Integrated Transcriptomic and Proteomic Analysis Identifies Plasma Biomarkers of Hepatocellular Failure in Alcohol-Associated Hepatitis.

Alcohol-associated hepatitis (AH) is a form of liver failure with high short-term mortality. Recent studies have shown that defective function of hepatocyte nuclear factor 4 alpha (HNF4a) and systemic inflammation are major disease drivers of AH. Plasma biomarkers of hepatocyte function could be useful for diagnostic and prognostic purposes. Herein, an integrative analysis of hepatic RNA sequencing and liquid chromatography-tandem mass spectrometry was performed to identify plasma protein signatures for patients with mild and severe AH. Alcohol-related liver disease cirrhosis, nonalcoholic fatty liver disease, and healthy subjects were used as comparator groups. Levels of identified proteins primarily involved in hepatocellular function were decreased in patients with AH, which included hepatokines, clotting factors, complement cascade components, and hepatocyte growth activators. A protein signature of AH disease severity was identified, including thrombin, hepatocyte growth factor α, clusterin, human serum factor H-related protein, and kallistatin, which exhibited large abundance shifts between severe and nonsevere AH. The combination of thrombin and hepatocyte growth factor α discriminated between severe and nonsevere AH with high sensitivity and specificity. These findings were correlated with the liver expression of genes encoding secreted proteins in a similar cohort, finding a highly consistent plasma protein signature reflecting HNF4A and HNF1A functions. This unbiased proteomic-transcriptome analysis identified plasma protein signatures and pathways associated with disease severity, reflecting HNF4A/1A activity useful for diagnostic assessment in AH.

Validation of a multiplexed and targeted lipidomics assay for accurate quantification of lipidomes.

A major challenge of lipidomics is to determine and quantify the precise content of complex lipidomes to the exact lipid molecular species. Often, multiple methods are needed to achieve sufficient lipidomic coverage to make these determinations. Multiplexed targeted assays offer a practical alternative to enable quantitative lipidomics amenable to quality control standards within a scalable platform. Herein, we developed a multiplexed normal phase liquid chromatography-hydrophilic interaction chromatography multiple reaction monitoring method that quantifies lipid molecular species across over 20 lipid classes spanning wide polarities in a single 20-min run. Analytical challenges such as in-source fragmentation, isomer separations, and concentration dynamics were addressed to ensure confidence in selectivity, quantification, and reproducibility. Utilizing multiple MS/MS product ions per lipid species not only improved the confidence of lipid identification but also enabled the determination of relative abundances of positional isomers in samples. Lipid class-based calibration curves were applied to interpolate lipid concentrations and guide sample dilution. Analytical validation was performed following FDA Bioanalytical Method Validation Guidance for Industry. We report repeatable and robust quantitation of 900 lipid species measured in NIST-SRM-1950 plasma, with over 700 lipids achieving inter-assay variability below 25%. To demonstrate proof of concept for biomarker discovery, we analyzed plasma from mice treated with a glucosylceramide synthase inhibitor, benzoxazole 1. We observed expected reductions in glucosylceramide levels in treated animals but, more notably, identified novel lipid biomarker candidates from the plasma lipidome. These data highlight the utility of this qualified lipidomic platform for enabling biological discovery.

Separation of ß-Amyloid Tryptic Peptide Species with Isomerized and Racemized l-Aspartic Residues with Ion Mobility in Structures for Lossless Ion Manipulations.

Accumulation of ß-amyloid (Aß) is one of the hallmarks of Alzheimer's disease. The deposition of ß-amyloid plaques is likely to start years in advance of manifestation of clinical symptoms, although the exact timing is unknown. Over the years, Aß peptides undergo both post-translational modification and stereoisomerization. Analysis of the resulting stereoisomers is particularly challenging because of their identical elemental composition and similar physicochemical properties. Herein, we have utilized our recently developed structures for lossless ion manipulations ion mobility-mass spectrometry platform (SLIM IM-MS), in conjunction with serpentine ultralong path with extended routing (SUPER), to baseline resolve four distinct sets of Aß17-28 tryptic peptide epimers on a rapid (∼1 s) time scale. We discovered that sodium adduct ions, [M + H + Na]2+, allowed baseline SLIM SUPER IM resolution for all Aß epimer sets assessed, while such baseline separations were unachievable for their [M + 2H]2+ doubly protonated ions.

