Hexapeptide Library Research Articles

Inhibition of avian influenza virus H9N2 infection by antiviral hexapeptides that target viral attachment to epithelial cells

The H9N2 subtype of influenza A virus circulates frequently among poultry in Asian and North African countries causing economic loss in the poultry sector. The antigenic variations of the H9N2 virus were at the origin of its genetic evolution through the emergence of viral strains transmissible to humans and resistant to chemical antivirals, which require a strengthening of the fight means against this virus. In this study, we used a random linear hexapeptide library fused to the gene III protein of M13 filamentous bacteriophage to select new antiviral peptides that inhibit the infectivity of H9N2 virus. After three rounds of stringent selection and amplification, polyclonal phage-peptides directed against H9N2 virus were assessed by ELISA, and the optimal phage-peptides were grown individually and characterized for binding to H9N2 virus by monoclonal phage ELISA. The DNA of 27 phage-peptides clones was amplified by PCR, sequenced, and their amino acid sequences were deduced. Sixteen different phage-peptides were able to bind specifically the H9N2 virus, among them, 13 phage-peptides interacted with the hemagglutinin H9. Two selected peptides, P1 (LSRMPK) and P2 (FAPRWR) have shown antiviral activity in ovo and P1 was more protective in vivo then P2 when co-administered with the H9N2 virus. Mechanistically, these peptides prevent infection by inhibiting the attachment of the H9N2 virus to the cellular receptor. Molecular docking revealed that the peptides LSRMPK and FAPRWR bind to hemagglutinin protein H9, but interact differently with the receptor binding site (RBS). The present study demonstrated that the peptide P1 (LSRMPK) could be used as a new inhibitory molecule directed against the H9N2 virus.

Identification of B Cell and T Cell Epitopes Using Synthetic Peptide Combinatorial Libraries.

This article presents a combinatorial library method that consists of the synthesis and screening of mixture-based synthetic combinatorial libraries of peptide molecules to identify B and T cell epitopes. The protocols employ peptide libraries to identify peptides recognized by MAbs and T cells. The first protocol uses a positional scanning peptide library made up of hexapeptides to identify antigenic determinants recognized by MAbs. The 120 mixtures in the hexapeptide library are tested for their inhibitory activity in a competitive ELISA. The second protocol uses a decapeptide library to identify T cell peptide ligands. The 200 mixtures of the decapeptide library are tested for their ability to induce T cell activation. Support protocols cover optimization of the assay conditions for each MAb or T cell, to achieve the best level of sensitivity and reproducibility, and preparation of a hexapeptide library, along with deconvolution approaches. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Screening peptide library for antibody inhibition Basic Protocol 2: Screening a peptide library to identify CD4+ Or CD8+ T cell ligands Support Protocol 1: Optimizing antigen and antibody concentrations for screening assay Support Protocol 2: Preparing a positional scanning peptide library.

In Silico Approach for Designing Potent Neuroprotective Hexapeptide.

Alzheimer's disease (AD) is a constantly recurring neurodegenerative disease that deteriorates over a period of time. In this pathology, connections between neurons become extremely damaged due to the deposition of senile plaques in the membrane region, which results in abnormal signal transduction processes. Also, the intracellular microtubule networks are disrupted in the hyperphosphorylated tau cascade of AD. Therefore, design and development of potent neuroprotective molecules that can instantaneously target multiple facets of AD pathogenesis are greatly needed to tackle this unmet medical need. Here, we have implemented a pharmacophore based in silico analysis of various neuroprotective peptides known for neurotherapeutic application in AD. Fascinatingly, we have identified an active core of these peptides and designed a library of hexapeptides. We observed that peptide "LETVNQ" (LE6) has shown significant protection ability against degeneration of neurons. Experimental evidence suggests that this peptide immensely reduced the aggregation rate of amyloid-β (Aβ) and helped in microtubule polymerization. Intriguingly, this newly designed peptide does not have any cytotoxicity toward differentiated PC12 neurons; rather it helps in neurite outgrowth. Further, LE6 helps to maintain the complex microtubule network in cells by promoting the polymerization rate of intracellular microtubules and mediates excellent protection of neurons even after removal of nerve growth factor (NGF). Finally, we observed that this LE6 peptide has substantial stability under physiological conditions and helps to retain healthy morphology of primary rat cortical neurons. This excellent piece of work identifies a potent hexapeptide, which has exceptional ability to protect neurons as well as microtubule from degeneration and may become potent therapeutics against AD pathogenesis in the future.

