The acute phase response protein SERPINA3 is increased in tear fluid from the unaffected eyes of patients with unilateral acute anterior uveitis.

BACKGROUND: To identify candidate tear fluid biomarkers in patients with unilateral acute anterior uveitis (AAU) that can aid in the differentiation between these patients and patients with bacterial keratitis or healthy controls. METHODS: Thirteen patients (40.1 ± 16.2 years of age) with unilateral AAU, seven patients with unilateral bacterial keratitis (40.2 ± 15.3 years of age), and 14 healthy subjects (41.1 ± 11.6 years of age) were included. The tear proteome of affected eyes was compared with that of the unaffected eye or healthy controls. Proteins were identified by liquid chromatography tandem mass spectrometry and enzyme-linked immunosorbent assay. RESULTS: Relative protein ratios were detected and calculated for 272 unique proteins. Compared with healthy controls and the unaffected eye, the top upregulated proteins in AAU eyes were submaxillary gland androgen regulated protein 3B (SMR3B) and SMR3A. Similarly, the top upregulated proteins in bacterial keratitis were S100 calcium-binding protein A9 and orosomucoid 2. The acute phase response protein Serpin Family A Member 3 (SERPINA3) was increased in the healthy eye of AAU patients (P = 0.019) compared with healthy controls. Laser flare measurements in affected eyes of AAU patients showed positive logarithmic correlation with SERPINA3 in tear samples of the unaffected eye (P = 0.022). The use of SERPINA3 as a tear biomarker yielded a sensitivity of 85% and a specificity of 71% in detecting patients with AAU in the study population. CONCLUSIONS: The acute phase response protein SERPINA3 was increased in tear samples of unaffected eyes of patients with unilateral AAU compared with healthy controls. This study highlights SERPINA3 as a potential biomarker for AAU. Future research should explore the dynamic properties of SERPINA3 in the tear fluid of active and quiescent uveitis eyes.

Altered protein levels in bone marrow lesions of hip osteoarthritis: Analysis by proteomics and multiplex immunoassays.

AIM: To assess tissue level changes of proteome and cytokine profiles of subchondral bone in hip osteoarthritis (OA) affected by bone marrow lesions (BMLs). We compared significant protein level differences in osteoarthritic bone with BMLs to control bone without bone marrow lesions. METHODS: Subchondral bone biopsies were taken from femoral heads of end-stage osteoarthritis patients with (BML, n = 21) and without (CON, n = 9) BMLs. Proteins were extracted through a standardized Trizol protocol and used in the subsequent analyses. Angiogenesis and bone markers were assessed using multiplex immunoassays (Luminex). Liquid chromatography tandem mass spectrometry (LC-MS/MS) was performed to detect significant differences in proteome and peptide profiles between BML and CON. RESULTS: Multiplex immunoassays revealed increased tissue contents of vascular endothelial growth factors (VEGF-A/C/D), endothelin-1, angiopoietin-2 and interleukin-6 (IL-6) in bone with BMLs compared to control bone, whereas osteoprotegerin levels were reduced. Mass spectrometry demonstrated pronounced increase in the levels of hemoglobin (73-fold), serum albumin (30-fold), alpha-1-antitrypsin (9-fold), apolipoprotein A1 (4.7-fold), pre-laminin-A/C (3.7-fold) and collagen-alpha1-XII (3-fold) in BMLs, while aggrecan core protein (ACAN) and hyaluronan and proteoglycan link protein 1 (HAPL1) decreased 37- and 29-fold respectively. CONCLUSION: Reduced osteoprotegerin, ACAN and HAPL1 are consistent with osteoclastic activation and high remodeling activity in BMLs. The pronounced increase in angiogenesis markers, hemoglobin and serum albumin support the presence of increased vascularity in subchondral bone affected by BMLs in OA. VEGFs and IL-6 are known nociceptive modulators, and increased levels are in keeping with pain being a clinical feature frequently associated with BMLs.

Unilateral acute anterior uveitis is associated with ipsilateral changes in the tear fluid proteome that involves the LXR/RXR pathway.