Global "omics" evaluation of human placental responses to preeclamptic conditions.

BACKGROUND: Preeclampsia (PE) is a leading cause of maternal death. Its cause is still debated but there is general agreement that the placenta plays a central role. Perhaps the most commonly proposed contributors to PE include placental hypoxia, oxidative stress, and increased proinflammatory cytokines. How the placenta responds to these abnormalities has been considered but not as part of a comprehensive analysis of low-molecular-weight biomolecules and their responses to these accepted PE conditions. OBJECTIVE: Using a peptidomic approach, we sought to identify a set of molecules exhibiting differential expression in consequence of provocative agents/chemical mediators of PE applied to healthy human placental tissue. STUDY DESIGN: Known PE conditions were imposed on normal placental tissue from 13 uncomplicated pregnancies and changes in the low-molecular-weight peptidome were evaluated. A t test was used to identify potential markers for each imposed stress. These markers were then submitted to a least absolute shrinkage and selection operator multinomial logistic regression model to identify signatures specific to each stressor. Estimates of model performance on external data were obtained through internal validation. RESULTS: A total of 146 markers were increased/decreased as a consequence of exposure to proposed mediators of PE. Of these 75 changed with hypoxia; 23 with hypoxia-reoxygenation/oxidative stress and 48 from exposure to tumor necrosis factor-α. These markers were chemically characterized using tandem mass spectrometry. Identification rates were: hypoxia, 34%; hypoxia-reoxygenation, 60%; and tumor necrosis factor-α, 50%. Least absolute shrinkage and selection operator modeling specified 16 markers that effectively distinguished all groups, ie, the 3 abnormal conditions and control. Bootstrap estimates of misclassification rates, multiclass area under the curve, and Brier score were 0.108, 0.944, and 0.160, respectively. CONCLUSION: Using this approach we found previously unknown molecular changes in response to individual PE conditions that allowed development biomolecular signatures for exposure to each accepted pathogenic condition.

Novel "omics" approach for study of low-abundance, low-molecular-weight components of a complex biological tissue: regional differences between chorionic and basal plates of the human placenta.

Tissue proteomics has relied heavily on two-dimensional gel electrophoresis, for protein separation and quantification, then single protein isolation, trypsin digestion, and mass spectrometric protein identification. Such methods are predominantly used for study of high-abundance, full-length proteins. Tissue peptidomics has recently been developed but is still used to study the most highly abundant species, often resulting in observation and identification of dozens of peptides only. Tissue lipidomics is likewise new, and reported studies are limited. We have developed an "omics" approach that enables over 7,000 low-molecular-weight, low-abundance species to be surveyed and have applied this to human placental tissue. Because the placenta is believed to be involved in complications of pregnancy, its proteomic evaluation is of substantial interest. In previous research on the placental proteome, abundant, high-molecular-weight proteins have been studied. Application of large-scale, global proteomics or peptidomics to the placenta have been limited, and would be challenging owing to the anatomic complexity and broad concentration range of proteins in this tissue. In our approach, involving protein depletion, capillary liquid chromatography, and tandem mass spectrometry, we attempted to identify molecular differences between two regions of the same placenta with only slightly different cellular composition. Our analysis revealed 16 species with statistically significant differences between the two regions. Tandem mass spectrometry enabled successful sequencing, or otherwise enabled chemical characterization, of twelve of these. The successful discovery and identification of regional differences between the expression of low-abundance, low-molecular weight biomolecules reveals the potential of our approach.

Leveraging High-Resolution Ion Mobility-Mass Spectrometry for Cyclic Peptide Soft Spot Identification.