High throughput protease profiling comprehensively defines active site specificity for thrombin and ADAMTS13

We have combined random 6 amino acid substrate phage display with high throughput sequencing to comprehensively define the active site specificity of the serine protease thrombin and the metalloprotease ADAMTS13. The substrate motif for thrombin was determined by >6,700 cleaved peptides, and was highly concordant with previous studies. In contrast, ADAMTS13 cleaved only 96 peptides (out of >107 sequences), with no apparent consensus motif. However, when the hexapeptide library was substituted into the P3-P3′ interval of VWF73, an exosite-engaging substrate of ADAMTS13, 1670 unique peptides were cleaved. ADAMTS13 exhibited a general preference for aliphatic amino acids throughout the P3-P3′ interval, except at P2 where Arg was tolerated. The cleaved peptides assembled into a motif dominated by P3 Leu, and bulky aliphatic residues at P1 and P1′. Overall, the P3-P2′ amino acid sequence of von Willebrand Factor appears optimally evolved for ADAMTS13 recognition. These data confirm the critical role of exosite engagement for substrates to gain access to the active site of ADAMTS13, and define the substrate recognition motif for ADAMTS13. Combining substrate phage display with high throughput sequencing is a powerful approach for comprehensively defining the active site specificity of proteases.

CycLS: Accurate, whole-library sequencing of cyclic peptides using tandem mass spectrometry

Cyclic peptides are of great interest as therapeutic compounds due to their potential for specificity and intracellular activity, but specific compounds can be difficult to identify from large libraries without resorting to molecular encoding techniques. Large libraries of cyclic peptides are often DNA-encoded or linearized before sequencing, but both of those deconvolution strategies constrain the chemistry, assays, and quantification methods which can be used. We developed an automated sequencing program, CycLS, to identify cyclic peptides contained within large synthetic libraries. CycLS facilitates quick and easy identification of all library-members via tandem mass spectrometry data without requiring any specific chemical moieties or modifications within the library. Validation of CycLS against a library of 400 cyclic hexapeptide peptoid hybrids (peptomers) of unique mass yielded a result of 95% accuracy when compared against a simulated library size of 234,256 compounds. CycLS was also evaluated by resynthesizing pure compounds from a separate 1800-member library of cyclic hexapeptides and hexapeptomers with high mass redundancy. Of 22 peptides resynthesized, 17 recapitulated the retention times and fragmentation patterns assigned to them from the whole-library bulk assay results. Implementing a database-matching approach, CycLS is fast and provides a robust method for sequencing cyclic peptides that is particularly applicable to the deconvolution of synthetic libraries.

Influence of different sample preparation strategies on the proteomic identification of stress biomarkers in porcine saliva

BackgroundThe influence of two different sample treatments comprising the enrichment of glycoproteins by boronic acid and dynamic range compression by hexapeptide libraries, on the detection of stress markers in saliva of pigs was evaluated in this study. For this purpose, saliva samples collected before and after the application of an acute stress model consisting of nasal restraining in pigs were processed without any treatment and with the two different treatments mentioned above. Protein separation by two-dimensional gel electrophoresis (2-DE) followed by identification of proteins using MALDI-TOF/TOF mass spectrometry (MS) was used as proteomic technique.ResultsThe application of each of the two different sample treatment protocols allowed the identification of unique proteins that could be potential salivary acute stress markers in pigs: lipocalin 1, protein S100-A8 and immunoglobulin M by enrichment of glycoproteins; protein S100-A9, double headed protease inhibitor submandibular gland, and haemoglobin by dynamic range compression; and protein S100-A12 by both protocols. Salivary lipocalin, prolactin inducible protein, light chain of immunoglobulins, adenosine deaminase and carbonic anhydrase VI were identified as potential markers in untreated saliva as well as one of the other treatments.ConclusionThe use of different procedures allowed the detection of different potential stress markers. Although from a practical point of view, the use of saliva without further treatment as well as the enrichment of glycoproteins are less expensive and easy to do procedures.