PURPOSE: To determine whether unilateral acute anterior uveitis (AAU) induces ipsilateral changes in the tear fluid proteome. METHODS: Five patients (25-77 years old) with unilateral AAU were included. Tear fluid samples were obtained using Schirmer's test strips. The healthy eye served as control. Proteins were identified by liquid chromatography tandem mass spectrometry. RESULTS: Two hundred forty-two tear fluid sample proteins were identified, of which 75 were present in at least three patients. Nine proteins were at least 1.5-fold increased, whereas eight were at least 1.5-fold decreased in tears from the diseased eye compared with the healthy eye. APOBEC3A was significantly increased (1.43-fold; P = 0.04), whereas TGM2 was significantly decreased (- 1.21-fold; P = 0.03) in tears from the diseased eye relative to the healthy eye. Ingenuity Pathway Analysis identified LXR/RXR (P < 1.02E-4) as a top canonical pathway. CONCLUSION: Unilateral AAU induced detectable changes in the ipsilateral tear fluid proteome and involvement of the inflammation-associated LXR/RXR pathway.

Vitamin D Increases Glucose Stimulated Insulin Secretion from Insulin Producing Beta Cells (INS1E).

BACKGROUND: Vitamin D affects the pancreatic beta cell function and in vitro studies have shown that vitamin D may influence insulin secretion, apoptosis, and gene regulation. However, the outcomes have differed and there has been uncertainty regarding the effect of different vitamin D metabolites on insulin secretion. OBJECTIVES: We hypothesized that vitamin D could increase insulin secretion in insulin producing beta cells and investigated the effect of 25(OH) vitamin D and 1,25(OH)2 vitamin D on insulin secretion. METHODS: The study was conducted in INS1E cells, an established insulinoma cell line from rat. The cells were divided into three groups; a control group, a group with 1,25(OH)2 vitamin D enriched medium (10 nM), and a group with 25(OH) vitamin D (10 nM) supplemented medium. After 72 hours of treatment, the cells underwent glucose stimulation at different concentrations (0, 5, 11, and 22 mM) for 60 minutes. RESULTS: INS1E cells treated with 1,25(OH)2 vitamin D showed a trend towards increased insulin secretion at all glucose concentrations compared to control cells and at 22 mM glucose, the difference was significant (18.40 +/- 1.97 vs 12.90 +/- 2.22 nmol/L, P < 0.05). However, pretreatment with 25(OH) vitamin D did not show any significant increase in insulin secretion compared to cells without vitamin D treatment. There was no difference in insulin secretion in cells not stimulated with glucose. CONCLUSIONS: Treatment with 1,25(OH)2 vitamin D combined with high levels of glucose increased insulin secretion in INS1E cells, whereas 25(OH) vitamin D had no effect. This suggests that glucose stimulated insulin secretion in INS1E beta cells appears to be related to the type of vitamin D metabolite treatment.

Establishment of a tear protein biomarker panel differentiating between Graves' disease with or without orbitopathy.

BACKGROUND: Graves' orbitopathy (GO) is an autoimmune inflammatory ocular complication and one of the most frequent manifestations of Graves' disease (GD). Clinical judgment of GO is subjective sometimes leading to clinical and therapeutic challenges. Better tools to diagnose this severe complication are warranted. PATIENTS AND METHODS: The aim of the present study was to evaluate tear levels of LYZ, LACRT and AZGP1 in GD patients with or without GO, as possible biomarkers for GO. Tear samples were collected from GD patients with moderate-to-severe GO (n = 21) and no clinical signs of GO (n = 21). Additionally, 18 GD patients with mild GO and 9 patients without GO were included in a further part of the study. RESULTS: Tear levels of LYZ (p < 0.001), LACRT (p = 0.004) and AZGP1 (p = 0.001) were significantly elevated in GD patients with moderate-to-severe GO compared to GD patients without GO. The discriminatory power of the three biomarkers, combined in a panel was confirmed by ROC plot analysis, with an AUC value of 0.93 (sensitivity of 95%; specificity of 80%). Since LYZ showed the best performance in discriminating between GD patients with (moderate-to-severe) and without GO (in combination with limited sample volume available), LYZ levels were also measured in tears from GD patients with mild GO and without GO. Significantly higher levels of LYZ were measured in GD patients with mild GO compared to those without GO (p = 0.003). CONCLUSIONS: We have established a novel three-protein biomarker panel that is able to discriminate between GD patients with and without GO, which might aid in diagnostic evaluation of GO as well as an indicator for disease activity.

Longitudinal plasma metabolic profiles, infant feeding, and islet autoimmunity in the MIDIA study.

