Analysis Of Tear Proteins Research Articles

Experimental design considerations for studies of human tear proteins

PurposeHuman tears contain abundant, diverse sets of proteins that may serve as biomarkers of ocular surface health. There is a need for reproducible methods that consider multiple factors influencing the tear proteome, in addition to the variable of interest. Here we examined a workflow for proteomic analysis of tear proteins without the need to pool tear samples from multiple individuals, thus allowing for analyses based on individual factors, and increasing opportunities for protein biomarker discovery. MethodsTears were collected by Schirmer strip following topical ocular anesthetic application then individually stored at −80 °C prior to processing for proteomics. Tear proteins were extracted from Schirmer strips, digested using suspension trapping spin columns (S-Trap), and labeled with high multiplicity tandem mass tags (TMT). Peptide digests were then extensively fractionated by two-dimensional chromatography and analyzed by mass spectrometry to identify and measure changes in protein abundance in each sample. Analysis of select samples was performed to test protocols and to compare the impact of clinically relevant parameters. To facilitate comparison of separate TMT experiments, common pool samples were included in each TMT instrument run and internal reference scaling (IRS) was performed. ResultsDifferences in subsets of tear proteins were noted for: geographic site of tear collection, contact lens use, and differences in tear fluid volume among individuals. ConclusionThese findings demonstrate that proteomic analysis of human tear proteins can be performed without the need to pool samples, and that development of analytic workflows must consider factors that may affect outcomes in studies focused on diverse clinical samples.

Analysis and comparison of tear protein profiles in dogs using different tear collection methods

BackgroundTear proteomic analysis has become an important tool in medical and veterinary research. The tear collection method could influence the tear protein profile. This study aims to evaluate the protein profiles of dog tears collected using microcapillary tubes (MT), Schirmer tear strips (ST), and ophthalmic sponges (OS).MethodsThe tear samples were collected using MT, ST, and OS. Tear protein profiles were analyzed using two-dimensional electrophoresis (2-DE) and the different protein spots’ expression was compared. Fourteen protein spots were identified using liquid chromatography-tandem mass spectrometry (LC-MS/MS).ResultsTear protein concentrations ranged from 2.80 to 4.03 μg/μL, with no statistically significant differences among collection methods. Protein expression in each collection method differed in terms of both the number and intensity of the spots. There were 249, 327, and 330 protein spots found from tears collected with MT, ST, and OS, respectively. The proteins albumin, haptoglobin, and lactoferrin identified from OS were found to have higher spot intensities than other methods of collection. The use of MT demonstrated the downregulation of nine proteins.ConclusionsThe recent study supported that tear protein analysis is affected by different tear collection methods. Although ST is commonly used for tear collection, it provides insufficient information to study particular tear proteins.

Longitudinal Tear Protein Changes Correlate with Ocular Chronic GVHD Development in Allogeneic Hematopoietic Stem Cell Transplant Patients

Ocular graft-versus-host disease (oGVHD) is a manifestation of chronic GVHD, frequently occurring in patients after allogeneic hematopoietic stem cell transplant (HSCT). We analyzed tear protein changes before and after allogeneic HSCT, and correlated their levels with the oGVHD development. This retrospective study included 102 patients, and data were recorded before the conditioning treatment, and after 3 to 6 months postoperatively. Tear protein analysis was performed with the Agilent-2100 Bioanalyzer on individual tears sampled by aspiration. Total protein (TP), Lysozyme-C (LYS-C), Lactoferrin (LACTO), Lipocalin-1 (LIPOC-1), Transferrin (TRANSF), Albumin (ALB), and Zinc-alpha-2-glycoprotein (ZAG-2) levels were retrieved and statistically analyzed. Following HSCT forty-three patients developed oGVHD. TP, LACTO, LYS-C, and ZAG-2 levels significantly decreased post-HSCT as compared to pre HSCT levels. In univariate analysis, TP, LACTO, and ZAG-2 decrease was associated with an increased development of oGVHD (OR = 4.49; 95% CI, 1.9 to 10.5; p < 0.001; OR = 3.08; 95% CI 1.3 to 7.6; p = 0.01; OR = 11.1; 95% CI 2.7 to 46.6; p < 0.001, respectively). TRANSF post-HSCT levels significantly increased (OR 15.7; 95% CI, 4.1 to 52.2; p = 0.0001). No pre-post-HSCT changes were shown in ALB and LIPOC-1 levels. Data suggest that TP content, LACTO, TRANSF, and ZAG-2 pre-post changes might be significant predictors of oGVHD development.

