Faculty and Staff

Insulin-like growth factor 1 (IGF1) is an important biomarker for the management of growth hormone disorders. Recently there has been rising interest in deploying mass spectrometric (MS) methods of detection for measuring IGF1. However, widespread clinical adoption of any MS-based IGF1 assay will require increased throughput and speed to justify the costs of analyses, and robust industrial platforms that are reproducible across laboratories. Presented here is an MS-based quantitative IGF1 assay with performance rating of >1,000 samples/day, and a capability of quantifying IGF1 point mutations and posttranslational modifications. The throughput of the IGF1 mass spectrometric immunoassay (MSIA) benefited from a simplified sample preparation step, IGF1 immunocapture in a tip format, and high-throughput MALDI-TOF MS analysis. The Limit of Detection and Limit of Quantification of the resulting assay were 1.5 μg/L and 5 μg/L, respectively, with intra- and inter-assay precision CVs of less than 10%, and good linearity and recovery characteristics. The IGF1 MSIA was benchmarked against commercially available IGF1 ELISA via Bland-Altman method comparison test, resulting in a slight positive bias of 16%. The IGF1 MSIA was employed in an optimized parallel workflow utilizing two pipetting robots and MALDI-TOF-MS instruments synced into one-hour phases of sample preparation, extraction and MSIA pipette tip elution, MS data collection, and data processing. Using this workflow, high-throughput IGF1 quantification of 1,054 human samples was achieved in approximately 9 hours. This rate of assaying is a significant improvement over existing MS-based IGF1 assays, and is on par with that of the enzyme-based immunoassays. Furthermore, a mutation was detected in ∼1% of the samples (SNP: rs17884626, creating an A→T substitution at position 67 of the IGF1), demonstrating the capability of IGF1 MSIA to detect point mutations and posttranslational modifications.

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.

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.

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.

Oppia Quadrangle Av-10 (288–360°E, ±22°) is a junction of key geologic features that preserve a rough history of Asteroid (4) Vesta and serves as a case study of using geologic mapping to define a relative geologic timescale. Clear filter images, stereo-derived topography, slope maps, and multispectral color-ratio images from the Framing Camera on NASA’s Dawn spacecraft served as basemaps to create a geologic map and investigate the spatial and temporal relationships of the local stratigraphy. Geologic mapping reveals the oldest map unit within Av-10 is the cratered highlands terrain which possibly represents original crustal material on Vesta that was then excavated by one or more impacts to form the basin Feralia Planitia. Saturnalia Fossae and Divalia Fossae ridge and trough terrains intersect the wall of Feralia Planitia indicating that this impact basin is older than both the Veneneia and Rheasilvia impact structures, representing Pre-Veneneian crustal material. Two of the youngest geologic features in Av-10 are Lepida (∼45 km diameter) and Oppia (∼40 km diameter) impact craters that formed on the northern and southern wall of Feralia Planitia and each cross-cuts a trough terrain. The ejecta blanket of Oppia is mapped as ‘dark mantle’ material because it appears dark orange in the Framing Camera ‘Clementine-type’ color-ratio image and has a diffuse, gradational contact distributed to the south across the rim of Rheasilvia. Mapping of surface material that appears light orange in color in the Framing Camera ‘Clementine-type’ color-ratio image as ‘light mantle material’ supports previous interpretations of an impact ejecta origin. Some light mantle deposits are easily traced to nearby source craters, but other deposits may represent distal ejecta deposits (emplaced >5 crater radii away) in a microgravity environment.

We produced two 1:250,000 scale geologic maps of the adjacent quadrangles Av-6 Gegania and Av-7 Lucaria, located in the equatorial region of (4) Vesta (0–144°E, 22°S to 22°N). The mapping is based on clear and color filter images of the Framing Camera (FC) onboard the Dawn spacecraft, which has captured the entire illuminated surface of Vesta with high spatial resolution (up to ∼20 m/pixel), and on a digital terrain model derived from FC imagery. Besides the geologic mapping itself, a secondary purpose of this work is to investigate one of the most prominent morphological features on Vesta, namely the aggregation of several giant equatorial troughs termed the Divalia Fossae, most probably formed during the Rheasilvia impact near Vesta’s south pole. The up to 465 km long and 22 km wide troughs show height differences of up to 5 km between adjacent troughs and ridges. Another imprint of the Rheasilvia impact is the >350 km long and ∼250 km wide swath of ejecta crossing quadrangle Av-6 Gegania. This lobe shows a distinct appearance in FC color ratios and a high albedo in FC images, indicating a mineralogical similarity to material typically found within the Rheasilvia basin, in particular composed of diogenite-rich howardites. Almost the entire northern half of the mapping area shows the oldest surface, being dominated by upper crustal basaltic material. To the south, increasingly younger formations related to the Rheasilvia impact occur, either indicated by the troughs formed by Rheasilvia or by the Rheasilvia ejecta itself. Only medium sized impact craters with diameters less than 22 km occur within the two mapped quadrangles. Some of the craters exhibit ejecta blankets and/or distinctly dark or bright ejecta material in ejecta rays outside and exposures within the crater, and mass-wasting deposits down crater slopes, forming the youngest surfaces.

