Wenbo Zhi

Proteomic technologies for the discovery of type 1 diabetes biomarkers

In this review, we discuss several important issues concerning the discovery of protein biomarkers for complex human diseases, with a focus on type 1 diabetes. Serum or plasma is the first choice of specimen due to its richness in biological information and relatively easy availability. It is a challenging task to comprehensively characterize the serum/plasma proteome because of the large dynamic range of protein concentration. Therefore, sample pretreatment is required in order to explore the low- to medium-abundance proteins contained in serum/ plasma. In this regard, enrichment of low-abundance proteins using random hexapeptide library beads has distinct advantages over the traditional immune-depletion methods, including higher efficiency, higher binding capacity, and lower cost. In-depth mining of serum/plasma proteome using different separation techniques have also been evaluated and are discussed in this review. Overall, the shotgun proteomics—multidimensional separation of digested peptides followed by mass spectrometry analysis—is highly Efficient and therefore has become a preferred method for protein biomarker discovery.

Discovery and Validation of Serum Protein Changes in Type 1 Diabetes Patients Using High Throughput Two Dimensional Liquid Chromatography-Mass Spectrometry and Immunoassays

Type 1 diabetes (T1D) is expected to cause significant changes in the serum proteome; however, few studies have systematically assessed the proteomic profile change associated with the disease. In this study, a semiquantitative spectral counting-based two dimensional liquid chromatography mass spectrometry platform was used to analyze serum samples from T1D patients and controls. In this discovery phase, significant differences were found for 21 serum proteins implicated in inflammation, oxidation, metabolic regulation, and autoimmunity. To assess the validity of these findings, six candidate proteins including adiponectin, insulin-like growth factor binding protein 2, serum amyloid protein A, C-reactive protein, myeloperoxidase, and transforming growth factor beta induced were selected for subsequent immune assays for 1139 T1D patients and 848 controls. A series of statistical analyses using cases and controls matched for age, sex, and genetic risk confirmed that T1D patients have significantly higher serum levels for four of the six proteins: adiponectin (odds ratio (OR) = 1.95, p = 10−27), insulin-like growth factor binding protein 2 (OR = 2.02, p < 10−20), C-reactive protein (OR = 1.13, p = 0.007), serum amyloid protein A (OR = 1.51, p < 10−16); whereas the serum levels were significantly lower in patients than controls for the two other proteins: transforming growth factor beta induced (OR = 0.74, p < 10−5) and myeloperoxidase (OR = 0.51, p < 10−41). Compared with subjects in the bottom quartile, subjects in the top quartile for adiponectin (OR = 6.29, p < 10−37), insulin-like growth factor binding protein 2 (OR = 7.95, p < 10−46), C-reactive protein (OR = 1.38, p = 0.025), serum amyloid protein A (OR = 3.36, p < 10−16) had the highest risk of T1D, whereas subjects in the top quartile of transforming growth factor beta induced (OR = 0.41, p < 10−11) and myeloperoxidase (OR = 0.10, p < 10−43) had the lowest risk of T1D. These findings provided valuable information on the proteomic changes in the sera of T1D patients.

Selected reaction monitoring (SRM) mass spectrometry without isotope labeling can be used for rapid protein quantification

The validation of putative biomarker candidates has become the major bottle-neck in protein biomarker development. Conventional immunoaffinity methods are limited by the availability of antibodies and kits. Here we demonstrate the feasibility of using selected reaction monitoring (SRM) without isotope labeling to achieve fast and reproducible quantification of serum proteins. The SRM/MRM assays for three standard serum proteins, including ceruloplasmin (CP), serum aymloid A (SAA) and sex hormone binding globulin (SHBG), have good linear ranges, generally 10(3) to 10(4) . There are almost perfect correlations between SRM intensities and the loaded peptide amounts (R(2) is usually ~0.99). Our data suggest that SRM/MRM is able to quantify proteins within the range of 0.2-2 fmol, which is comparable to the commercial ELISA/LUMINEX kits for these proteins. Excellent correlations between SRM/MRM and ELISA/LUMINEX assays were observed for SAA and SHBG (R(2)=0.928 and 0.851, respectively). However, the correlation between SRM/MRM and ELISA for CP is less desirable (R(2)=0.565). The reproducibility for SRM/MRM assays is generally very good but may depend on the proteins/peptides being analyzed (R(2)=0.931 and 0.882 for SAA and SHBG, and 0.723 for CP). The SRM/MRM assay without isotope labeling is a rapid and useful method for protein biomarker validation in a modest number of samples and is especially useful when other assays such as ELISA or LUMINEX are not available.