Tear protein analysis in patients with primary acquired nasolacrimal duct obstruction treated with lacrimal passage intubation.

The pathophysiology of nasolacrimal duct obstruction (NLDO) may involve quantitative as well as qualitative changes in tears. We measured tear protein concentrations in patients with primary acquired NLDO and compared them with the tear proteins in healthy individuals and patients with dry eye disease. Case-control study. Twenty-four patients diagnosed with primary acquired NLDO who underwent endoscopic lacrimal passage intubation were included in the study. Tear fluid was collected with Schirmer's test strips three times: before intubation, after intubation while the nasolacrimal tube was in place, and after extubation. At the same time, 24 age-matched normal controls and 24 patients with dry eye were selected from subjects whose tears were collected during the same study period. We measured the following components of tear fluid: total protein, lactoferrin, albumin, and interleukin 6 (IL-6) levels. Total proteins and albumin levels in tears of the NLDO group were significantly lower than those of the control group (P < 0.001 and P = 0.014, respectively, Mann-Whitney U test). Total proteins, albumin, and lactoferrin levels were significantly increased after extubation of nasolacrimal tube (P < 0.001, P = 0.004, and P = 0.029, respectively, Wilcoxon matched-pairs signed-rank test). There were no statistically significant differences in total protein, albumin, lactoferrin and IL-6 levels between patients with NLDO after lacrimal passage extubation and controls. Our study shows distinct differences in protein composition of tear fluid in NLDO eyes and demonstrates that these can be normalized by nasolacrimal tube placement.

Tear proteome in uveitis

AbstractPurposeUveitis represents the fourth commonest cause of legal blindness in adult patients. Analysis of tear fluid proteins in acute anterior uveitis (AAU) patients could represent a novel diagnostic modality. This study aimed at detecting potential tear fluid biomarkers in patients with AAU and bacterial keratitis (BK).MethodsFourteen patients with unilateral AAU, seven patients with bacterial keratitis (BK) and 14 healthy age‐ and sex‐matched control subjects were included. Tear fluid samples were obtained by inserting two Schirmer’s test strips behind the lower eyelid of each eye for five minutes without anesthesia. Proteins were then identified by liquid chromatography tandem mass spectrometry. Pooled tear fluid samples from healthy controls were compared with samples from AAU and BK patients, respectively. Ingenuity Pathway Analysis (IPA) was used to explore biological function.ResultsA relative protein level was obtained for 235 proteins in AAU patients and 198 proteins in BK patients, while using healthy control subjects as reference. In AAU patients, nine proteins were at least two‐fold up‐regulated, whereas in BK patients, 12 proteins were up‐regulated more than two‐fold. None of the top ten upregulated proteins in AAU and BK overlapped. The top canonical pathway identified by IPA was ‘Acute Phase Response Signaling’ in AAU and ‘LXR/RXR activation’ in BK. Top upstream activated regulators included peroxisome proliferator activated receptor delta in AAU and interleukin 6 in BK.ConclusionsTear fluid protein analysis represents a promising diagnostic modality for ocular inflammatory diseases and demonstrate differentially expressed proteins in acute anterior uveitis and bacterial keratitis.

Combined Targeted Analysis of Metabolites and Proteins in Tear Fluid With Regard to Clinical Applications.

