Profiles of histidine-rich glycoprotein associate with age and risk of all-cause mortality.

Despite recognizing aging as a common risk factor of many human diseases, little is known about its molecular traits. To identify age-associated proteins circulating in human blood, we screened 156 individuals aged 50-92 using exploratory and multiplexed affinity proteomics assays. Profiling eight additional study sets (N = 3,987), performing antibody validation, and conducting a meta-analysis revealed a consistent age association (P = 6.61 × 10-6) for circulating histidine-rich glycoprotein (HRG). Sequence variants of HRG influenced how the protein was recognized in the immunoassays. Indeed, only the HRG profiles affected by rs9898 were associated with age and predicted the risk of mortality (HR = 1.25 per SD; 95% CI = 1.12-1.39; P = 6.45 × 10-5) during a follow-up period of 8.5 yr after blood sampling (IQR = 7.7-9.3 yr). Our affinity proteomics analysis found associations between the particular molecular traits of circulating HRG with age and all-cause mortality. The distinct profiles of this multipurpose protein could serve as an accessible and informative indicator of the physiological processes related to biological aging.

Molecular Profiling for Predictors of Radiosensitivity in Patients with Breast or Head-and-Neck Cancer.

Nearly half of all cancers are treated with radiotherapy alone or in combination with other treatments, where damage to normal tissues is a limiting factor for the treatment. Radiotherapy-induced adverse health effects, mostly of importance for cancer patients with long-term survival, may appear during or long time after finishing radiotherapy and depend on the patient's radiosensitivity. Currently, there is no assay available that can reliably predict the individual's response to radiotherapy. We profiled two study sets from breast (n = 29) and head-and-neck cancer patients (n = 74) that included radiosensitive patients and matched radioresistant controls.. We studied 55 single nucleotide polymorphisms (SNPs) in 33 genes by DNA genotyping and 130 circulating proteins by affinity-based plasma proteomics. In both study sets, we discovered several plasma proteins with the predictive power to find radiosensitive patients (adjusted p < 0.05) and validated the two most predictive proteins (THPO and STIM1) by sandwich immunoassays. By integrating genotypic and proteomic data into an analysis model, it was found that the proteins CHIT1, PDGFB, PNKD, RP2, SERPINC1, SLC4A, STIM1, and THPO, as well as the VEGFA gene variant rs69947, predicted radiosensitivity of our breast cancer (AUC = 0.76) and head-and-neck cancer (AUC = 0.89) patients. In conclusion, circulating proteins and a SNP variant of VEGFA suggest that processes such as vascular growth capacity, immune response, DNA repair and oxidative stress/hypoxia may be involved in an individual's risk of experiencing radiation-induced toxicity.

Targeted Analysis of Serum Proteins Encoded at Known Inflammatory Bowel Disease Risk Loci.

Background: Few studies have investigated the blood proteome of inflammatory bowel disease (IBD). We characterized the serum abundance of proteins encoded at 163 known IBD risk loci and tested these proteins for their biomarker discovery potential. Methods: Based on the Human Protein Atlas (HPA) antibody availability, 218 proteins from genes mapping at 163 IBD risk loci were selected. Targeted serum protein profiles from 49 Crohn's disease (CD) patients, 51 ulcerative colitis (UC) patients, and 50 sex- and age-matched healthy individuals were obtained using multiplexed antibody suspension bead array assays. Differences in relative serum abundance levels between disease groups and controls were examined. Replication was attempted for CD-UC comparisons (including disease subtypes) by including 64 additional patients (33 CD and 31 UC). Antibodies targeting a potentially novel risk protein were validated by paired antibodies, Western blot, immuno-capture mass spectrometry, and epitope mapping. Results: By univariate analysis, 13 proteins mostly related to neutrophil, T-cell, and B-cell activation and function were differentially expressed in IBD patients vs healthy controls, 3 in CD patients vs healthy controls and 2 in UC patients vs healthy controls (q < 0.01). Multivariate analyses further differentiated disease groups from healthy controls and CD subtypes from UC (P < 0.05). Extended characterization of an antibody targeting a novel, discriminative serum marker, the laccase (multicopper oxidoreductase) domain containing 1 (LACC1) protein, provided evidence for antibody on-target specificity. Conclusions: Using affinity proteomics, we identified a set of IBD-associated serum proteins encoded at IBD risk loci. These candidate proteins hold the potential to be exploited as diagnostic biomarkers of IBD.

Affinity Proteomics Exploration of Melanoma Identifies Proteins in Serum with Associations to T-Stage and Recurrence.