AIMS: The aim of this study was to investigate the longitudinal plasma metabolic profiles in healthy infants and the potential association with breastfeeding duration and islet autoantibodies predictive of type 1 diabetes. METHOD: Up to four longitudinal plasma samples from age 3 months from case children who developed islet autoimmunity (n = 29) and autoantibody-negative control children (n = 29) with the HLA DR4-DQ8/DR3-DQ2 genotype were analyzed using two-dimensional gas chromatography coupled to a time-of-flight mass spectrometer for detection of small polar metabolites. RESULTS: Plasma metabolite levels were found to depend strongly on age, with fold changes varying up to 50% from age 3 to 24 months (p < 0.001 after correction for multiple testing). Tyrosine levels tended to be lower in case children, but this was not significant after correction for multiple testing. Ornithine levels were lower in case children compared with the controls at the time of seroconversion, but the difference was not statistically significant after correcting for multiple testing. Breastfeeding for at least 3 months as compared with shorter duration was associated with higher plasma levels of isoleucine, and lower levels of methionine and 3,4-dihydroxybutyric acid at 3 months of age. CONCLUSIONS: Plasma levels of several small, polar metabolites changed with age during early childhood, independent of later islet autoimmunity status and sex. Breastfeeding was associated with higher levels of branched-chain amino acids, and lower levels of methionine and 3,4-dihydroxybutyric acid.

Proteomic analysis of the INS-1E secretome identify novel vitamin D-regulated proteins.

BACKGROUND: Experimental evidence indicates that vitamin D may have a beneficial role in pancreatic ß-cell function. METHODS: In the present study, stable isotope labelling by amino acids in cell culture (SILAC) in combination with liquid chromatography-tandem mass spectrometry was used to quantitatively assess the impact of the active vitamin D metabolite, 1,25-(OH)2 D3 , on global protein expression in INS-1E cell secretome. RESULTS: Twenty-one proteins were found up-regulated (≥1.5 fold changes) and three down-regulated (≤0.67) after treatment of INS-1E cells with 1,25-(OH)2 D3 . Up-regulation of proteins implicated in ß-cell growth and proliferation, such as IGF2, IGFBP7 and gelsolin, suggest that 1,25-(OH)2 D3 has a positive effect on ß-cell growth and proliferation. Moreover, modulations of several proteins implicated in prohormone processing and insulin exocytosis (IGF2, IGFBP7, Scg5, ProSAAS, Fabp5, Ptprn2 and gelsolin) appear to support the hypothesis that 1,25-(OH)2 D3 plays positive modulatory role in insulin processing and secretion. CONCLUSIONS: Together, we reveal a number of novel vitamin D-regulated proteins that may contribute to a better understanding of the reported beneficial effects of vitamin D on pancreatic ß-cells. Copyright © 2016 John Wiley & Sons, Ltd.

Single unit filter-aided method for fast proteomic analysis of tear fluid.

Human tear fluid is a complex mixture containing high concentrations of proteins and is increasingly becoming an important source for studying protein biomarkers of eye-related diseases such as Graves' ophthalmopathy. Today, the Schirmer tear test is the most widely used technique for tear collection. However, sample handling and protein extraction from these strips have been highly challenging. Cutting and removal of the Schirmer strips after extraction, which may lead to sample loss prior to downstream analysis, are some of the challenges to consider. To address some of these limitations, we have developed a single-unit filter-aided method for both sample handling and protein extraction. In addition, we systematically investigated the most suitable conditions for protein extraction from these strips. Among the different extraction conditions applied, extraction with 100 mM ammonium bicarbonate containing 50 mM NaCl resulted in the highest number of identified proteins using one-dimensional liquid chromatography tandem mass spectrometry (LC-MS/MS). Moreover, 1526 proteins were identified when the optimized extraction method was combined with two-dimensional LC-MS/MS analysis, demonstrating the applicability of this novel approach to the study of the tear proteome. This dataset of identified proteins represents a comprehensive catalogue of the tear proteome and may serve as a list for future biomarker research.

Discovery of novel vitamin D-regulated proteins in INS-1 cells: a proteomic approach.