PurposeTo establish a robust workflow for combined mass spectrometry–based analysis of metabolites and proteins in tear fluid with regard to clinical applicability.MethodsTear fluid was taken from 12 healthy volunteers at different time points using specially designed Schirmer strips. Following the liquid extraction of metabolites from standardized punches, the remaining material was processed for bottom-up proteomics. Targeted metabolite profiling was performed adapting a metabolomics kit, which targets 188 metabolites from four different analyte classes. Proteomics was performed of the identical samples targeting 15 tear proteins relevant to ocular health.ResultsSixty metabolites could be consistently determined in all tear samples (98 metabolites were detectable in average) covering acylcarnitines, amino acids, biogenic amines, and glycerophospholipids. Following normalization, the majority of metabolites exhibited intraindividual variances of less than 20%, both regarding different times of sampling, and the individual eye. The targeted analysis of tear proteins revealed a mean intraindividual variation of 23% for the three most abundant proteins. Even extreme differences in tear secretion rates resulted in interindividual variability below 30% for 65 metabolites and two proteins.ConclusionsThe newly established workflow can be used for combined targeted detection of metabolites and proteins in one punch of a Schirmer strip in a clinical setting.Translational RelevanceOur data about intra- and interindividual as well as intereye variation provide a valuable basis for the design of clinical studies, and for the applicability of multiplexed “omics” to well accessible tear fluid with regard to future routine use.

Tear protein analysis in presumed congenital alacrima.

ObjectivesIt is often hard to reach a definitive diagnosis of congenital alacrima because of the difficultly in proving the lack of lacrimal tissue. We report here the distinct tear protein profile in presumed congenital alacrima.Patients and methodsA 13-year-old girl with presumed congenital alacrima and 15 healthy volunteers aged 23–35 years were included in this study. Tears were collected using Schirmer strips, and tear protein analyses were performed. Tear samples were collected from the patient with alacrima before and after surgical punctal occlusion of both the upper and lower puncta, and tear protein extract was fractionated using HPLC. Lactoferrin and albumin concentrations in the tears were separately determined using ELISA.ResultsThe chromatogram in the patient greatly differed from that in normal subjects with low concentrations of secretory IgA, lipocalin-1, and lysozyme. ELISA results indicated that the tear fluid in the patient contained a high concentration of albumin but a minimal concentration of lactoferrin. Punctal occlusion improved the ocular surface findings, but major lacrimal protein concentrations remained low and tear albumin concentrations further increased.ConclusionThe tear protein profile of a patient with presumed congenital alacrima greatly differed from that of normal subjects. Tear protein analysis may be useful in making a diagnosis of congenital alacrima and in assessing the efficacy and mechanism of punctal occlusion.

Predictive role of tear protein expression in the early diagnosis of Sjögren's syndrome.

Background The contribution of tear protein expression in patients with presumed diagnosis of Sjögren syndrome is underestimated. We aimed to evaluate the role of tear proteins in the Sjögren syndrome early diagnosis. Methods Charts from 110 patients suspected of Sjögren syndrome were analysed and the subsequent diagnosis retrieved. Subjective symptoms (ocular surface disease index, OSDI), tear film break-up time (TFBUT), Schirmer test, Jones test, tear clearance (TC), corneal (NEI score) and conjunctival staining (van Bjerstelveldt score), esthesiometry, cytology, tear protein analysis (total protein [TP] content, lysozyme-C [LYS-C], lactoferrin [LACTO], lipocalin-1 [LIPOC-1] and albumin [ALB]) were analysed. The diagnostic performance with area under the curve (AUC) and odds ratio (OR) for each parameter were calculated. Results Thirty-five patients (31.8%) had been diagnosed as affected by Sjögren syndrome. Clinical tests showed lower diagnostic performance (OSDI > 44 [AUC 0.57], Schirmer ≤ 5 mm [0.59], TFBUT ≤ 3 s [0.72], TC > 1/16 [0.68], Jones ≤ 4 mm [0.68], corneal staining > 2 [0.51], conjunctival staining > 2 [0.78]) compared with tear proteins (LYS-C ≤ 1.5 mg/mL [0.79], LACTO ≤ 20% [0.94], LIPOC-1 ≤ 10% [0.89], ALB ≥ 15% [0.79]). LYS-C, LACTO, LIPOC-1 and ALB showed a significant association in predicting Sjögren syndrome vs. not-Sjögren syndrome dry eye (OR, respectively, 4.9, 5.5, 7.2, 6.7). Conclusions Tear proteins' concentrations showed a significant higher accuracy compared with the traditional ocular clinical tests for reaching Sjögren syndrome's diagnosis. In particular, LACTO and LIPOC-1 provided an excellent diagnostic performance and thus could likely be considered promising biomarkers of Sjögren syndrome.