BACKGROUND: Blood-based proteomic profiling may aid and expand our understanding of diseases and their different phenotypes. The aim of the presented study was to profile serum samples from patients with malignant melanoma using affinity proteomic assays to describe proteins in the blood stream that are associated to stage or recurrence of melanoma. MATERIAL AND METHODS: Multiplexed protein analysis was conducted using antibody suspension bead arrays. A total of 232 antibodies against 132 proteins were selected from (i) a screening with 4595 antibodies and 32 serum samples from melanoma patients and controls, (ii) antibodies used for immunohistochemistry, (iii) protein targets previously related with melanoma. The analysis was performed with 149 serum samples from patients with malignant melanoma. Antibody selectivity was then assessed by Western blot, immunocapture mass spectrometry, and epitope mapping. Lastly, indicative antibodies were applied for IHC analysis of melanoma tissues. RESULTS: Serum levels of regucalcin (RGN) and syntaxin 7 (STX7) were found to be lower in patients with both recurring tumors and a high Breslow's thickness (T-stage 3/4) compared to low thickness (T-stage 1/2) without disease recurrence. Serum levels of methylenetetrahydrofolate dehydrogenase 1-like (MTHFD1L) were instead elevated in sera of T3/4 patients with recurrence. The analysis of tissue sections with S100A6 and MTHFD1L showed positive staining in a majority of patients with melanoma, and S100A6 was significantly associated to T-stage. CONCLUSIONS: Our findings provide a starting point to further study RGN, STX7, MTHFD1L and S100A6 in serum to elucidate their involvement in melanoma progression and to assess a possible contribution to support clinical indications.

Elevated levels of circulating CDH5 and FABP1 in association with human drug-induced liver injury.

BACKGROUND & AIMS: The occurrence of drug-induced liver injury (DILI) is a major issue in all phases of drug development. To identify novel biomarker candidates associated with DILI, we utilised an affinity proteomics strategy, where antibody suspension bead arrays were applied to profile plasma and serum samples from human DILI cases and controls. METHODS: An initial screening was performed using 4594 randomly selected antibodies, representing 3450 human proteins. Resulting candidate proteins together with proposed DILI biomarker candidates generated a DILI array of 251 proteins for subsequent target analysis and verifications. In total, 1196 samples from 241 individuals across four independent cohorts were profiled: healthy volunteers receiving acetaminophen, patients with human immunodeficiency virus and/or tuberculosis receiving treatment, DILI cases originating from a wide spectrum of drugs, and healthy volunteers receiving heparins. RESULTS: We observed elevated levels of cadherin 5, type 2 (CDH5) and fatty acid-binding protein 1 (FABP1) in DILI cases. In the two longitudinal cohorts, CDH5 was elevated already at baseline. FABP1 was elevated after treatment initiation and seemed to respond more rapidly than alanine aminotransferase (ALT). The elevations were verified in the DILI cases treated with various drugs. In the heparin cohort, CDH5 was stable over time whereas FABP1 was elevated. CONCLUSIONS: These results suggest that CDH5 may have value as a susceptibility marker for DILI. FABP1 was identified as a biomarker candidate with superior characteristics regarding tissue distribution and kinetics compared to ALT but likely with limited predictive value for the development of severe DILI. Further studies are needed to determine the clinical utility of the proposed markers.

Affinity proteomics discovers decreased levels of AMFR in plasma from Osteoporosis patients.

PURPOSE: Affinity proteomic approaches by antibody bead arrays enable multiplexed analysis of proteins in body fluids. In the presented study, we investigated blood plasma within osteoporosis to discovery differential protein profiles and to propose novel biomarkers candidates for subsequent studies. EXPERIMENTAL DESIGN: Starting with 4608 antibodies and plasma samples from 22 women for an untargeted screening, a set of 72 proteins were suggested for further analysis. Complementing these with targets from literature and other studies, a targeted bead array of 180 antibodies was built to profile for 92 proteins in plasma samples of 180 women from two independent population-based studies. RESULTS: Differential profiles between osteoporosis patients and matched controls were discovered for 12 proteins in at least one of the two study sets. Among these targets, the levels of autocrine motility factor receptor (AMFR) were concordantly lower in plasma of female osteoporosis patients. Subsequently, verification of anti-AMFR antibody selectivity was conducted using high-density peptide and protein arrays, and Western blotting. CONCLUSIONS AND CLINICAL RELEVANCE: Further validation in additional study sets will be needed to determine the clinical value of the observed decrease in AMFR plasma levels in osteoporosis patients, but AMFR may aid our understanding of disease mechanisms and could support existing tools for diagnosis and monitoring of patient mobility within osteoporosis.