BACKGROUND: Experimental evidence indicates that vitamin D may have a beneficial role in pancreatic ß-cell function. Global gene expression studies have shown that the active metabolite 1,25-dihydroxyvitamin D3 [1,25-(OH)2 D3 ] modulates genes involved in ion transport, lipid metabolism and insulin secretion. METHODS: We employed stable isotope labelling by amino acids in cell culture in combination with liquid chromatography-tandem mass spectrometry to quantitatively assess the impact of two vitamin D metabolites, 1,25-(OH)2 D3 and 25-hydroxyvitamin D3 [25-(OH)D3 ], on global protein expression on a model rat ß-cell line, insulinoma-derived INS-1 cells. RESULTS: Although treatment with 1,25-(OH)2 D3 resulted in 31 differentially expressed proteins, 25-(OH)D3 had no impact on protein expression. Of these 31 proteins, 29 were upregulated, whereas two showed a decrease in abundance. Proteins whose expression levels markedly increased in the presence of 1,25-(OH)2 D3 included Crat, Hmgn2, Protein Tmsbl1 and Gdap1. One of the most important findings in this study is upregulation of proteins implicated in insulin granule motility and insulin exocytosis, suggesting a positive effect on insulin secretion. Moreover, modulation of several membrane transport proteins suggests that 1,25-(OH)2 D3 has an impact on the homeostatic regulation of ions, which is critical for most functions in the ß-cell. CONCLUSIONS: In this study, we discovered a number of novel 1,25-(OH)2 D3 -regulated proteins, which may contribute to a better understanding of the reported beneficial effects of vitamin D on pancreatic ß-cells. All in all, our findings should pave the way for future studies providing insights into molecular mechanisms by which 1,25-(OH)2 D3 regulates protein expression in pancreatic ß-cells.

Tmem27 is upregulated by vitamin D in INS-1 cells and its serum concentrations are low in patients with autoimmune diabetes.

Transmembrane protein 27 (Tmem27), which is expressed in pancreatic ß-cells, plays an important role in insulin secretion and pancreatic ß-cell proliferation. Analysis of the INS-1 cell proteome using stable isotope labeling by amino acids in cell culture (SILAC) in combination with LC-MS identified Tmem27 as the one of most robustly (up to seven-fold) upregulated proteins after treatment with the active metabolite of vitamin D, 1,25-(OH)2D3. Furthermore, we report that Tmem27 which is cleaved and released from, i.e. pancreatic ß-cells, is present in human serum and its levels are significantly lower in subjects with autoimmune diabetes as compared to healthy individuals (13% of the levels). Additionally, Tmem27 correlated positively (0.70) with C-peptide serum levels in healthy subjects. Our data indicate that Tmem27 could be of potential value as a serum marker for the pathogenesis of diabetes and as such may warrant the development of measurement methods with lower limit of detection for its further validation.

Fibrin(ogen) may be an important target for methylglyoxal-derived AGE modification in elastic arteries of humans.

Diabetes is associated with increased risk of cardiovascular disease. Advanced glycation end-products (AGEs) are considered to be a major pathogenic factor for diabetic vascular complications. The levels of AGEs are increased in diabetic patients. We have studied the presence of the major AGE methylglyoxal (MGO)-derived hydroimidazolone in human aorta and carotid arteries, using immunohistochemistry (IHC), western blotting and mass spectrometry. By IHC, MGO-derived modifications were detected mainly associated with cells in intimal thickenings and cells in microvessels in adventitia. In type V lesions MGO-derived AGE was also present, extracellular in the necrotic core and in cells at the border of the core. The highest degree of modification was probably associated with cell nuclei. By western blotting and mass spectrometry fibrin(ogen), the cytoskeleton-associated protein moesin and the nuclear proteins lamin A and C were identified as putative main targets for MGO-derived modification. LC-MS/MS studies of fibrin(ogen) modified in vitro with low concentrations of MGO identified the sites that were most prone to modification. These results indicate that AGE modifications occur preferentially on specific proteins. The modification of these proteins may play a role in vascular dysfunction and development of atherosclerosis in diabetes.

Identification of CJC-1295, a growth-hormone-releasing peptide, in an unknown pharmaceutical preparation.

Several peptide drugs are being manufactured illicitly, and in some cases they are being made available to the public before entering or completing clinical trials. At the request of Norwegian police and customs authorities, unknown pharmaceutical preparations suspected to contain peptide drugs are regularly subjected to analysis. In 2009, an unknown pharmaceutical preparation was submitted for analysis by liquid chromatography-high resolution tandem mass spectrometry (LC-HRMS/MS). The preparation was found to contain a 29 amino acid peptide with a C-terminal amide function. Based on the interpretation of mass spectrometric data, an amino acid sequence was proposed. The sequence is consistent with a peptide currently marketed under the name CJC-1295. CJC-1295 is a releasing factor for growth hormone and is therefore considered a Prohibited Substance under Section S2 of the WADA Prohibited List. This substance has potential performance-enhancing effects, it is readily available, and there is reason to believe that it is being used within the bodybuilding community.