Effects of punctal occlusion on global tear proteins in patients with dry eye

PurposeTo investigate effects of punctal occlusion on global tear protein levels in patients with dry eye. MethodsIn this prospective, longitudinal, single-center study, nonabsorbable punctal plugs were inserted bilaterally into the lower punctum of 30 patients with moderate dry eye. Dry eye symptoms, fluorescein corneal staining, Schirmer I test, tear film break-up time, and safety were assessed in the more severely affected eye. Tear proteins at weeks 1 and 3 were quantified by iTRAQ relative to baseline preocclusion levels. ResultsOf 29 patients who completed the study, 23 (mean age 49.8 years) had sufficient tear samples for analysis. After 3 weeks, punctal occlusion significantly upregulated tear proteins, including glutathione synthase (mean of 1.6-fold, P = 0.01) and interleukin-1 receptor antagonist (1.7-fold, P = 0.032) and downregulated cholinergic receptor (neuronal) alpha-7 (0.79-fold, P = 0.039) and lymphocyte cytosolic protein-1 (0.66-fold, P = 0.012). Clustering analysis of global tear proteins revealed two clear profile changes; the first group of patients (cluster 1, n = 10) had a reduction in the inflammatory proteins (e.g., S100A8) and rise in lacrimal proteins supporting the ocular surface (e.g., lysozyme), whereas the second group (cluster 2, n = 13) had an increase in inflammatory proteins and a decrease in lacrimal proteins. Logistic regression analysis revealed that cluster 1 patients had significantly (P = 0.006) lower Schirmer scores at baseline (mean [standard deviation]: 4.3 [4.3] mm) than cluster 2 (6.8 [2.6] mm). ConclusionsPunctal plugs produced a beneficial pattern of tear protein change in patients with relatively low Schirmer scores within 3 weeks of punctal occlusion.

Intolerant contact lens wearers exhibit ocular surface impairment despite 3 months wear discontinuation.

To evaluate ocular surface (OS) parameters recovery in intolerant contact lens (CL) wearers after a period of discontinuation. This is a retrospective analysis of data from 87 intolerant CL wearers who had discontinued their use for an average period of 12weeks because of associated discomfort and failure to successfully refit. Data were collected from clinical charts. Data from 50 matched healthy volunteers served as controls. Clinical tests included subjective discomfort symptoms questionnaire (Ocular Surface Disease Index, OSDI), Schirmer test, break-up time (BUT), corneal esthesiometry and corneo-conjunctival staining. Laboratory tests included scraping and imprint cytology. Tear protein analysis included dosage of total tear protein (TP), lysozyme-C (LYS-C), lactoferrin (LACTO), zinc-α2-glycoprotein (ZAG-2), IgA heavy chain bands (Ig-A), and serum albumin (ALB). Data were correlated to wear parameters. All values were significantly worse in intolerant CL wearers group (p always <0.001). In particular, lower values compared to controls were found for BUT, corneal esthesiometry, goblet cell density, LYS-C, LACTO, ZAG-2, and TP. On the contrary, higher values compared to controls were found for OSDI, staining, imprint cytology, scraping cytology, ALB, IgA-heavy chain. The IgA/LYS-C ratio calculated as an index of the increased activity of the IgA-producing cell was found significantly higher in the intolerant group and in correlation with discomfort symptoms. Ocular surface parameters were altered in intolerant CL wearers, even after a prolonged discontinuation period. Our data suggest that OS recovery necessary to successfully refit lenses may need a discontinuation time longer than 3months.

Specific immunological tear protein analysis in patients with keratoconus and dry eye disease

Specific immunological tear protein analysis in patients with keratoconus and dry eye disease This study involves the analysis of immunological tears proteins (sIgA, MMP-1, TNF-a) in patients with dry eye disease and keratoconus. sIgA and MMP-1 were significantly decreased in KC. sIgA decrease and TNF-α increase significantly in DED.