Minocycline in Acute Cerebral Hemorrhage: An Early Phase Randomized Trial

Background and Purpose— Minocycline is under investigation as a neurovascular protective agent for stroke. This study evaluated the pharmacokinetic, anti-inflammatory, and safety profile of minocycline after intracerebral hemorrhage. Methods— This study was a single-site, randomized controlled trial of minocycline conducted from 2013 to 2016. Adults ≥18 years with primary intracerebral hemorrhage who could have study drug administered within 24 hours of onset were included. Patients received 400 mg of intravenous minocycline, followed by 400 mg minocycline oral daily for 4 days. Serum concentrations of minocycline after the last oral dose and biomarkers were sampled to determine the peak concentration, half-life, and anti-inflammatory profile. Results— A total of 16 consecutive eligible patients were enrolled, with 8 randomized to minocycline. Although the literature supports a time to peak concentration (Tmax) of 1 hour for oral minocycline, the Tmax was estimated to be at least 6 hours in this cohort. The elimination half-life (available on 7 patients) was 17.5 hours (SD±3.5). No differences were observed in inflammatory biomarkers, hematoma volume, or perihematomal edema. Concentrations remained at neuroprotective levels (>3 mg/L) throughout the dosing interval in 5 of 7 patients. Conclusions— In intracerebral hemorrhage, a 400 mg dose of minocycline was safe and achieved neuroprotective serum concentrations. However, oral administration led to delayed absorption in these critically ill patients and should not be used when rapid, high concentrations are desired. Given the safety and pharmacokinetic profile of minocycline in intracerebral hemorrhage and promising data in the treatment of ischemic stroke, intravenous minocycline is an excellent candidate for a prehospital treatment trial. Clinical Trial Registration— URL: http://www.clinicaltrials.gov. Unique identifier: NCT01805895.

Twelve serum proteins progressively increase with disease stage in squamous cell cervical cancer patients

Objective This study aimed to reliably identify serum protein profile alterations that may be useful for elucidation of the disease mechanism and/or finding new targets for treatment and intervention. Materials and Methods A total of 1057 women at 4 different squamous cell cervical cancer stages (noninvasive, invasive International Federation of Gynecology and Obstetrics stages I, II, and III) were included in this cross-sectional study. Forty-seven serum proteins were profiled using multiplex Luminex immunoassays. Results Serum concentration of serum amyloid A (SAA), C-reactive protein (CRP), soluble tumor necrosis factor receptor I and II (sTNFRI and sTNFRII), soluble interleukin 2 receptor &agr; (sIL2R&agr;), CXCL1, CXCL9, hepatocyte growth factor, squamous cell carcinoma antigen (SCCA), insulin-like growth factor binding protein 2, CA125, and carcinoembryonic antigen (CEA) were elevated significantly as disease progressed in cervical cancer patients. Serum levels are significantly different at early stage (I) for SAA, CRP, sIL2R&agr;, sTNFRII, SCCA, and CEA (P values ranged from 0.02 for CEA to 0.0001 for CRP and SCCA) and at late stages (II and III) for all 12 proteins (P values ranged from 8.78E-5 for CA125 to 3.49E-47 for SAA), as compared to the noninvasive stage. The areas under the curves of these proteins for disease state separation also improved with the advancement of the disease. The correlations between serum concentrations of these proteins also show different patterns at different clinical stages. These proteins are involved in multiple mechanisms including inflammation and immunity, angiogenesis, growth promotion, and metastasis. Conclusions A number of serum proteins are significantly different between patients at different stages of cervical cancer.

