outcomes in patients receiving neoadjuvant versus adjuvant therapy; however, subset analysis in 2 combined trials (B-18 and B-27) demonstrated a trend in DFS improvement in young patients ( 5 months from diagnosis. Results: 409 patients with Stage II-III disease with were identified: 124 received neoadjuvant and 285 received adjuvant chemotherapy. Out of this group, 81 patients had TN breast cancer (60 Stage II and 21 Stage III). Median age was 49 years. Median follow-up was 72 months. Chemotherapy consisted of adriamycin, taxol and cytoxan for a median of 9 cycles. Stage III patients who completed chemotherapy within 5 months had a statistically significant improvement in OS and DFS (p=0.03), and had a trend towards improvement in DMFS (p=0.10) when compared with those who took longer than 5 months to complete chemotherapy. Conclusion: Completion of chemotherapy in a shorter time interval in patients with Stage III, TN breast cancers was associated with an improvement in DFS and OS. Consideration of timing of chemotherapy warrants further study. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P5-10-19.