Analysis of tear fluid proteins: use of multiplex assays in profiling biomarkers of dry eye disease

Analysis of tear fluid proteins: use of multiplex assays in profiling biomarkers of dry eye disease

Analysis of tear fluid proteins: use of multiplex assays in profiling biomarkers of dry eye disease

Aims Dry eye disease (DED) is a distressing condition that is commonly associated with ageing, contact lens wear and autoimmune disease. DED is one of the fastest-growing eye disorders in older populations (over-50s) and is significantly underdiagnosed. Multiplex studies of tear fluids from DED patient have suggested a role for inflammatory cytokines in its pathology. In this study, we investigated tear fluids from DED and normal subjects for a panel of 7 cytokines, using the multiplex bead assay.

Tear Proteomics in Young Malays with Dry Eyes

Introduction: Changes in tear protein concentrations may reflect ocular surface health. This study analyzes changes in tear protein concentrations of young Malays with dry eye (DE) and determines its association with the clinical findings. Methods: Subjects were screened using McMonnies questionnaire (MDEQ) and flourescein tear break up time (TBUT). Total tear protein concentration (TTPC) was determined using Bradford's technique and specific tear protein (sIgA, lysozyme, lactoferrin and human serum albumin (HSA)) concentrations were determined using SDS-PAGE. Parametric and nonparametric tests were used to compare means between groups. Spearman correlation was used to determine the association between variables measured. Results: A total of 42 subjects (21 DE and 21 NDE) were included. Mean MDEQ score for DE was 16.00±1.48 and NDE was 8.47±3.47. Mean TBUT for DE was 3.47±0.47s and NDE was 4.98±0.43s. Mean TTPC for DE and NDE was 9.84±2.40mg/ml and 8.96±1.84mg/ml respectively. Mean sIgA, lysozyme, lactoferrin and HSA for DE was 0.54±0.10mg/ml, 1.68±0.17mg/ml, 1.47±0.25mg/ml, 0.06±0.03mg/ml and for NDE was 0.57±0.09mg/ ml, 2.04±0.19mg/ml, 1.75±0.23mg/ml, 0.06±0.03mg/ml accordingly. Significant differences were noted in MDEQ score (p=0.01), TBUT (p=0.01), lactoferrin (p=0.01) and lysozyme (p=0.01) but not in TTPC (p=0.19), HSA (p=0.74) and sIgA (p=0.24) between groups. Significant correlations were noted between TBUT with lactoferrin (r=0.02, p=0.02) and lysozyme (r=0.63, p=0.01) and between MDEQ score with lactoferrin (r=-0.34, p=0.02) and lysozyme (r=-0.64, p=0.01). Conclusions: There are changes in specific tear protein in dry eye patients, which correlate well with clinical results. Tear protein analysis may play an important role in the diagnosis of the dry eye.

Chapter Four - Tear Fluid Protein Biomarkers

The tear film covers and protects the ocular surface. It contains various molecules including a large variety of proteins. The protein composition of the tear fluid can change with respect to various local and systemic diseases. Prior to the advent of the proteomic era, tear protein analysis was limited to a few analytical techniques, the most common of which was immunoelectrophoresis, an approach dependent on antibody availability. Using proteomics, hundreds of tear proteins could potentially be identified and subsequently studied. Although detection of low-abundance proteins in the complex tear proteome remains a challenge, advances in sample fractionation and mass spectrometry have greatly enhanced our ability to detect these proteins. With increasing proteomic applications, tears show great potential as biomarkers in the development of clinical assays for various human diseases. In this chapter, we discuss the structure and functions of the tear film and methods for its collection. We also summarize potential tear protein biomarkers identified using proteomic techniques for both ocular and systemic diseases. Finally, modern proteomic techniques for tear biomarker research and future challenges are explored.

