ASU Scholarship Showcase

Human protein diversity arises as a result of alternative splicing, single nucleotide polymorphisms (SNPs) and posttranslational modifications. Because of these processes, each protein can exists as multiple variants in vivo. Tailored strategies are needed to study these protein variants and understand their role in health and disease. In this work we utilized quantitative mass spectrometric immunoassays to determine the protein variants concentration of beta-2-microglobulin, cystatin C, retinol binding protein, and transthyretin, in a population of 500 healthy individuals. Additionally, we determined the longitudinal concentration changes for the protein variants from four individuals over a 6 month period. Along with the native forms of the four proteins, 13 posttranslationally modified variants and 7 SNP-derived variants were detected and their concentration determined. Correlations of the variants concentration with geographical origin, gender, and age of the individuals were also examined. This work represents an important step toward building a catalog of protein variants concentrations and examining their longitudinal changes.

Plasmonic and metamaterial based nano/micro-structured materials enable spectrally selective resonant absorption, where the resonant bandwidth and absorption intensity can be engineered by controlling the size and geometry of nanostructures. Here, we demonstrate a simple, lithography-free approach for obtaining a resonant and dynamically tunable broadband absorber based on vanadium dioxide (VO2) phase transition. Using planar layered thin film structures, where top layer is chosen to be an ultrathin (20 nm) VO2 film, we demonstrate broadband IR light absorption tuning (from similar to 90% to similar to 30% in measured absorption) over the entire mid-wavelength infrared spectrum. Our numerical and experimental results indicate that the bandwidth of the absorption bands can be controlled by changing the dielectric spacer layer thickness. Broadband tunable absorbers can find applications in absorption filters, thermal emitters, thermophotovoltaics, and sensing.

Proteins can exist as multiple proteoforms in vivo, as a result of alternative splicing and single-nucleotide polymorphisms (SNPs), as well as posttranslational processing. To address their clinical significance in a context of diagnostic information, proteoforms require a more in-depth analysis. Mass spectrometric immunoassays (MSIA) have been devised for studying structural diversity in human proteins. MSIA enables protein profiling in a simple and high-throughput manner, by combining the selectivity of targeted immunoassays, with the specificity of mass spectrometric detection. MSIA has been used for qualitative and quantitative analysis of single and multiple proteoforms, distinguishing between normal fluctuations and changes related to clinical conditions. This mini review offers an overview of the development and application of mass spectrometric immunoassays for clinical and population proteomics studies. Provided are examples of some recent developments, and also discussed are the trends and challenges in mass spectrometry-based immunoassays for the next-phase of clinical applications.

Introduction: Apolipoprotein C-III (apoC-III) regulates triglyceride (TG) metabolism. In plasma, apoC-III exists in non-sialylated (apoC-III_0a without glycosylation and apoC-III[subscript 0b] with glycosylation), monosialylated (apoC-III₁) or disialylated (apoC-III₂) proteoforms. Our aim was to clarify the relationship between apoC-III sialylation proteoforms with fasting plasma TG concentrations.

Methods: In 204 non-diabetic adolescent participants, the relative abundance of apoC-III plasma proteoforms was measured using mass spectrometric immunoassay.

Results: Compared with the healthy weight subgroup (n = 16), the ratios of apoC-III_0a, apoC-III_0b, and apoC-III₁ to apoC-III₂ were significantly greater in overweight (n = 33) and obese participants (n = 155). These ratios were positively correlated with BMI z-scores and negatively correlated with measures of insulin sensitivity (S[subscript i]). The relationship of apoC-III₁ / apoC-III₂ with S_i persisted after adjusting for BMI (p = 0.02). Fasting TG was correlated with the ratio of apoC-III_0a / apoC-III₂ (r = 0.47, p<0.001), apoC-III_0b / apoC-III₂ (r = 0.41, p<0.001), apoC-III₁ / apoC-III₂ (r = 0.43, p<0.001). By examining apoC-III concentrations, the association of apoC-III proteoforms with TG was driven by apoC-III_0a (r = 0.57, p<0.001), apoC-III_0b (r = 0.56. p<0.001) and apoC-III₁ (r = 0.67, p<0.001), but not apoC-III₂ (r = 0.006, p = 0.9) concentrations, indicating that apoC-III relationship with plasma TG differed in apoC-III₂ compared with the other proteoforms.

Conclusion: We conclude that apoC-III_0a, apoC-III_0b, and apoC-III₁, but not apoC-III₂ appear to be under metabolic control and associate with fasting plasma TG. Measurement of apoC-III proteoforms can offer insights into the biology of TG metabolism in obesity.

The impetus for discovery and evaluation of protein biomarkers has been accelerated by recent development of advanced technologies for rapid and broad proteome analyses. Mass spectrometry (MS)-based protein assays hold great potential for in vitro biomarker studies. Described here is the development of a multiplex mass spectrometric immunoassay (MSIA) for quantification of apolipoprotein C-I (apoC-I), apolipoprotein C-II (apoC-II), apolipoprotein C-III (apoC-III) and their proteoforms. The multiplex MSIA assay was fast (∼40 min) and high-throughput (96 samples at a time). The assay was applied to a small cohort of human plasma samples, revealing the existence of multiple proteoforms for each apolipoprotein C. The quantitative aspect of the assay enabled determination of the concentration for each proteoform individually. Low-abundance proteoforms, such as fucosylated apoC-III, were detected in less than 20% of the samples. The distribution of apoC-III proteoforms varied among samples with similar total apoC-III concentrations. The multiplex analysis of the three apolipoproteins C and their proteoforms using quantitative MSIA represents a significant step forward toward better understanding of their physiological roles in health and disease.

