An aptamer triple helix molecular switch for sensitive electrochemical assay of lipocalin 1 biomarker via dual signal amplifications.

Sensitively monitoring the concentration change of lipocalin 1 (LCN1) can provide data support for accurately diagnosing diabetic retinopathy and efficacy of treatment. Using a new aptamer triplex switch (ATS) probe and catalytic hairpin assembly (CHA) and hybridization chain reaction (HCR) dual signal amplifications, a highly sensitive electrochemical and enzyme-free biosensor for detecting LCN1 is reported. The ATS probes recognize and bind LCN1 to change its structure and release ssDNA sequences, which trigger the capture of methylene blue (MB)-tagged DNA on the sensor electrode via the CHA and HCR reactions. These MB tags are then subjected to electrochemical measurements to obtain highly amplified current outputs to sensitively detect LCN1 from 5 pM to 1 nM with a detection limit of 0.85 pM. This sensor also exhibits high interfering protein discrimination capability and can be employed to monitor low levels of LCN1 in diluted tear samples. Our established sensor significantly outperforms current major LCN1 detection methods based on immunoassays and thus holds promising potential for evaluating the severity of diabetic retinopathy and monitoring treatment response.

Lipocalin-1 is the acceptor protein for phospholipid transfer protein in tears.

Phospholipid transfer protein, ∼80 kDa, transfers phospholipids from micelles to lipid binding proteins. The acceptor protein in plasma is apolipoprotein-A1, 28 kDa. Previously, phospholipid transfer protein was found in tears but an acceptor protein was not identified. To search for the acceptor protein(s) in tears a fluorescent phospholipid transfer assay was altered to omit the extrinsic acceptor. Human tears were incubated with fluorescent micelles and showed marked transfer activity verifying a native acceptor protein must be present. Reconstituted tears without tear lipocalin (lipocalin-1) eliminated the transfer of phospholipids. To determine if phospholipid transfer protein is involved in carrying phospholipid to the surface of tears from tear lipocalin, a fraction enriched in phospholipid transfer protein was injected into the subphase of a tear mimicking buffer in which tear lipocalin was present. The addition of phospholipid transfer protein did not increase the thickness of the surface layer regardless of the presence of lipid bearing tear lipocalin. The data show that phospholipid transfer protein transfers phospholipid from micelles to tear lipocalin. Phospholipid transfer protein does not transport the phospholipid. While tear lipocalin has no intrinsic transfer activity from micelles, it is the acceptor protein for phospholipid transfer protein in tears.

Interaction of ceramides and tear lipocalin.

The distribution of lipids in tears is critical to their function. Lipids in human tears may retard evaporation by forming a surface barrier at the air interface. Lipids complexed with the major lipid binding protein in tears, tear lipocalin, reside in the bulk (aqueous) and may have functions unrelated to the surface. Many new lipids species have been revealed through recent mass spectrometric studies. Their association with lipid binding proteins has not been studied. Squalene, (O-acyl) omega-hydroxy fatty acids (OAHFA) and ceramides are examples. Even well-known lipids such as wax and cholesteryl esters are only presumed to be unbound because extracts of protein fractions of tears were devoid of these lipids. Our purpose was to determine by direct binding assays if the aforementioned lipids can bind tear lipocalin. Lipids were screened for ability to displace DAUDA from tear lipocalin in a fluorescence displacement assay. Di- and tri-glycerides, squalene, OAHFA, wax and cholesterol esters did not displace DAUDA from tear lipocalin. However, ceramides displaced DAUDA. Apparent dissociation constants for ceramide-tear lipocalin complexes using fluorescent analogs were measured consistently in the submicromolar range with 3 methods, linear spectral summation, high speed centrifugal precipitation and standard fluorescence assays. At the relatively small concentrations in tears, all ceramides were complexed to tear lipocalin. The lack of binding of di- and tri-glycerides, squalene, OAHFA, as well as wax and cholesterol esters to tear lipocalin is consonant with residence of these lipids near the air interface.

Ligand binding complexes in lipocalins: Underestimation of the stoichiometry parameter (n).