Affinity proteomic profiling of plasma, cerebrospinal fluid, and brain tissue within multiple sclerosis.

The brain is a vital organ and because it is well shielded from the outside environment, possibilities for noninvasive analysis are often limited. Instead, fluids taken from the spinal cord or circulatory system are preferred sources for the discovery of candidate markers within neurological diseases. In the context of multiple sclerosis (MS), we applied an affinity proteomic strategy and screened 22 plasma samples with 4595 antibodies (3450 genes) on bead arrays, then defined 375 antibodies (334 genes) for targeted analysis in a set of 172 samples and finally used 101 antibodies (43 genes) on 443 plasma as well as 573 cerebrospinal spinal fluid (CSF) samples. This revealed alteration of protein profiles in relation to MS subtypes for IRF8, IL7, METTL14, SLC30A7, and GAP43. Respective antibodies were subsequently used for immunofluorescence on human post-mortem brain tissue with MS pathology for expression and association analysis. There, antibodies for IRF8, IL7, and METTL14 stained neurons in proximity of lesions, which highlighted these candidate protein targets for further studies within MS and brain tissue. The affinity proteomic translation of profiles discovered by profiling human body fluids and tissue provides a powerful strategy to suggest additional candidates to studies of neurological disorders.

Identification of candidate serum proteins for classifying well-differentiated small intestinal neuroendocrine tumors.

BACKGROUND: Patients with well-differentiated small intestine neuroendocrine tumors (WD-SI-NETs) are most often diagnosed at a metastatic stage of disease, which reduces possibilities for a curative treatment. Thus new approaches for earlier detection and improved monitoring of the disease are required. MATERIALS AND METHODS: Suspension bead arrays targeting 124 unique proteins with antibodies from the Human Protein Atlas were used to profile biotinylated serum samples. Discoveries from a cohort of 77 individuals were followed up in a cohort of 132 individuals both including healthy controls as well as patients with untreated primary WD-SI-NETs, lymph node metastases and liver metastases. RESULTS: A set of 20 antibodies suggested promising proteins for further verification based on technically verified statistical significance. Proceeding, we assessed the classification performance in an independent cohort of patient serum, achieving, classification accuracy of up to 85% with different subsets of antibodies in respective pairwise group comparisons. The protein profiles of nine targets, namely IGFBP2, IGF1, SHKBP1, ETS1, IL1α, STX2, MAML3, EGR3 and XIAP were verified as significant contributors to tumor classification. CONCLUSIONS: We propose new potential protein biomarker candidates for classifying WD-SI-NETs at different stage of disease. Further evaluation of these proteins in larger sample sets and with alternative approaches is needed in order to further improve our understanding of their functional relation to WD-SI-NETs and their eventual use in diagnostics.

Highly multiplexed antibody suspension bead arrays for plasma protein profiling.

Alongside the increasing availability of affinity reagents, antibody microarrays have become a powerful tool to screen for target proteins in complex samples. Applying directly labeled samples onto arrays instead of using sandwich assays offers an approach to facilitate a systematic, high-throughput, and flexible exploration of protein profiles in body fluids such as serum or plasma. As an alternative to planar arrays, a system based on color-coded beads for the creation of antibody arrays in suspension has become available to offer a microtiter plate-based option for screening larger number of samples with variable sets of capture reagents. A procedure was established for analyzing biotinylated samples without the necessity to remove excess labeling substance. We have shown that this assay system allows detecting proteins down into lower pico-molar and higher pg/ml levels with dynamic ranges over three orders of magnitude. Presently, this workflow enables the profiling of 384 samples for up to 384 proteins per assay.

Classification of protein profiles from antibody microarrays using heat and detergent treatment.

Antibody microarrays offer new opportunities for exploring the proteome and to identify biomarker candidates in human serum and plasma. Here, we have investigated the effect of heat and detergents on an antibody-based suspension bead array (SBA) assay using polyclonal antibodies and biotinylated plasma samples. With protein profiles from more than 2300 antibodies generated in 384-plex antibody SBAs, three major classes of heat and detergent susceptibility could be described. The results show that washing of the beads with SDS (rather than Tween) after target binding lowered intensity levels of basically all profiles and that about 50% of the profiles appeared to be lowered to a similar extent by heating of the sample. About 33% of the profiles appeared to be insensitive to heat treatment while another 17% showed a positive influence of heat to yield elevated profiles. The results suggest that the classification of antibodies is driven by the molecular properties of the antibody-antigen interaction and can generally not be predicted based on protein class or Western blot data. The experimental scheme presented here can be used to systematically categorize antibodies and thereby combine antibodies with similar properties into targeted arrays for analysis of plasma and serum.