Clinical interpretation of serum hepcidin-25 in inflammation and renal dysfunction.

Introduction: Hepcidin is a hormone that regulates systemic iron homeostasis. Serum hepcidin levels are under the influence of various stimuli, particularly inflammation and renal dysfunction. The measurement of hepcidin in circulation is a potentially useful clinical tool in the diagnosis, monitoring and treatment of iron metabolism disorder, although clinical interpretation of hepcidin level remains difficult. We evaluated he diagnostic potential and limitations of hepcidin-25 by investigating its relationship with iron and hematological indices, inflammation, and renal dysfunction. Methods: This retrospective study included 220 adult patients not requiring dialysis. Variations of biologically active hepcidin-25 were examined using a mass spectrometry-based assay in various inflammatory and renal states. The log[hepcidin]:log[ferritin] ratio was calculated as an hepcidin index. Results: In 220 adult patients not requiring dialysis, variation in hepcidin-25 level was significantly larger once CRP exceeded 10 mg/l (p < 0.001). Inflammation was not a determinant of hepcidin-25 in the setting of renal dysfunction. Hepcidin-25 median (7.37 nM) and variance were significantly higher (p < 0.001), once estimated glomerular filtration rate (eGFR) dropped below 30 ml/min/1.73 m2. The log[hepcidin]:log[ferritin] index normalized hepcidin levels. Patients with iron deficiency have a notably lower index when compared to controls (-0.66 vs 0.3). Conclusion: Severe renal dysfunction (eGFR < 30) affected hepcidin-25 expression and clearance to variable degree between individuals. Although, hepcidin-25 testing is not warranted in patients with infection, inflammatory autoimmune conditions (CRP > 10 mg/l) and/or severe renal dysfunction (eGFR < 30), the hepcidin index may serve as a potential biomarker for iron deficiency in complex cases.

Affinity Purification Coupled to Stable Isotope Dilution LC-MS/MS Analysis to Discover IgG4 Glycosylation Profiles for Autoimmune Pancreatitis.

Type 1 autoimmune pancreatitis (AIP) is categorized as an IgG4-related disease (IgG4-RD), where a high concentration of plasma IgG4 is one of the common biomarkers among patients. IgG Fc-glycosylation has been reported to be potential biosignatures for diseases. However, human IgG3 and IgG4 Fc-glycopeptides from populations in Asia were found to be isobaric ions when using LC-MS/MS as an analytical tool. In this study, an analytical workflow that coupled affinity purification and stable isotope dilution LC-MS/MS was developed to dissect IgG4 glycosylation profiles for autoimmune pancreatitis. Comparing the IgG4 and glycosylation profiles among healthy controls, patients with pancreatic ductal adenocarcinoma (PDAC), and AIP, the IgG4 glycosylations from the AIP group were found to have more digalactosylation (compared to PDAC) and less monogalactosylation (compared to HC). In addition, higher fucosylation and sialylation profiles were also discovered for the AIP group. The workflow is efficient and selective for IgG4 glycopeptides, and can be used for clinical biosignature discovery.

A multiplexed, automated immuno-matrix assisted laser desorption/ionization mass spectrometry assay for simultaneous and precise quantitation of PTEN and p110α in cell lines and tumor tissues.

The PI3-kinase/AKT/mTOR pathway plays a central role in cancer signaling. While p110α is the catalytic α-subunit of PI3-kinase and a major drug target, PTEN is the main negative regulator of the PI3-kinase/AKT/mTOR pathway. PTEN is often down-regulated in cancer, and there are conflicting data on PTEN's role as breast cancer biomarker. PTEN and p110α protein expression in tumors is commonly analyzed by immunohistochemistry, which suffers from poor multiplexing capacity, poor standardization, and antibody crossreactivity, and which provides only semi-quantitative data. Here, we present an automated, and standardized immuno-matrix-assisted laser desorption/ionization mass spectrometry (iMALDI) assay that allows precise and multiplexed quantitation of PTEN and p110α concentrations, without the limitations of immunohistochemistry. Our iMALDI assay only requires a low-cost benchtop MALDI-TOF mass spectrometer, which simplifies clinical translation. We validated our assay's precision and accuracy, with simultaneous enrichment of both target proteins not significantly affecting the precision and accuracy of the quantitation when compared to the PTEN- and p110α-singleplex iMALDI assays (<15% difference). The multiplexed assay's linear range is from 0.6-20 fmol with accuracies of 90-112% for both target proteins, and the assay is free of matrix-related interferences. The inter-day reproducibility over 5-days was high, with an overall CV of 9%. PTEN and p110α protein concentrations can be quantified down to 1.4 fmol and 0.6 fmol per 10 µg of total tumor protein, respectively, in various tumor tissue samples, including fresh-frozen breast tumors and colorectal cancer liver metastases, and patient-derived xenograft (PDX) tumors.

Systematic Optimization of the iMALDI Workflow for the Robust and Straightforward Quantification of Signaling Proteins in Cancer Cells.