The stoichiometry of a ligand binding reaction to a protein is given by a parameter (n). The value of this parameter may indicate the presence of protein monomer or dimers in the binding complex. Members of the lipocalin superfamily show variation in the stoichiometry of binding to ligands. In some cases the stoichiometry parameter (n) has been variously reported for the same protein as mono- and multimerization of the complex. Prime examples include retinol binding protein, ß lactoglobulin and tear lipocalin, also called lipocalin-1(LCN1). Recent work demonstrated the stoichiometric ratio for ceramide:tear lipocalin varied (range n = 0.3-0.75) by several different methods. The structure of ceramide raises the intriguing possibility of a lipocalin dimer complex with each lipocalin molecule attached to one of the two alkyl chains of ceramide. The stoichiometry of the ceramide-tear lipocalin binding complex was explored in detail using size exclusion chromatography and time resolved fluorescence anisotropy. Both methods showed consistent results that tear lipocalin remains monomeric when bound to ceramide. Delipidation experiments suggest the most likely explanation is that the low 'n' values result from prior occupancy of the binding sites by native ligands. Lipocalins such as tear lipocalin that have numerous binding partners are particularly prone to an underestimated apparent stoichiometry parameter.

Allergenic Can f 1 and its human homologue Lcn-1 direct dendritic cells to induce divergent immune responses.

Why and when the immune system skews to Th2 mediated allergic immune responses is still poorly characterized. With two homologous lipocalins, the major respiratory dog allergen Can f 1 and the human endogenous, non-allergenic Lipocalin-1, we investigated their impact on human monocyte-derived dendritic cells (DC). The two lipocalins had differential effects on DC according to their allergenic potential. Compared to Lipocalin-1, Can f 1 persistently induced lower levels of the Th1 skewing maturation marker expression, tryptophan breakdown and interleukin (IL)-12 production in DC. As a consequence, T cells stimulated by DC treated with Can f 1 produced more of the Th2 signature cytokine IL-13 and lower levels of the Th1 signature cytokine interferon-γ than T cells stimulated by Lipocalin-1 treated DC. These data were partially verified by a second pair of homologous lipocalins, the cat allergen Fel d 4 and its putative human homologue major urinary protein. Our data indicate that the crosstalk of DC with lipocalins alone has the potential to direct the type of immune response to these particular antigens. A global gene expression analysis further supported these results and indicated significant differences in intracellular trafficking, sorting and antigen presentation pathways when comparing Can f 1 and Lipocalin-1 stimulated DC. With this study we contribute to a better understanding of the induction phase of a Th2 immune response.

Targeting iron acquisition blocks infection with the fungal pathogens Aspergillus fumigatus and Fusarium oxysporum.

Filamentous fungi are an important cause of pulmonary and systemic morbidity and mortality, and also cause corneal blindness and visual impairment worldwide. Utilizing in vitro neutrophil killing assays and a model of fungal infection of the cornea, we demonstrated that Dectin-1 dependent IL-6 production regulates expression of iron chelators, heme and siderophore binding proteins and hepcidin in infected mice. In addition, we show that human neutrophils synthesize lipocalin-1, which sequesters fungal siderophores, and that topical lipocalin-1 or lactoferrin restricts fungal growth in vivo. Conversely, we show that exogenous iron or the xenosiderophore deferroxamine enhances fungal growth in infected mice. By examining mutant Aspergillus and Fusarium strains, we found that fungal transcriptional responses to low iron levels and extracellular siderophores are essential for fungal growth during infection. Further, we showed that targeting fungal iron acquisition or siderophore biosynthesis by topical application of iron chelators or statins reduces fungal growth in the cornea by 60% and that dual therapy with the iron chelator deferiprone and statins further restricts fungal growth by 75%. Together, these studies identify specific host iron-chelating and fungal iron-acquisition mediators that regulate fungal growth, and demonstrate that therapeutic inhibition of fungal iron acquisition can be utilized to treat topical fungal infections.

Delineation of solution burst-phase protein folding events by encapsulating the proteins in silica gels.