Epitope identification of specific naturally occurring human anti-avidin antibodies

Human serum contains natural antibodies against avidin. Affinity purified natural anti-avidin human IgG exhibits affinity constants comparable to those of antibodies produced by active immunization of rabbits. Using a random hexapeptide library displayed on the filamentous M13 phage, and rabbit anti-avidin purified antibodies as a selector, we searched for epitopes shared by both selector and natural human anti-avidin IgG. This approach, enabled the isolation and identification of phagotopes bearing consensus motifs similar to sequence stretches of the avidin loops and β-sheet regions. These phagotopes were recognized by the natural human anti-avidin antibodies. The fact that natural anti-avidin antibodies in human serum have similar epitopes to those of IgG elicited by active immunization of animals, led us to suggest that small peptide epitopes may prevent deleterious effects caused by antibodies formed against food proteins as well as therapeutic proteins.

Targeting Leishmania major parasite with peptides derived from a combinatorial phage display library

Cutaneous leishmaniasis (CL) is a global problem caused by intracellular protozoan pathogens of the genus Leishmania for which there are no suitable vaccine or chemotherapy options. Thus, de novo identification of small molecules binding to the Leishmania parasites by direct screening is a promising and appropriate alternative strategy for the development of new drugs. In this study, we used a random linear hexapeptide library fused to the gene III protein of M13 filamentous bacteriophage to select binding peptides to metacyclic promastigotes from a highly virulent strain of Leishmania major (Zymodeme MON-25; MHOM/TN/94/GLC94). After four rounds of stringent selection and amplification, polyclonal and monoclonal phage-peptides directed against L. major metacyclic promastigotes were assessed by ELISA, and the optimal phage-peptides were grown individually and characterized for binding to L. major by monoclonal phage ELISA. The DNA of 42 phage-peptides clones was amplified by PCR, sequenced, and their amino acid sequences deduced. Six different peptide sequences were obtained with frequencies of occurrence ranging from 2.3% to 85.7%. The biological effect of the peptides was assessed in vitro on human monocytes infected with L. major metacyclic promastigotes, and in vivo on susceptible parasite-infected BALB/c mice. The development of cutaneous lesions in the right hind footpads of infected mice after 13 weeks post-infection showed a protection rate of 81.94% with the injected peptide P2. Moreover, Western blots revealed that the P2 peptide interacted with the major surface protease gp63, a protein of 63kDa molecular weight. Moreover, bioinformatics were used to predict the interaction between peptides and the major surface molecule of the L. major. The molecular docking showed that the P2 peptide has the minimum interaction energy and maximum shape complimentarity with the L. major gp63 active site. Our study demonstrated that the P2 peptide occurs at high frequency during the screening procedure, best inhibits L. major growth kinetics in vitro, and reduces cutaneous lesions in BALB/c mice, thus showing great promise in the development of new therapeutic molecules.

A new proteinase 3 substrate with improved selectivity over human neutrophil elastase

We report the synthesis and enzymatic studies on a new proteinase 3 intermolecular quenched substrate with enhanced selectivity over neutrophil elastase. Using combinatorial chemistry methods, we were able to synthesize the hexapeptide library with the general formula ABZ-Tyr-Tyr-Abu-X1′-X2′-X3′-Tyr(3-NO2)-NH2 using the mix and split method. The iterative deconvolution of such a library allowed us to obtain the sequence ABZ-Tyr-Tyr-Abu-Asn-Glu-Pro-Tyr(3-NO2)-NH2 with a high specificity constant (kcat/KM=1534×103M−1s−1) and superior selectivity over neutrophil elastase and other neutrophil-derived serine proteases. Moreover, using the obtained substrate, we were able to detect a picomolar concentration of proteinase 3 (PR3). Incubation of the above-mentioned substrate with neutrophil lysate resulted in a strong fluorescent signal that was significantly reduced in the presence of a PR3 selective inhibitor.

