ASU Scholarship Showcase

S-cysteinylated albumin and methionine-oxidized apolipoprotein A-I (apoA-I) have been posed as candidate markers of diseases associated with oxidative stress. Here, a dilute-and-shoot form of LC–electrospray ionization–MS requiring half a microliter of blood plasma was employed to simultaneously quantify the relative abundance of these oxidized proteoforms in samples stored at −80 °C, −20 °C, and room temperature and exposed to multiple freeze-thaw cycles and other adverse conditions in order to assess the possibility that protein oxidation may occur as a result of poor sample storage or handling. Samples from a healthy donor and a participant with poorly controlled type 2 diabetes started at the same low level of protein oxidation and behaved similarly; significant increases in albumin oxidation via S-cysteinylation were found to occur within hours at room temperature and days at −20 °C. Methionine oxidation of apoA-I took place on a longer time scale, setting in after albumin oxidation reached a plateau. Freeze–thaw cycles had a minimal effect on protein oxidation. In matched collections, protein oxidation in serum was the same as that in plasma. Albumin and apoA-I oxidation were not affected by sample headspace or the degree to which vials were sealed. ApoA-I, however, was unexpectedly found to oxidize faster in samples with lower surface-area-to-volume ratios. An initial survey of samples from patients with inflammatory conditions normally associated with elevated oxidative stress-including acute myocardial infarction and prostate cancer—demonstrated a lack of detectable apoA-I oxidation. Albumin S-cysteinylation in these samples was consistent with known but relatively brief exposures to temperatures above −30 °C (the freezing point of blood plasma). Given their properties and ease of analysis, these oxidized proteoforms, once fully validated, may represent the first markers of blood plasma specimen integrity based on direct measurement of oxidative molecular damage that can occur under suboptimal storage conditions.

Swinging arms are a key functional component of multistep catalytic transformations in many naturally occurring multi-enzyme complexes. This arm is typically a prosthetic chemical group that is covalently attached to the enzyme complex via a flexible linker, allowing the direct transfer of substrate molecules between multiple active sites within the complex. Mimicking this method of substrate channelling outside the cellular environment requires precise control over the spatial parameters of the individual components within the assembled complex. DNA nanostructures can be used to organize functional molecules with nanoscale precision and can also provide nanomechanical control. Until now, protein–DNA assemblies have been used to organize cascades of enzymatic reactions by controlling the relative distance and orientation of enzymatic components or by facilitating the interface between enzymes/cofactors and electrode surfaces. Here, we show that a DNA nanostructure can be used to create a multi-enzyme complex in which an artificial swinging arm facilitates hydride transfer between two coupled dehydrogenases. By exploiting the programmability of DNA nanostructures, key parameters including position, stoichiometry and inter-enzyme distance can be manipulated for optimal activity.

A structurally and compositionally well-defined and spectrally tunable artificial light-harvesting system has been constructed in which multiple organic dyes attached to a three-arm-DNA nanostructure serve as an antenna conjugated to a photosynthetic reaction center isolated from Rhodobacter sphaeroides 2.4.1. The light energy absorbed by the dye molecules is transferred to the reaction center, where charge separation takes place. The average number of DNA three-arm junctions per reaction center was tuned from 0.75 to 2.35. This DNA-templated multichromophore system serves as a modular light-harvesting antenna that is capable of being optimized for its spectral properties, energy transfer efficiency, and photostability, allowing one to adjust both the size and spectrum of the resulting structures. This may serve as a useful test bed for developing nanostructured photonic systems.

Background: Cysteine sulfenic acid (Cys-SOH) plays important roles in the redox regulation of numerous proteins. As a relatively unstable posttranslational protein modification it is difficult to quantify the degree to which any particular protein is modified by Cys-SOH within a complex biological environment. The goal of these studies was to move a step beyond detection and into the relative quantification of Cys-SOH within specific proteins found in a complex biological setting--namely, human plasma.

Results: This report describes the possibilities and limitations of performing such analyses based on the use of thionitrobenzoic acid and dimedone-based probes which are commonly employed to trap Cys-SOH. Results obtained by electrospray ionization-based mass spectrometric immunoassay reveal the optimal type of probe for such analyses as well as the reproducible relative quantification of Cys-SOH within albumin and transthyretin extracted from human plasma--the latter as a protein previously unknown to be modified by Cys-SOH.

Conclusions: The relative quantification of Cys-SOH within specific proteins in a complex biological setting can be accomplished, but several analytical precautions related to trapping, detecting, and quantifying Cys-SOH must be taken into account prior to pursuing its study in such matrices.

Serum Amyloid A (SAA) is an acute phase protein complex consisting of several abundant isoforms. The N- terminus of SAA is critical to its function in amyloid formation. SAA is frequently truncated, either missing an arginine or an arginine-serine dipeptide, resulting in isoforms that may influence the capacity to form amyloid. However, the relative abundance of truncated SAA in diabetes and chronic kidney disease is not known.

Methods: Using mass spectrometric immunoassay, the abundance of SAA truncations relative to the native variants was examined in plasma of 91 participants with type 2 diabetes and chronic kidney disease and 69 participants without diabetes.