Many studies have shown that during the early stages of the folding of a protein, chain collapse and secondary structure formation lead to a partially folded intermediate. Thus, direct observation of these early folding events is crucial if we are to understand protein-folding mechanisms. Notably, these events usually manifest as the initial unresolvable signals, denoted the burst phase, when monitored during conventional mixing experiments. However, folding events can be substantially slowed by first trapping a protein within a silica gel with a large water content, in which the trapped native state retains its solution conformation. In this study, we monitored the early folding events involving secondary structure formation of five globular proteins, horse heart cytochrome c, equine ß-lactoglobulin, human tear lipocalin, bovine α-lactalbumin, and hen egg lysozyme, in silica gels containing 80% (w/w) water by CD spectroscopy. The folding rates decreased for each of the proteins, which allowed for direct observation of the initial folding transitions, equivalent to the solution burst phase. The formation of each initial intermediate state exhibited single exponential kinetics and Arrhenius activation energies of 14-31 kJ/mol.

Expression, characterization and ligand specificity of lipocalin-1 interacting membrane receptor (LIMR).

Human lipocalin-1 interacting membrane receptor (LIMR) was the first lipocalin receptor to be identified, as a specific receptor for lipocalin-1 (Lcn1). Subsequently LIMR has been reported to interact with other ligands as well, notably with the bovine lipocalin ß-lactoglobulin (BLG) and with the unrelated secretoglobin uteroglobin (UG). To study the ligand-binding behaviour of this prototypic lipocalin receptor in more detail, a system was developed for the recombinant expression of LIMR in Drosophila Schneider 2 (S2) cells, and for the subsequent solubilization and purification of the protein. The receptor forms dimers or larger oligomers when solubilized in n-dodecyl ß-D-maltoside (DDM). The full-length, functional receptor was captured onto a surface plasmon resonance (SPR) chip via an α-V5 antibody, and the binding of various potential ligands was followed in time. In this way, LIMR was shown to be highly specific for Lcn1, binding the lipocalin with low micromolar to high nanomolar affinity. No interactions with any of the other putative ligands could be detected, raising doubts about the physiological relevance of the reported binding of BLG and UG to the receptor.

Quantification of individual proteins in silicone hydrogel contact lens deposits.

PURPOSE: The aim of this study was to quantify specific proteins deposited on daily wear silicone hydrogel lenses used in combination with multipurpose disinfecting solutions (MPDSs) by applying multiple-reaction-monitoring mass spectrometry (MRM-MS). METHODS: Balafilcon A or senofilcon A contact lenses used with different MPDSs on a daily wear schedule were collected. Each worn lens was extracted and then digested with trypsin. MRM-MS was applied to quantify the amounts of lysozyme, lactoferrin, lipocalin-1, proline-rich protein-4, and keratin-1 in the extracts. RESULTS: The amount of protein extracted from the contact lenses was affected by the individual wearers, lens material, and type of care system used. Higher amounts of proteins were extracted from lenses after wear when they were used with an MPDS containing polyhexamethylene biguanide (PHMB) and poloxamer 407 compared with MPDSs containing polyquaternium-1 (PQ-1)/alexidine dihydrochloride with Tetronic 904 or PQ-1/ PHMB with poloxamine and sulfobetaine (p < 0.05). There was a correlation between the amount of lipocalin-1 or keratin-1 extracted from lenses and symptoms of ocular dryness. CONCLUSIONS: The MRM-MS technique is a promising approach that could be used to reveal associations of individual proteins deposited on lenses with performance of contact lenses during wear.

Lacritin and the tear proteome as natural replacement therapy for dry eye.