Peptides Trapping Dioxins: A Docking‐Based Inverse Screening Approach

A rapid and cost‐effective computational methodology for designing and rationalizing the selection of small peptides as receptors for dioxin‐like compounds was proposed. The backbone of the dioxin Ah receptor binding site was used to design a series of penta‐ and hexapeptide libraries, with 1400 elements in total. Peptide flexibility was considered and 10 conformers were found to be a good option to represent peptide conformational space with fair speed‐accuracy ratio. Each peptide conformer was treated as a possible receptor, generating a dedicated box and then running a docking process using as ligands a family of 76 dibenzo‐p‐dioxins and 113 dibenzofurans mono‐ and polychlorinated. Significant predictions were confirmed by comparing primary structure of top and bottom ranked peptides binding dioxins confirming that scrambled positions of the same amino acids gave completely different predicted binding. The hexapeptide EWFQPW, with the best binding score, was chosen as selective sorbent material in solid‐phase extraction. The retention performances were tested using the 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin and two polychlorinated biphenyls in order to verify the hexapeptide specificity. The solid‐phase extraction experimental procedure was optimized, and analytical parameters of hexapeptide sorbent material were compared with the resin without hexapeptide and a commercial reversed phase cartridge.

Chemokine CCR3 ligands-binding peptides derived from a random phage-epitope library

Eosinophils are major effectors cells implicated in a number of chronic inflammatory diseases in humans, particularly bronchial asthma and allergic rhinitis. The human chemokine receptor C-C receptor 3 (hCCR3) provides a mechanism for the recruitment of eosinophils into tissue and thus has recently become an attractive biological target for therapeutic intervention. In order to develop peptides antagonists of hCCR3-hCCL11 (human eotaxin) interactions, a random bacteriophage hexapeptide library was used to map structural features of hCCR3 by determining the epitopes of neutralizing anti-hCCR3 mAb 7B11. This mAb t is selective for hCCR3 and exhibit potent antagonist activity in receptor binding and functional assays. After three rounds of biopanning, four mAb7B11-binding peptides were identified from a 6-mer linear peptide library. The phage bearing the peptides showed specific binding to immobilized mAb 7B11 with over 94% of phages bound being competitively inhibited by free synthetic peptides. In FACScan analysis all selected phage peptides were able to strongly inhibit the binding of mAb 7B11 to hCCR3-transfected preB-300-19 murine cells. Furthermore, synthetic peptides of the corresponding phage epitopes were effective in blocking the antibody–hCCR3 interactions and to inhibit the binding of hCCL11 to hCCR3 transfectants. Chemically synthesized peptides CKGERF, FERKGK, SSMKVK and RHVSSQ, effectively competed for 125I-hCCL11 binding to hCCR3 with IC50 ranging from 3.5 to 9.7μM. Calcium release and chemotaxis of hCCR3 transfectants or human eosinophils were inhibited by all peptides in a dose-dependent manner. Furthermore, they showed inhibitory effects on chemotaxis of human eosinophils induced by hCCL11, hCCL5, hCCL7, hCCL8, and hCCL24. Specificities of all selected peptides were assessed with hCXCR1, hCXCR2, hCXCR3, and hCCR5 receptors. Peptides CKGERF and FERKGK showed inhibitory effects on eosinophil chemotaxis in a murine model of mCCL11-induced peritoneal eosinophilia. The development of peptides inhibiting the interactions between hCCR3 and its chemokine ligands will facilitate the development of small peptides antagonists with the hope of ameliorating chronic inflammatory diseases in humans.

Identification of B cell and T cell epitopes using synthetic peptide combinatorial libraries.

This unit presents a combinatorial library method that consists of the synthesis and screening of mixture-based synthetic combinatorial libraries of peptide molecules. The protocols employ peptide libraries to identify peptides recognized by MAbs and T cells. The first protocol uses a positional scanning peptide library made up of hexapeptides to identify antigenic determinants recognized by MAbs. The 120 mixtures in the hexapeptide library are tested for their inhibitory activity in a competitive ELISA. The second protocol uses a decapeptide library to identify T cell peptide ligands. The 200 mixtures of the decapeptide library are tested for their ability to induce T cell activation. Support protocols cover optimization of the assay conditions for each MAb or T cell, to achieve the best level of sensitivity and reproducibility, and preparation of a hexapeptide library, along with deconvolution approaches.