Serum Protein Profile at Remission Can Accurately Assess Therapeutic Outcomes and Survival for Serous Ovarian Cancer

Background Biomarkers play critical roles in early detection, diagnosis and monitoring of therapeutic outcome and recurrence of cancer. Previous biomarker research on ovarian cancer (OC) has mostly focused on the discovery and validation of diagnostic biomarkers. The primary purpose of this study is to identify serum biomarkers for prognosis and therapeutic outcomes of ovarian cancer. Experimental Design Forty serum proteins were analyzed in 70 serum samples from healthy controls (HC) and 101 serum samples from serous OC patients at three different disease phases: post diagnosis (PD), remission (RM) and recurrence (RC). The utility of serum proteins as OC biomarkers was evaluated using a variety of statistical methods including survival analysis. Results Ten serum proteins (PDGF-AB/BB, PDGF-AA, CRP, sFas, CA125, SAA, sTNFRII, sIL-6R, IGFBP6 and MDC) have individually good area-under-the-curve (AUC) values (AUC = 0.69–0.86) and more than 10 three-marker combinations have excellent AUC values (0.91–0.93) in distinguishing active cancer samples (PD & RC) from HC. The mean serum protein levels for RM samples are usually intermediate between HC and OC patients with active cancer (PD & RC). Most importantly, five proteins (sICAM1, RANTES, sgp130, sTNFR-II and sVCAM1) measured at remission can classify, individually and in combination, serous OC patients into two subsets with significantly different overall survival (best HR = 17, p<10−3). Conclusion We identified five serum proteins which, when measured at remission, can accurately predict the overall survival of serous OC patients, suggesting that they may be useful for monitoring the therapeutic outcomes for ovarian cancer.

IGF-Binding Proteins in Type-1 Diabetes Are More Severely Altered in the Presence of Complications.

Aims Reduced levels of free and total insulin-like growth factor 1 (IGF-I) have been observed in type-1 diabetes (T1D) patients. The bioavailability of IGF-I from the circulation to the target cells is controlled by multifunctional IGF-binding proteins (IGFBPs). The aim of this study was to profile serum IGFBPs in T1D and its complications. Design We measured the IGFBP levels in 3662 patient serum samples from our ongoing Phenome and Genome of Diabetes Autoimmunity (PAGODA) study. IGFBP levels of four different groups of T1D patients (with 0, 1, 2, and ≥3 complications) were compared with healthy controls. Results Three serum IGFBPs (IGFBP-1, -2, and -6) are significantly higher in T1D patients, and these alterations are greater in the presence of diabetic complications. IGFBP-3 is lower in patients with diabetic complications. Analyses using quintiles revealed that risk of T1D complications increases with increasing concentrations of IGFBP-2 (fifth quintile ORs: 18–60, p < 10−26), IGFBP-1 (fifth quintile ORs: 8–20, p < 10−15), and IGFBP-6 (fifth quintile ORs: 3–148, p < 10−3). IGFBP-3 has a negative association with T1D complications (fifth quintile ORs: 0.12–0.25, p < 10−5). Conclusion We found that elevated serum levels of IGFBP-1, -2, and -6 were associated with T1D, and its complications and IGFBP-3 level was found to be decreased in T1D with complications. Given the known role of these IGFBPs, the overall impact of these alterations suggests a negative effect on IGF signaling.

Novel function of ceramide for regulation of mitochondrial ATP release in astrocytes

We reported that amyloid β peptide (Aβ42) activated neutral SMase 2 (nSMase2), thereby increasing the concentration of the sphingolipid ceramide in astrocytes. Here, we show that Aβ42 induced mitochondrial fragmentation in wild-type astrocytes, but not in nSMase2-deficient cells or astrocytes treated with fumonisin B1 (FB1), an inhibitor of ceramide synthases. Unexpectedly, ceramide depletion was concurrent with rapid movements of mitochondria, indicating an unknown function of ceramide for mitochondria. Using immunocytochemistry and super-resolution microscopy, we detected ceramide-enriched and mitochondria-associated membranes (CEMAMs) that were codistributed with microtubules. Interaction of ceramide with tubulin was confirmed by cross-linking to N-[9-(3-pent-4-ynyl-3-H-diazirine-3-yl)-nonanoyl]-D-erythro-sphingosine (pacFACer), a bifunctional ceramide analog, and binding of tubulin to ceramide-linked agarose beads. Ceramide-associated tubulin (CAT) translocated from the perinuclear region to peripheral CEMAMs and mitochondria, which was prevented in nSMase2-deficient or FB1-treated astrocytes. Proximity ligation and coimmunoprecipitation assays showed that ceramide depletion reduced association of tubulin with voltage-dependent anion channel 1 (VDAC1), an interaction known to block mitochondrial ADP/ATP transport. Ceramide-depleted astrocytes contained higher levels of ATP, suggesting that ceramide-induced CAT formation leads to VDAC1 closure, thereby reducing mitochondrial ATP release, and potentially motility and resistance to Aβ42. Our data also indicate that inhibiting ceramide generation may protect mitochondria in Alzheimer’s disease.

