pI-separation of proteins by capillary pH-gradient ion-exchange chromatography

In the present work, pI-separation of proteins by pH-gradient ion-exchange chromatography (IEC) on packed capillary columns is demonstrated. An important technical challenge for adapting this technique to capillary dimensions, i.e. the development of a miniaturized flow-through pH probe for reliable pH monitoring of the column effluent, was solved by designing a low ÝL/min flow rate housing to a commercially available micro pH probe. Highly linear outlet pH gradients within the pH range 8.5 to 4.0 were obtained when applying simple buffering species consisting solely of piperazine, N-methylpiperazine and imidazole on 0.32 mm ID x 10 cm fused silica capillaries packed with anion-exchange polystyrene divinylbenzene (PS-DVB)-based macroporous materials, i.e. 10 Ým Mono P and 10 Ým PL-SAX. When using a pH gradient from pH 6.8 to 4.0, both columns were able to baseline separate the A and B genetic variants of £]-lactoglobulin, which differ with two amino acid residues only. However, the PL-SAX column provided almost a two-fold decrease in peak width compared to the Mono P column. The influence of varying the buffer concentration, injection volume and column temperature on peak widths and resolution of £]-lactoglobulins was investigated, e.g., a 100 ÝL sample of dilute £]-lactoglobulins was injected directly on the column with practically no increase in peak width as compared to what obtained with conventional injection volumes. In addition, slightly better resolution was obtained with 10 mM buffers than with the other concentrations, and the narrowest peaks and best resolution were obtained between 30 and 50 oC. Furthermore, a pH gradient, which was set to decrease from the pH of the start buffer (i.e. 10 mM piperazine and N-methylpiperazine, pH = 6.8) to the pH of the eluting buffer (i.e. 10 mM piperazine and N-methylpiperazine, pH = 4.3) in 25 min, was used to fractionate 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 (5 ÝL), and the within-day repeatability (n = 6) of the retention time, peak width and peak area ranged from 0.3-3.0%, 3.1-6.9%, and 4.0-11.5%, respectively. Finally, heart-cut 2D separation was performed, where bovine milk pI-fractions from the pH-gradient IEC column (first dimension) were injected on-line to a C8 reversed phase column (second dimension) where further separation according to hydrophobicity was achieved

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 (moderateto-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)

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