Cyclic peptides are an important class of molecules that gained significant attention in the field of drug discovery due to their unique pharmacological characteristics and enhanced proteolytic stability. Yet, gastrointestinal degradation remains a major hurdle in the discovery of orally bioavailable cyclic peptides. Soft spot identification (SSID) of the regions in the cyclic peptide sequence susceptible to amide hydrolysis by proteases is used in the discovery stage to guide medicinal chemistry design. SSID can be an arduous task, traditionally performed using liquid chromatography-tandem mass spectrometry (LC-MS/MS), often resulting in complex and time-consuming manual analysis, particularly when isomeric linear peptide metabolites chromatographically coelute. Here, we present an alternative orthogonal approach that entails a high-resolution ion mobility (HRIM) system based on Structures for Lossless Ion Manipulation (SLIM) technology interfaced with quadrupole time-of-flight (QTOF) mass spectrometry to address some of the challenges associated with SSID. Two strategies were used to resolve linear isomeric peptide metabolites: labeled and label-free, both utilizing the HRIM platform. The label-free strategy leverages negative polarity to ionize the isomers which achieves better separation of the gas phase ions in the ion mobility (IM) dimension as compared to positive polarity, which is a more conventional approach when studying proteins and peptides. The second approach uses an isotope-labeled dimethyl tag on the terminal amine group, acting as a "shift reagent" to influence the mobility of isomers in the positive mode. This method resulted in baseline separation for the isomers of interest and produced unique product ions in the fragmentation spectra for unambiguous soft spot identification. Both label-free and labeled strategies demonstrated the ability to solve the challenges associated with SSID for cyclic peptides.

Investigating Performance of the SLIM-Based High Resolution Ion Mobility Platform for Separation of Isomeric Phosphatidylcholine Species.

Lipids are structurally diverse molecules that play a pivotal role in a plethora of biological processes. However, deciphering the biological roles of the specific lipids is challenging due to the existence of numerous isomers. This high chemical complexity of the lipidome is one of the major challenges in lipidomics research, as the traditional liquid chromatography-mass spectrometry (LC-MS) based approaches are often not powerful enough to resolve these isomeric and isobaric nuances within complex samples. Thus, lipids are uniquely suited to the benefits provided by multidimensional liquid chromatography-ion mobility-mass spectrometry (LC-IM-MS) analysis. However, many forms of lipid isomerism, including double-bond positional isomers and regioisomers, are structurally similar such that their collision cross section (CCS) differences are unresolvable via conventional IM approaches. Here we evaluate the performance of a high resolution ion mobility (HRIM) system based on structures for lossless ion manipulation (SLIM) technology interfaced to a high resolution quadrupole time-of-flight (QTOF) analyzer to address the noted lipidomic isomerism challenge. SLIM implements the traveling wave ion mobility technique along an ∼13 m ion path, providing longer path lengths to enable improved separation of isomeric features. We demonstrate the power of HRIM-MS to dissect isomeric PC standards differing only in double bond (DB) and stereospecific number (SN) positions. The partial separation of protonated DB isomers is significantly enhanced when they are analyzed as metal adducts. For sodium adducts, we achieve close to baseline separation of three different PC 18:1/18:1 isomers with different cis-double bond locations. Similarly, PC 18:1/18:1 (cis-9) can be resolved from the corresponding PC 18:1/18:1 (trans-9) form. The separation capacity is further enhanced when using silver ion doping, enabling the baseline separation of regioisomers that cannot be resolved when measured as sodium adducts. The sensitivity and reproducibility of the approach were assessed, and the performance for more complex mixtures was benchmarked by identifying PC isomers in total brain and liver lipid extracts.

Nutritional markers and proteome in patients undergoing treatment for pulmonary tuberculosis differ by geographic region.

INTRODUCTION: Contemporary phase 2 TB disease treatment clinical trials have found that microbiologic treatment responses differ between African versus non-African regions, the reasons for which remain unclear. Understanding host and disease phenotypes that may vary by region is important for optimizing curative treatments. METHODS: We characterized clinical features and the serum proteome of phase 2 TB clinical trial participants undergoing treatment for smear positive, culture-confirmed TB, comparing host serum protein expression in clinical trial participants enrolled in African and Non-African regions. Serum samples were collected from 289 participants enrolled in the Centers for Disease Control and Prevention TBTC Study 29 (NCT00694629) at time of enrollment and at the end of the intensive phase (after 40 doses of TB treatment). RESULTS: After a peptide level proteome analysis utilizing a unique liquid chromatography IM-MS platform (LC-IM-MS) and subsequent statistical analysis, a total of 183 core proteins demonstrated significant differences at both baseline and at week 8 timepoints between participants enrolled from African and non-African regions. The majority of the differentially expressed proteins were upregulated in participants from the African region, and included acute phase proteins, mediators of inflammation, as well as coagulation and complement pathways. Downregulated proteins in the African population were primarily linked to nutritional status and lipid metabolism pathways. CONCLUSIONS: We have identified differentially expressed nutrition and lipid pathway proteins by geographic region in TB patients undergoing treatment for pulmonary tuberculosis, which appear to be associated with differential treatment responses. Future TB clinical trials should collect expanded measures of nutritional status and further evaluate the relationship between nutrition and microbiologic treatment response.