Tear protein analysis: the influence of tear sampling techniques

Tear protein analysis: the influence of tear sampling techniques

Tear analysis in ocular surface diseases

The thin layer of tears covering the ocular surface are a complex body fluid containing thousands of molecules of varied form and function of several origins. In this review, we have discussed some key issues in the analysis of tears in the context of understanding and diagnosing eye disease using current technologies of proteomics and metabolomics, and for their potential for clinical application. In the last several years, advances in proteomics/metabolomics/lipidomics technologies have greatly expanded our knowledge of the chemical composition of tear fluid. The quickened pace of studies has shown that tears as a complex extra-cellular fluid of the ocular surface contains a great deal of molecular information useful for the diagnosis, prognosis, and treatment of ocular surface diseases that has the ability to addresses the emphasis on personalized medicine and biomarkers of disease. Future research directions will likely include (1) standardize tear collection, storage, extraction, and sample preparation; (2) quantitative proteomic analysis of tear proteins using multiple reaction monitoring (MRM)-based mass spectrometry; (3) population based studies of human tear proteomics/metabolomics; (4) tear proteomics/metabolomics for systemic diseases; and (5) functional studies of tear proteins.

Using soybean trypsin inhibitor as an external loading control for Western blot analysis of tear proteins: Application to corneal disease

Using soybean trypsin inhibitor as an external loading control for Western blot analysis of tear proteins: Application to corneal disease

Human Tear Protein Analysis Enabled by an Alkaline Microfluidic Homogeneous Immunoassay

The ability to probe the protein content of human tear fluid has enormous potential for deepening our understanding of ocular and systemic disease pathology and enabling novel noninvasive tear-based diagnostic technologies. To overcome current challenges in tear proteomic measurements, we report on the first microfluidic homogeneous immunoassay capable of making rapid, quantitative, and specific measurements of endogenous tear protein biomarkers in human tear fluid. Lactoferrin (Lf) is a tear-specific biomarker for Sjögren's syndrome (SS), a serious systemic autoimmune disease currently diagnosed through rudimentary volumetric and surface chemistry measurements and an invasive lip biopsy. We detail optimization of a homogeneous electrophoretic immunoassay for Lf in <1 μL of tear fluid at clinically relevant concentrations. In particular, we present assay development details and a final assay that enables quantification of Lf in <5 s in a clinically relevant range for SS diagnostics. Characterization suggests the on-chip assay is accurate to within 15% of ELISA, specific (<15% nonspecific signal), and with a lower limit of detection of 3 ± 2 nM Lf in human tear matrix. Additionally, we develop and characterize a protocol for eluting proteins from nitrocellulose Schirmer strips, the clinical de facto standard for tear collection and storage. We relate on-chip measured Lf concentrations back to ocular surface concentrations for the first time to our knowledge. Taken in sum, this work details important steps toward (1) expanding the set of proteins quantified by electrophoretic immunoassays to encompass a wider range of isoelectric points than has been reported, (2) creating a first-in-kind translatable assay with clinical relevance to SS diagnostics, and (3) expanding the analytical toolkit available for rapid tear protein measurements, as is relevant to the advancement of basic research and clinical medicine.

Tear Film Volume and Protein Analysis in Full-term Newborn Infants

To investigate infant tear film secretion and protein profile, and to compare major tear proteins, such as immunoglobulin A, lactoferrin, and lysozyme, with those of adult controls. Tears were collected, with a cellulose rod, from 40 healthy infants (19 female infants and 21 male infants, gestational duration: 39.71 ± 1.27 weeks) within 48 hours of birth and 22 adults (10 female infants and 12 male infants, mean age: 24.95 ± 3.63 years). A second collection was obtained from 14 of the infants (8 female infants and 6 male infants, postnatal age: 7.76 ± 6.14 weeks). The tear volume was measured, and protein in the samples was analyzed by Bradford assay and gel electrophoresis. Median tear volume (interquartile range) was 0.5 μL (0.6-2 μL) for newborn infants, 2.5 μL (1.4-7.75 μL) for these infants at an older age, and 6 μL (2.73-12.75 μL) in adults (P < 0.001, Kruskall-Wallis test). Immunoglobulin A concentration was significantly lower in newborns (P < 0.001, analysis of variance). Lipocalin was present in 36% of the newborn tear samples, whereas serum albumin was found in 86%. Mean protein concentration (μg/μL ± SD) was 10.95 ± 5.51 in the newborns, 12.93 ± 3.99 in the older infants, and 13.04 ± 3.46 in the adults (P > 0.5, analysis of variance). This is the first study reporting an investigation of unstimulated infant tears, using a noninvasive collection method. Tear protein content demonstrated that the infant tear film is different to that in adults.