Tear proteins are potential biomarkers, drug targets, and even biotherapeutics. As a biotherapeutic, a recombinant tear protein might physiologically rescue the ocular surface when a deficiency is detected. Such a strategy pays more attention to the natural prosecretory and protective properties of the tear film and seeks to alleviate symptoms by addressing cause, rather than the current palliative, non-specific and temporary approaches. Only a handful of tear proteins appear to be selectively downregulated in dry eye, the most common eye disease. Lacritin and lipocalin-1 are two tear proteins selectively deficient in dry eye. Both proteins influence ocular surface health. Lacritin is a prosecretory mitogen that promotes basal tearing when applied topically. Levels of active monomeric lacritin are negatively regulated by tear tissue transglutaminase, whose expression is elevated in dry eye with ocular surface inflammation. Lipocalin-1 is the master lipid sponge of the ocular surface, without which residual lipids could interfere with epithelial wetting. It also is a carrier for vitamins and steroid hormones, and is a key endonuclease. Accumulation of DNA in tears is thought to be proinflammatory. Functions of these and other tear proteins may be influenced by protein-protein interactions. Here we discuss new advances in lacritin biology and provide an overview on lipocalin-1, and newly identified members of the tear proteome.

Protein deposition and its effect on bacterial adhesion to contact lenses.

PURPOSE: Bacterial adhesion to contact lenses is believed to be the initial step for the development of several adverse reactions that occur during lens wear such as microbial keratitis. This study examined the effect of combinations of proteins on the adhesion of bacteria to contact lenses. METHODS: Unworn balafilcon A and senofilcon A lenses were soaked in commercially available pure protein mixtures to achieve the same amount of various proteins as found ex vivo. These lenses were then exposed to Pseudomonas aeruginosa and Staphylococcus aureus. Following incubation, the numbers of P. aeruginosa or S. aureus that adhered to the lenses were measured. The possible effect of proteins on bacterial growth was investigated by incubating bacteria in medium containing protein. RESULTS: Although there was a significant (p < 0.003) increase in the total or viable counts of one strain of S. aureus (031) on balafilcon A lenses soaked in the lysozyme/lactoferrin combination, the protein adhered to lenses did not alter the adhesion of any other strains of P. aeruginosa or S. aureus (p > 0.05). Growth of S. aureus 031 (p < 0.0001) but not of P. aeruginosa 6294 was stimulated by addition of lysozyme/lactoferrin combination (2.8/0.5 mg/mL). Addition of lipocalin did not affect the growth of any strains tested (p > 0.05). CONCLUSIONS: Adsorption of amounts of lysozyme and lactoferrin or lipocalin equivalent to those extracted from worn contact lenses did not affect the adhesion of most strains of S. aureus or P. aeruginosa to lens surfaces.

Cation-π interactions in lipocalins: structural and functional implications.

The cation-π interaction impacts protein folding, structural stability, specificity, and molecular recognition. Cation-π interactions have been overlooked in the lipocalin family. To fill this gap, these interactions were analyzed in the 113 crystal and solution structures from the lipocalin family. The cation-π interactions link previously identified structurally conserved regions and reveal new motifs, which are beyond the reach of a sequence alignment algorithm. Functional and structural significance of the interactions were tested experimentally in human tear lipocalin (TL). TL, a prominent and promiscuous lipocalin, has a key role in lipid binding at the ocular surface. Ligand binding modulation through the loop AB at the "open" end of the barrel has been erroneously attributed solely to electrostatic interactions. Data revealed that the interloop cation-π interaction in the pair Phe28-Lys108 contributes significantly to stabilize the holo-conformation of the loop AB. Numerous energetically significant and conserved cation-π interactions were uncovered in TL and throughout the lipocalin family. Cation-π interactions, such as the highly conserved Trp17-Arg118 pair in TL, were educed in low temperature experiments of mutants with Trp to Tyr substitutions.

The conserved disulfide bond of human tear lipocalin modulates conformation and lipid binding in a ligand selective manner.