Workflow for analysis of high mass accuracy salivary data set using MaxQuant and ProteinPilot search algorithm

LTQ Orbitrap data analyzed with ProteinPilot can be further improved by MaxQuant raw data processing, which utilizes precursor-level high mass accuracy data for peak processing and MGF creation. In particular, ProteinPilot results from MaxQuant-processed peaklists for Orbitrap data sets resulted in improved spectral utilization due to an improved peaklist quality with higher precision and high precursor mass accuracy (HPMA). The output and postsearch analysis tools of both workflows were utilized for previously unexplored features of a three-dimensional fractionated and hexapeptide library (ProteoMiner) treated whole saliva data set comprising 200 fractions. ProteinPilot's ability to simultaneously predict multiple modifications showed an advantage from ProteoMiner treatment for modified peptide identification. We demonstrate that complementary approaches in the analysis pipeline provide comprehensive results for the whole saliva data set acquired on an LTQ Orbitrap. Overall our results establish a workflow for improved protein identification from high mass accuracy data.

Introducing an Asp-Pro Linker in the Synthesis of Random One-Bead-One-Compound Hexapeptide Libraries Compatible with ESI-MS Analysis

A random hexapeptide library (one-bead-one-compound), containing sixteen amino acids (16(6) different sequences) was synthesized on a Tentagel resin previously modified with a dipeptide linker (Asp-Pro). This peptide bond is highly susceptible to cleavage under mild acidic conditions in a salt-free solution prepared with H(2)(16)O/H(2)(18)O (60/40% v/v). In the hydrolysis, hexapeptides are released with an additional Asp residue partially labeled with (18)O at the C-terminus. These conditions are fully compatible with ESI-MS analysis and facilitate sequencing by MS, as N- and C-terminal ions can be easily differentiated in MS/MS spectra. The peptides were sequenced manually and also with de novo sequencing programs, and identifying them in a database containing all possible heptapeptide sequences or in a filtered database. The proposed strategy is also compatible with stepwise Edman degradation using either intact beads or the released free peptides.

Changes in Serum Protein Profiles of Chickens with Tibial Dyschondroplasia

Differences in serum protein profiles were analyzed to identify possible biomarkers associated with a poultry leg problem named tibial dyschondroplasia (TD) that can lead to lameness. A bead-based affinity matrix (ProteoMiner TM ) containing a combinatorial library of hexapeptides was used to deplete high abundant proteins and enrich the less abundant ones to compare between the sera of six-week old normal and TD affected chickens. Equal amounts of proteins in ProteoMiner depleted serum from control and TD-affected birds were subject to 2D gel electrophoresis, image analysis, and compared to identify the differentially expressed protein spots. The protein spots were characterized using in-gel trypsin digestion followed by mass spectrometry (MS). Of 46 matched protein spots in the gels, 33 were identified by peptide mass finger printing (PMF) and tandem mass spectrometry (MS/MS). Eight spots corresponding to immunoglobulins (Ig) were up-regulated in birds with TD and two spots down regulated. The up-regulated Ig proteins belonged to IgM and IgY(IgG) classes indicated by the identification of 'mu' chain and Fc fragment associated peptides respectively. Enzyme linked immunosorbent assay corroborated an increase in serum IgM levels but not IgG. Although the significance of the increase in IgM proteins in the serum of TD-affected chickens is not understood, it is likely that IgM plays some role in the removal of apoptotic chondrocytes which abound within TD lesions.

Non-Biased Enrichment Does Not Improve Quantitative Proteomic Delineation of Reovirus T3D-Infected HeLa Cell Protein Alterations