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.

The oxidative modification of apolipoprotein A-I’s methionine148 (M148) is associated with defective HDL function in vitro. Multiple reaction monitoring (MRM) is a mass spectrometric technique that can be used to quantitate post-translational modifications. In this study, we developed an MRM assay to monitor the abundance ratio of the peptide containing oxidized M148 to the native peptide in ApoA-I. Measurement of the oxidized-to-unoxidized-M148 ratio was reproducible (CV < 5%). The extent of methionine M148 oxidation in the HDL of healthy controls, and type 2 diabetic participants with and without prior cardiovascular events (CVD) were then examined. The results suggest a significant increase in the relative ratio of the peptide containing oxidized M148 to the unmodified peptide in the HDL of participants with diabetes and CVD (p < 0.001), compared to participants without CVD. Monitoring the abundance ratio of the peptides containing oxidized and unoxidized M148 by MRM provides a means of examining the relationship between M148 oxidation and vascular complications in CVD.

The strong light-matter interaction and the valley selective optical selection rules make monolayer (ML) MoS₂ an exciting 2D material for fundamental physics and optoelectronics applications. But, so far, optical transition linewidths even at low temperature are typically as large as a few tens of meV and contain homogeneous and inhomogeneous contributions. This prevented in-depth studies, in contrast to the better-characterized ML materials MoSe₂ and WSe₂. In this work, we show that encapsulation of ML MoS₂ in hexagonal boron nitride can efficiently suppress the inhomogeneous contribution to the exciton linewidth, as we measure in photoluminescence and reflectivity a FWHM down to 2 meV at T = 4 K. Narrow optical transition linewidths are also observed in encapsulated WS₂, WSe₂, and MoSe₂ MLs. This indicates that surface protection and substrate flatness are key ingredients for obtaining stable, high-quality samples. Among the new possibilities offered by the well-defined optical transitions, we measure the homogeneous broadening induced by the interaction with phonons in temperature-dependent experiments. We uncover new information on spin and valley physics and present the rotation of valley coherence in applied magnetic fields perpendicular to the ML.

The valley degree of freedom in two-dimensional (2D) crystals recently emerged as a novel information carrier in addition to spin and charge. The intrinsic valley lifetime in 2D transition metal dichalcogenides (TMD) is expected to be markedly long due to the unique spin-valley locking behavior, where the intervalley scattering of the electron simultaneously requires a large momentum transfer to the opposite valley and a flip of the electron spin. However, the experimentally observed valley lifetime in 2D TMDs has been limited to tens of nanoseconds thus far. We report efficient generation of microsecond-long-lived valley polarization in WSe₂/MoS₂ heterostructures by exploiting the ultrafast charge transfer processes in the heterostructure that efficiently creates resident holes in the WSe₂ layer. These valley-polarized holes exhibit near-unity valley polarization and ultralong valley lifetime: We observe a valley-polarized hole population lifetime of more than 1 μs and a valley depolarization lifetime (that is, intervalley scattering lifetime) of more than 40 μs at 10 K. The near-perfect generation of valley-polarized holes in TMD heterostructures, combined with ultralong valley lifetime, which is orders of magnitude longer than previous results, opens up new opportunities for novel valleytronics and spintronics applications.

Modulated reflectance (contactless electroreflectance (CER), photoreflectance (PR), and piezoreflectance (PzR)) has been applied to study direct optical transitions in bulk MoS₂, MoSe₂, WS₂, and WSe₂. In order to interpret optical transitions observed in CER, PR, and PzR spectra, the electronic band structure for the four crystals has been calculated from the first principles within the density functional theory for various points of Brillouin zone including K and H points. It is clearly shown that the electronic band structure at H point of Brillouin zone is very symmetric and similar to the electronic band structure at K point, and therefore, direct optical transitions at H point should be expected in modulated reflectance spectra besides the direct optical transitions at the K point of Brillouin zone. This prediction is confirmed by experimental studies of the electronic band structure of MoS₂, MoSe₂, WS₂, and WSe₂ crystals by CER, PR, and PzR spectroscopy, i.e., techniques which are very sensitive to critical points of Brillouin zone. For the four crystals besides the A transition at K point, an A_H transition at H point has been observed in CER, PR, and PzR spectra a few tens of meV above the A transition. The spectral difference between A and A_H transition has been found to be in a very good agreement with theoretical predictions. The second transition at the H point of Brillouin zone (B_H transition) overlaps spectrally with the B transition at K point because of small energy differences in the valence (conduction) band positions at H and K points. Therefore, an extra resonance which could be related to the B_H transition is not resolved in modulated reflectance spectra at room temperature for the four crystals.