We used Dawn spacecraft data to identify and delineate geological units and landforms in the Marcia quadrangle of Vesta as a means to assess the role of the large, relatively young impact craters Marcia (∼63 km diam.) and Calpurnia (∼53 km diam.) and their surrounding ejecta field on the local geology. We also investigated a local topographic high with a dark-rayed crater named Aricia Tholus, and the impact crater Octavia that is surrounded by a distinctive diffuse mantle. Crater counts and stratigraphic relations suggest that Marcia is the youngest large crater on Vesta, in which a putative impact melt on the crater floor ranges in age between ∼40 and 60 Ma (depending upon choice of chronology system), and Marcia’s ejecta blanket ranges in age between ∼120 and 390 Ma (depending upon choice of chronology system).

We interpret the geologic units in and around Marcia crater to mark a major vestan time-stratigraphic event, and that the Marcia Formation is one of the geologically youngest formations on Vesta. Marcia crater reveals pristine bright and dark material in its walls and smooth and pitted terrains on its floor. The smooth unit we interpret as evidence of flow of impact melts and (for the pitted terrain) release of volatiles during or after the impact process. The distinctive dark ejecta surrounding craters Marcia and Calpurnia is enriched in OH- or H-bearing phases and has a variable morphology, suggestive of a complex mixture of impact ejecta and impact melts including dark materials possibly derived from carbonaceous chondrite-rich material. Aricia Tholus, which was originally interpreted as a putative vestan volcanic edifice based on lower resolution observations, appears to be a fragment of an ancient impact basin rim topped by a dark-rayed impact crater. Octavia crater has a cratering model formation age of ∼280–990 Ma based on counts of its ejecta field (depending upon choice of chronology system), and its ejecta field is the second oldest unit in this quadrangle. The relatively young craters and their related ejecta materials in this quadrangle are in stark contrast to the surrounding heavily cratered units that are related to the billion years old or older Rheasilvia and Veneneia impact basins and Vesta’s ancient crust preserved on Vestalia Terra.

The purpose of this paper is to introduce the Geologic Mapping of Vesta Special Issue/Section of Icarus, which includes several papers containing geologic maps of the surface of Vesta made to support data analysis conducted by the Dawn Science Team during the Vesta Encounter (July 2011–September 2012). In this paper we briefly discuss pre-Dawn knowledge of Vesta, provide the goals of our geologic mapping campaign, discuss the methodologies and materials used for geologic mapping, review the global geologic context of Vesta, discuss the challenges of mapping the geology of Vesta as a small airless body, and describe the content of the papers in this Special Issue/Section. We conclude with a discussion of lessons learned from our quadrangle-based mapping effort and provide recommendations for conducting mapping campaigns as part of planetary spacecraft nominal missions.

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.

Background: The cytokine MIF (Macrophage Migration Inhibitory Factor) has diverse physiological roles and is present at elevated concentrations in numerous disease states. However, its molecular heterogeneity has not been previously investigated in biological samples. Mass Spectrometric Immunoassay (MSIA) may help elucidate MIF post-translational modifications existing in vivo and provide additional clarity regarding its relationship to diverse pathologies.

Results: In this work, we have developed and validated a fully quantitative MSIA assay for MIF, and used it in the discovery and quantification of different proteoforms of MIF in serum samples, including cysteinylated and glycated MIF. The MSIA assay had a linear range of 1.56-50 ng/mL, and exhibited good precision, linearity, and recovery characteristics. The new assay was applied to a small cohort of human serum samples, and benchmarked against an MIF ELISA assay.

Conclusions: The quantitative MIF MSIA assay provides a sensitive, precise and high throughput method to delineate and quantify MIF proteoforms in biological samples.