Fractionation and separation of human salivary proteins by pH-gradient ion exchange and reversed phase chromatography coupled to mass spectrometry.

In the present work, a 2-D capillary liquid chromatography method for fractionation and separation of human salivary proteins is demonstrated. Fractionation of proteins according to their pI values was performed in the 1-D employing a strong anion exchange (SAX) column subjected to a wide-range descending pH gradient. Polystyrene-divinylbenzene (PS-DVB) RP columns were used for focusing and subsequent separation of the proteins in the 2-D. The SAX column was presaturated with a high pH buffer (A) consisting of 10 mM amine buffering species, pH 9.0, and elution was performed with a low pH elution buffer (B) having the same buffer composition and concentration as buffer A, but pH 3.5. Isoelectric point fractions eluting from the 1-D column were trapped on PS-DVB trap columns prior to back-flushed elution onto the PS-DVB analytical column for separation of the proteins. The 1-D fraction eluting at pH 9.0-8.7 was chosen for further analysis. After separation on the RP analytical column, nine RP protein fractions were collected and tryptic digested for subsequent analyses by MALDI TOF MS and column switching capillary LC coupled to ESI TOF MS and ESI QTOF MS. Eight proteins and two peptides were identified in the pH 9.0-8.7 fraction using peptide mass fingerprinting and uninterpreted MS/MS data.

Two-dimensional capillary liquid chromatography: pH gradient ion exchange and reversed phase chromatography for rapid separation of proteins.

In the present work, an orthogonal two-dimensional (2D) capillary liquid chromatography (LC) method for fractionation and separation of proteins using wide range pH gradient ion exchange chromatography (IEC) in the first dimension and reversed phase (RP) in the second dimension, is demonstrated. In the first dimension a strong anion exchange (SAX) column subjected to a wide range (10.5-3.5) descending pH gradient was employed, while in the second dimension, a large pore (4,000 A) polystyrene-divinylbenzene (PS-DVB) RP analytical column was used for separation of the protein pH-fractions from the first dimension. The separation power of the off-line 2D method was demonstrated by fractionation and separation of human plasma proteins. Seventeen pH-fractions were manually collected and immediately separated in the second dimension using a column switching capillary RP-LC system. Totally, more than 200 protein peaks were observed in the RP chromatograms of the pH-fractions. On-line 2D analysis was performed for fractionation and separation of ten standard proteins. Two pH-fractions (basic and acidic) from the first dimension were trapped on PS-DVB RP trap columns prior to back-flushed elution onto the analytical RP column for fast separation of the proteins with UV/MS detection.

Isoelectric point separation of proteins by capillary pH-gradient ion-exchange chromatography.

In the present work, isoelectric point (pl) separation of proteins by pH-gradient ion-exchange chromatography (IEC) on packed capillary columns is demonstrated. The development of a miniaturized flow-through pH probe for reliable pH monitoring of the column effluent, which was an important technical challenge for adapting this technique to capillary dimensions, was solved by designing a low microliter per minute flow rate housing to a commercially available micro pH probe. Highly linear outlet pH-gradients within the pH range 8.5-4.0 were obtained when applying simple inexpensive buffers consisting solely of piperazine, N-methylpiperazine and imidazole on 10 cm x 0.32 mm i.d. fused silica capillaries packed with anion-exchange poly(styrene divinylbenzene)-based macroporous materials, i.e. 10 microm Mono P from Amersham Biosciences and 10 microm PL-SAX from PolymerLabs. Furthermore, when using a pH-gradient from 6.8 to 4.3, both columns were able to baseline separate the A and B genetic variants of beta-lactoglobulin, which differ with two amino acid residues only, but the PL-SAX column provided almost a two-fold decrease in peak widths compared to the Mono P column. The influence of varying the buffer concentration, injection volume and column temperature on the peak widths and resolution of the beta-lactoglobulins was investigated, e.g. a 100 microl sample of dilute beta-lactoglobulins was injected directly on the column with practically no increase in peak width as compared to what obtained with conventional injection volumes. Finally, a pH-gradient from 6.8 to 4.3 was used to separate proteins in skimmed bovine milk on the PL-SAX column. The milk was simply diluted 1:10 (v/v) with water and filtrated before injection.