Mass spectrometry-based methods have allowed elucidation of alterations in complex proteomes, such as eukaryotic cells. Such studies have identified and measured relative abundances of thousands of host proteins after cells are infected with a virus. One of the potential limitations in such studies is that generally only the most abundant proteins are identified, leaving the deep richness of the cellular proteome largely unexplored. We differentially labeled HeLa cells with light and heavy stable isotopic forms of lysine and arginine and infected cells with reovirus strain T3D. Cells were harvested at 24 h post-infection. Heavy-labeled infected and light-labeled mock-infected cells were mixed together 1:1. Cells were then divided into cytosol and nuclear fractions and each fraction analyzed, both by standard 2D-HPLC/MS, and also after each fraction had been reacted with a random hexapeptide library (Proteominer® beads) to attempt to enrich for low-abundance cellular proteins. A total of 2,736 proteins were identified by two or more peptides at >99% confidence, of which 66 were significantly up-regulated and 67 were significantly down-regulated. Up-regulated proteins included those involved in antimicrobial and antiviral responses, GTPase activity, nucleotide binding, interferon signaling, and enzymes associated with energy generation. Down-regulated proteins included those involved in cell and biological adhesion, regulation of cell proliferation, structural molecule activity, and numerous molecular binding activities. Comparisons of the r2 correlations, degree of dataset overlap, and numbers of peptides detected suggest that non-biased enrichment approaches may not provide additional data to allow deeper quantitative and comparative mining of complex proteomes.

Hexapeptide library as a universal tool for sample preparation in protein glycosylation analysis

Recent analytical advancements allow for large-scale glycomics and glycan-biomarker research with N-glycans released from complex protein mixtures of e.g. plasma with a wide range of protein concentrations. Protein enrichment techniques to obtain samples with a better representation of low-abundance proteins are hardy applied. In this study, hexapeptide ligands previously described for enrichment of low-abundance proteins in proteomics are evaluated for glycan analysis. A repeatable on-bead glycan release strategy was developed, and glycans were analyzed using capillary sieving electrophoresis on a DNA analyzer. Binding of proteins to the hexapeptide library occurred via the protein backbone. At neutral pH no discrimination between protein glycoforms was observed. Interestingly, glycan profiles of plasma with and without hexapeptide library enrichment revealed very similar patterns, despite the vast changes in protein concentrations in the samples. The most significant differences in glycosylation profiles were ascribed to a reduction in immunoglobulin-derived glycans. These results suggest that specific and sensitive biomarkers will be hard to access on the full plasma level using protein enrichment in combination with glycan analysis. Instead, fractionation techniques or profiling strategies on the glycopeptide level after enrichment are proposed for in-depth glycoproteomics research.

Decreased concentrations of retinol-binding protein 4 in sera of epithelial ovarian cancer patients: A potential biomarker identified by proteomics

Ovarian cancer is the fifth leading cause of cancer death in women. Absence of a reliable biomarker precludes early diagnosis of the disease. To identify new proteins with potential diagnostic or prognostic value for the therapy of ovarian cancer we performed comparative proteomic analysis of sera from ovarian cancer patients and healthy women. We analyzed serum samples from 10 patients diagnosed with epithelial ovarian cancer and 10 age-matched healthy women. To decrease the extremely wide dynamic range of protein concentrations in serum we used combinatorial hexapeptide libraries. Serum samples were then subjected to proteomic 2-DE analysis. Three proteins with differential abundance were found and identified by mass spectrometry: α-1-antitrypsin, apolipoprotein A-IV and retinol-binding protein4. Identification of α-1-antitrypsin and apolipoprotein A-IV confirms previous studies but the identification of significantly decreased levels of RBP4 in ovarian cancer patients represents a novel observation. We verified the decrease of RBP4 levels in ovarian cancer patient sera by two independent methods and determined absolute RBP4 concentrations in patients and healthy women. We excluded possible non-cancer factors that could be responsible for the observed RBP4 decrease. We propose a connection of RBP4 with epithelial ovarian cancer and advocate the potential of RBP4 as a candidate diagnostic or prognostic biomarker.

Metabolically Stable Cellular Adhesion to Inert Surfaces

The structure of D-amino acid hexapeptides that promote cellular adhesion was determined by screening D-amino acid hexapeptide libraries synthesized on otherwise inert beaded PEGA resin. These new adhesion molecules provide a completely stable cellular environment and facilitate the maintenance of a monolayer of cells on beads for extended periods. The presence of the peptides promotes spreading of the cells on the bead surface. Not surprisingly, the molecules contained a significant number of arginines and/or lysines. However, the exact structure of each peptide is quite important for the degree of adhesion observed, and a motif with three or four basic amino acids spaced within amino acids of intermediate polarity clearly prevailed, for example, k-l/r-h-r-i/v-r-a; this maintains a polar/hydrophobic balance.

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.