Kidney-targeted inhibition of protein kinase C-α ameliorates nephrotoxic nephritis with restoration of mitochondrial dysfunction

To investigate the role of protein kinase C-α (PKC-α) in glomerulonephritis, the capacity of PKC-α inhibition to reverse the course of established nephrotoxic nephritis (NTN) was evaluated. Nephritis was induced by a single injection of nephrotoxic serum and after its onset, a PKC-α inhibitor was administered either systemically or by targeted glomerular delivery. By day seven, all mice with NTN had severe nephritis, whereas mice that received PKC-α inhibitors in either form had minimal evidence of disease. To further understand the underlying mechanism, label-free shotgun proteomic analysis of the kidney cortexes were performed, using quantitative mass spectrometry. Ingenuity pathway analysis revealed 157 differentially expressed proteins and mitochondrial dysfunction as the most modulated pathway. Functional protein groups most affected by NTN were mitochondrial proteins associated with respiratory processes. These proteins were down-regulated in the mice with NTN, while their expression was restored with PKC-α inhibition. This suggests a role for proteins that regulate oxidative phosphorylation in recovery. In cultured glomerular endothelial cells, nephrotoxic serum caused a decrease in mitochondrial respiration and membrane potential, mitochondrial morphologic changes and an increase in glycolytic lactic acid production; all normalized by PKC-α inhibition. Thus, PKC-α has a critical role in NTN progression, and the results implicate mitochondrial processes through restoring oxidative phosphorylation, as an essential mechanism underlying recovery. Importantly, our study provides additional support for targeted therapy to glomeruli to reverse the course of progressive disease.

Proteomic alterations in aqueous humor from patients with primary open angle glaucoma

Purpose Primary open angle glaucoma (POAG) is the most prevalent form of glaucoma, accounting for approximately 90% of all cases. The aqueous humor (AH), a biological fluid in the anterior and posterior chambers of the eye, is involved in a multitude of functions including the maintenance of IOP and ocular homeostasis. This fluid is very close to the pathologic site and is also known to have a significant role in glaucoma pathogenesis. The purpose of this study was to identify proteomic alterations in AH from patients with POAG. Methods AH samples were extracted from 47 patients undergoing cataract surgery (controls: n = 32; POAG: n = 15). Proteomic analysis of the digested samples was accomplished by liquid-chromatography-mass spectrometry. The identified proteins were evaluated using a variety of statistical and bioinformatics methods. Results A total of 33 proteins were significantly altered in POAG subjects compared with the controls. The most abundant proteins in POAG subjects are IGKC (13.56-fold), ITIH4 (4.1-fold), APOC3 (3.36-fold), IDH3A (3.11-fold), LOC105369216 (2.98-fold). SERPINF2 (2.94-fold), NPC2 (2.88-fold), SUCLG2 (2.70-fold), KIAA0100 (2.29-fold), CNOT4 (2.23-fold), AQP4 (2.11-fold), COL18A1 (2.08-fold), NWD1 (2.07-fold), and TMEM120B (2.06-fold). A significant increasing trend in the odds ratios of having POAG was observed with increased levels of these proteins. Conclusion Proteins identified in this study are implicated in signaling, glycosylation, immune response, molecular transport, and lipid metabolism. The identified candidate proteins may be potential biomarkers associated with POAG development and may lead to more insight in understanding the mechanisms underlying the pathogenesis of this disease.