Proteomic Insights into Phycobilisome Degradation, A Selective and Tightly Controlled Process in The Fast-Growing Cyanobacterium Synechococcus elongatus UTEX 2973.

Phycobilisomes (PBSs) are large (3-5 megadalton) pigment-protein complexes in cyanobacteria that associate with thylakoid membranes and harvest light primarily for photosystem II. PBSs consist of highly ordered assemblies of pigmented phycobiliproteins (PBPs) and linker proteins that can account for up to half of the soluble protein in cells. Cyanobacteria adjust to changing environmental conditions by modulating PBS size and number. In response to nutrient depletion such as nitrogen (N) deprivation, PBSs are degraded in an extensive, tightly controlled, and reversible process. In Synechococcus elongatus UTEX 2973, a fast-growing cyanobacterium with a doubling time of two hours, the process of PBS degradation is very rapid, with 80% of PBSs per cell degraded in six hours under optimal light and CO2 conditions. Proteomic analysis during PBS degradation and re-synthesis revealed multiple proteoforms of PBPs with partially degraded phycocyanobilin (PCB) pigments. NblA, a small proteolysis adaptor essential for PBS degradation, was characterized and validated with targeted mass spectrometry. NblA levels rose from essentially 0 to 25,000 copies per cell within 30 min of N depletion, and correlated with the rate of decrease in phycocyanin (PC). Implications of this correlation on the overall mechanism of PBS degradation during N deprivation are discussed.

Application of multiplexed ion mobility spectrometry towards the identification of host protein signatures of treatment effect in pulmonary tuberculosis.

RATIONALE: The monitoring of TB treatments in clinical practice and clinical trials relies on traditional sputum-based culture status indicators at specific time points. Accurate, predictive, blood-based protein markers would provide a simpler and more informative view of patient health and response to treatment. OBJECTIVE: We utilized sensitive, high throughput multiplexed ion mobility-mass spectrometry (IM-MS) to characterize the serum proteome of TB patients at the start of and at 8 weeks of rifamycin-based treatment. We sought to identify treatment specific signatures within patients as well as correlate the proteome signatures to various clinical markers of treatment efficacy. METHODS: Serum samples were collected from 289 subjects enrolled in CDC TB Trials Consortium Study 29 at time of enrollment and at the end of the intensive phase (after 40 doses of TB treatment). Serum proteins were immunoaffinity-depleted of high abundant components, digested to peptides and analyzed for data acquisition utilizing a unique liquid chromatography IM-MS platform (LC-IM-MS). Linear mixed models were utilized to identify serum protein changes in the host response to antibiotic treatment as well as correlations with culture status end points. RESULTS: A total of 10,137 peptides corresponding to 872 proteins were identified, quantified, and used for statistical analysis across the longitudinal patient cohort. In response to TB treatment, 244 proteins were significantly altered. Pathway/network comparisons helped visualize the interconnected proteins, identifying up regulated (lipid transport, coagulation cascade, endopeptidase activity) and down regulated (acute phase) processes and pathways in addition to other cross regulated networks (inflammation, cell adhesion, extracellular matrix). Detection of possible lung injury serum proteins such as HPSE, significantly downregulated upon treatment. Analyses of microbiologic data over time identified a core set of serum proteins (TTHY, AFAM, CRP, RET4, SAA1, PGRP2) which change in response to treatment and also strongly correlate with culture status. A similar set of proteins at baseline were found to be predictive of week 6 and 8 culture status. CONCLUSION: A comprehensive host serum protein dataset reflective of TB treatment effect is defined. A repeating set of serum proteins (TTHY, AFAM, CRP, RET4, SAA1, PGRP2, among others) were found to change significantly in response to treatment, to strongly correlate with culture status, and at baseline to be predictive of future culture conversion. If validated in cohorts with long term follow-up to capture failure and relapse of TB, these protein markers could be developed for monitoring of treatment in clinical trials and in patient care.