Results: The ratio of SAA 1.1 (missing N-terminal arginine) to native SAA 1.1 was lower in diabetics compared to non-diabetics (p = 0.004), and in males compared to females (p<0.001). This ratio was negatively correlated with glycated hemoglobin (r = −0.32, p<0.001) and triglyceride concentrations (r = −0.37, p<0.001), and positively correlated with HDL cholesterol concentrations (r = 0.32, p<0.001).

Conclusion: The relative abundance of the N-terminal arginine truncation of SAA1.1 is significantly decreased in diabetes and negatively correlates with measures of glycemic and lipid control.

There is an increasing awareness that health care must move from post-symptomatic treatment to presymptomatic intervention. An ideal system would allow regular inexpensive monitoring of health status using circulating antibodies to report on health fluctuations. Recently, we demonstrated that peptide microarrays can do this through antibody signatures (immunosignatures). Unfortunately, printed microarrays are not scalable. Here we demonstrate a platform based on fabricating microarrays (~10 M peptides per slide, 330,000 peptides per assay) on silicon wafers using equipment common to semiconductor manufacturing. The potential of these microarrays for comprehensive health monitoring is verified through the simultaneous detection and classification of six different infectious diseases and six different cancers. Besides diagnostics, these high-density peptide chips have numerous other applications both in health care and elsewhere.

Quiescin sulfhydryl oxidase 1 (QSOX1) is a highly conserved disulfide bond-generating enzyme that is overexpressed in diverse tumor types. Its enzymatic activity promotes the growth and invasion of tumor cells and alters extracellular matrix composition. In a nude mouse-human tumor xenograft model, tumors containing shRNA for QSOX1 grew significantly more slowly than controls, suggesting that QSOX1 supports a proliferative phenotype in vivo. High throughput screening experiments identified ebselen as an in vitro inhibitor of QSOX1 enzymatic activity. Ebselen treatment of pancreatic and renal cancer cell lines stalled tumor growth and inhibited invasion through Matrigel in vitro. Daily oral treatment with ebselen resulted in a 58% reduction in tumor growth in mice bearing human pancreatic tumor xenografts compared to controls. Mass spectrometric analysis of ebselen-treated QSOX1 mechanistically revealed that C165 and C237 of QSOX1 covalently bound to ebselen. This report details the anti-neoplastic properties of ebselen in pancreatic and renal cancer cell lines. The results here offer a “proof-of-principle” that enzymatic inhibition of QSOX1 may have clinical relevancy.

Significance: Modification of cysteine thiols dramatically affects protein function and stability. Hence, the abilities to quantify specific protein sulfhydryl groups within complex biological samples and map disulfide bond structures are crucial to gaining greater insights into how proteins operate in human health and disease. Recent Advances: Many different molecular probes are now commercially available to label and track cysteine residues at great sensitivity. Coupled with mass spectrometry, stable isotope-labeled sulfhydryl-specific reagents can provide previously unprecedented molecular insights into the dynamics of cysteine modification. Likewise, the combined application of modern mass spectrometers with improved sample preparation techniques and novel data mining algorithms is beginning to routinize the analysis of complex protein disulfide structures. Critical Issues: Proper application of these modern tools and techniques, however, still requires fundamental understanding of sulfhydryl chemistry as well as the assumptions that accompany sample preparation and underlie effective data interpretation. Future Directions: The continued development of tools, technical approaches, and corresponding data processing algorithms will, undoubtedly, facilitate site-specific protein sulfhydryl quantification and disulfide structure analysis from within complex biological mixtures with ever-improving accuracy and sensitivity. Fully routinizing disulfide structure analysis will require an equal but balanced focus on sample preparation and corresponding mass spectral dataset reproducibility.

The relationship between sequence and binding properties of an aptamer for immunoglobulin E (IgE) was investigated using custom DNA microarrays. Single, double and some triple mutations of the aptamer sequence were created to evaluate the importance of specific base composition on aptamer binding. The majority of the positions in the aptamer sequence were found to be immutable, with changes at these positions resulting in more than a 100-fold decrease in binding affinity. Improvements in binding were observed by altering the stem region of the aptamer, suggesting that it plays a significant role in binding. Results obtained for the various mutations were used to estimate the information content and the probability of finding a functional aptamer sequence by selection from a random library. For the IgE-binding aptamer, this probability is on the order of 10^-10 to 10^-9. Results obtained for the double and triple mutations also show that there are no compensatory mutations within the space defined by those mutations. Apparently, at least for this particular aptamer, the functional sequence space can be represented as a rugged landscape with sharp peaks defined by highly constrained base compositions. This makes the rational optimization of aptamer sequences using step-wise mutagenesis approaches very challenging.

There are an increasing variety of applications in which peptides are both synthesized and used attached to solid surfaces. This has created a need for high throughput sequence analysis directly on surfaces. However, common sequencing approaches that can be adapted to surface bound peptides lack the throughput often needed in library-based applications. Here we describe a simple approach for sequence analysis directly on solid surfaces that is both high speed and high throughput, utilizing equipment available in most protein analysis facilities. In this approach, surface bound peptides, selectively labeled at their N-termini with a positive charge-bearing group, are subjected to controlled degradation in ammonia gas, resulting in a set of fragments differing by a single amino acid that remain spatially confined on the surface they were bound to. These fragments can then be analyzed by MALDI mass spectrometry, and the peptide sequences read directly from the resulting spectra.