PURPOSE: Immuno-MALDI (iMALDI) combines immuno-enrichment of biomarkers with MALDI-MS for fast, precise, and specific quantitation, making it a valuable tool for developing clinical assays. iMALDI assays are optimized for the PI3-kinase signaling pathway members phosphatase and tensin homolog (PTEN) and PI3-kinase catalytic subunit alpha (p110α), with regard to sensitivity, robustness, and throughput. A standardized template for developing future iMALDI assays, including automation protocols to streamline assay development and translation, is provided. EXPERIMENTAL DESIGN: Conditions for tryptic digestion and immuno-enrichment (beads, bead:antibody ratios, incubation times, direct vs. indirect immuno-enrichment) are rigorously tested. Different strategies for calibration and data readout are compared. RESULTS: Digestion using 1:2 protein:trypsin (wt:wt) for 1 h yielded high and consistent peptide recoveries. Direct immuno-enrichment (antibody-bead coupling prior to antigen-enrichment) yielded 30% higher peptide recovery with a 1 h shorter incubation time than indirect enrichment. Immuno-enrichment incubation overnight yielded 1.5-fold higher sensitivities than 1 h incubation. Quantitation of the endogenous target proteins is not affected by the complexity of the calibration matrix, further simplifying the workflow. CONCLUSIONS AND CLINICAL RELEVANCE: This optimized and automated workflow will facilitate the clinical translation of high-throughput sensitive iMALDI assays for quantifying cell-signaling proteins in individual tumor samples, thereby improving patient stratification for targeted treatment.

Development of efficient on-bead protein elution process coupled to ultra-high performance liquid chromatography-tandem mass spectrometry to determine immunoglobulin G subclass and glycosylation for discovery of bio-signatures in pancreatic disease.

Type 1 autoimmune pancreatitis (AIP) is a kind of IgG4-related disease in which higher IgG4 and total IgG levels have been found in patient serum. Due to the similar imaging features and laboratory parameters between AIP and pancreatic ductal adenocarcinoma (PDAC), a differential diagnosis is still challenging. Since IgG profiles can be potential bio-signatures for disease, we developed and validated a method which coupled on-bead enzymatic protein elution process to an efficient UHPLC-MS/MS method to determine IgG subclass and glycosylation. A stable-isotope labeled IgG was incorporated as internal standard to achieve accurate quantification. For calibration curves, the correlation coefficients for total IgG and the four IgG subclasses were higher than 0.995. Intraday (n = 5) and interday (n = 3) precisions of the peak area ratios of LLOQ, low, medium, and high QC samples were all less than 6.6% relative standard deviation (% RSD), and the accuracies were between 93.5 and 114.9%. Calibration curves, precision, and accuracy were also evaluated for 26 IgG glycopeptides. The method was applied to samples from healthy controls and patients with AIP and PDAC. Distinct IgG patterns were discovered among the groups, and 7 glycopeptides showed high potential in differentiating AIP and PDAC. The results demonstrated that the developed method is suitable for multi-feature analysis of human IgG, and the discovered IgG profiles can be used as bio-signatures for AIP and PDAC.

Metabolome analysis for investigating host-gut microbiota interactions.

Dysbiosis of the gut microbiome is associated with host health conditions. Many diseases have shown to have correlations with imbalanced microbiota, including obesity, inflammatory bowel disease, cancer, and even neurodegeneration disorders. Metabolomics studies targeting small molecule metabolites that impact the host metabolome and their biochemical functions have shown promise for studying host-gut microbiota interactions. Metabolome analysis determines the metabolites being discussed for their biological implications in host-gut microbiota interactions. To facilitate understanding the critical aspects of metabolome analysis, this article reviewed (1) the sample types used in host-gut microbiome studies; (2) mass spectrometry (MS)-based analytical methods and (3) useful tools for MS-based data processing/analysis. In addition to the most frequently used sample type, feces, we also discussed others biosamples, such as urine, plasma/serum, saliva, cerebrospinal fluid, exhaled breaths, and tissues, to better understand gut metabolite systemic effects on the whole organism. Gas chromatography-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry (LC-MS), and capillary electrophoresis-mass spectrometry (CE-MS), three powerful tools that can be utilized to study host-gut microbiota interactions, are included with examples of their applications. After obtaining big data from MS-based instruments, noise removal, peak detection, missing value imputation, and data analysis are all important steps for acquiring valid results in host-gut microbiome research. The information provided in this review will help new researchers aiming to join this field by providing a global view of the analytical aspects involved in gut microbiota-related metabolomics studies.

Peptide and Protein Quantification Using Automated Immuno-MALDI (iMALDI).

Mass spectrometry (MS) is one of the most commonly used technologies for quantifying proteins in complex samples, with excellent assay specificity as a result of the direct detection of the mass-to-charge ratio of each target molecule. However, MS-based proteomics, like most other analytical techniques, has a bias towards measuring high-abundance analytes, so it is challenging to achieve detection limits of low ng/mL or pg/mL in complex samples, and this is the concentration range for many disease-relevant proteins in biofluids such as human plasma. To assist in the detection of low-abundance analytes, immuno-enrichment has been integrated into the assay to concentrate and purify the analyte before MS measurement, significantly improving assay sensitivity. In this work, the immuno- Matrix-Assisted Laser Desorption/Ionization (iMALDI) technology is presented for the quantification of proteins and peptides in biofluids, based on immuno-enrichment on beads, followed by MALDI-MS measurement without prior elution. The anti-peptide antibodies are functionalized on magnetic beads, and incubated with samples. After washing, the beads are directly transferred onto a MALDI target plate, and the signals are measured by a MALDI-Time of Flight (MALDI-TOF) instrument after the matrix solution has been applied to the beads. The sample preparation procedure is simplified compared to other immuno-MS assays, and the MALDI measurement is fast. The whole sample preparation is automated with a liquid handling system, with improved assay reproducibility and higher throughput. In this article, the iMALDI assay is used for determining the peptide angiotensin I (Ang I) concentration in plasma, which is used clinically as readout of plasma renin activity for the screening of primary aldosteronism (PA).