The primary aim of this study is the elucidation of the mechanism of disulfide induced alteration of ligand binding in human tear lipocalin (TL). Disulfide bonds may act as dynamic scaffolds to regulate conformational changes that alter protein function including receptor-ligand interactions. A single disulfide bond, (Cys61-Cys153), exists in TL that is highly conserved in the lipocalin superfamily. Circular dichroism and fluorescence spectroscopies were applied to investigate the mechanism by which disulfide bond removal effects protein stability, dynamics and ligand binding properties. Although the secondary structure is not altered by disulfide elimination, TL shows decreased stability against urea denaturation. Free energy change (ΔG(0)) decreases from 4.9±0.2 to 2.1±0.3kcal/mol with removal of the disulfide bond. Furthermore, ligand binding properties of TL without the disulfide vary according to the type of ligand. The binding of a bulky ligand, NBD-cholesterol, has a decreased time constant (from 11.8±0.2 to 3.3s). In contrast, the NBD-labeled phospholipid shows a moderate decrease in the time constant for binding, from 33.2±0.2 to 22.2±0.4s. FRET experiments indicate that the hairpin CD is directly involved in modulation of both ligand binding and flexibility of TL. In TL complexed with palmitic acid (PA-TL), the distance between the residues 62 of strand D and 81 of loop EF is decreased by disulfide bond reduction. Consequently, removal of the disulfide bond boosts flexibility of the protein to reach a CD-EF loop distance (24.3Å, between residues 62 and 81), which is not accessible for the protein with an intact disulfide bond (26.2Å). The results suggest that enhanced flexibility of the protein promotes a faster accommodation of the ligand inside the cavity and an energetically favorable ligand-protein complex.

Targeted proteomics using parallel reaction monitoring confirms salivary proteins indicative of metastatic triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is the most aggressive subtype due to the absence of hormonal receptors. Our study aimed to identify and determine the effectiveness of salivary proteins as candidate markers for metastatic TNBC subtype using parallel reaction monitoring mass spectrometry (PRM-MS). Three salivary proteins (lipocalin-1, SMR3B, and plastin-2) that showed significant differential expression in label-free quantitation (LFQ) between TNBC (N = 6) and health subjects (HS; N = 6) were selected for further validation. The developed PRM assay was used to quantify peptides GLST and NNLE (lipocalin-1), VYAL and MINL (Plastin-2) and GPYP, and IPPP (SMR3B) on a different cohort of TNBC patients (N = 20) and HS (N = 20) for evaluating their discriminating performances. Quantitative validation using PRM correlated well with the LFQ results, and 5 peptides from three proteins showed a similar up-or down-regulation. Subsequently, these proteins were validated by Western blot analysis. Compared to one protein's performance as an individual marker, the five-signature panel with salivary GLST, VYAL, MINL, GPYP, and IPPP achieved better performance in differentiating aggressive TNBC and HS with sensitivity (80%) and specificity (95%). Targeted proteomic analysis of the prioritized proteins highlights a peptide-based signature in saliva as the potential predictor to distinguish between TNBC and HS. SIGNIFICANCE OF THE STUDY: This study was designed to identify and quantify potential markers in saliva from the triple-negative breast cancer (TNBC) patients using parallel reaction monitoring assay. Three salivary proteins, Lipocalin-1 (LCN-1), Submaxillary androgen-regulated protein 3B (SMR3B), and Plastin-2 (LCP-1) selected in the discovery-phase were further quantified by targeted proteomics and Western blots. The salivary proteins successfully differentiated TNBC patients from healthy subjects with a sensitivity (80%) and specificity (95%).

The evolutionary history of the SAL1 gene family in eutherian mammals.

BACKGROUND: SAL1 (salivary lipocalin) is a member of the OBP (Odorant Binding Protein) family and is involved in chemical sexual communication in pig. SAL1 and its relatives may be involved in pheromone and olfactory receptor binding and in pre-mating behaviour. The evolutionary history and the selective pressures acting on SAL1 and its orthologous genes have not yet been exhaustively described. The aim of the present work was to study the evolution of these genes, to elucidate the role of selective pressures in their evolution and the consequences for their functions. RESULTS: Here, we present the evolutionary history of SAL1 gene and its orthologous genes in mammals. We found that (1) SAL1 and its related genes arose in eutherian mammals with lineage-specific duplications in rodents, horse and cow and are lost in human, mouse lemur, bushbaby and orangutan, (2) the evolution of duplicated genes of horse, rat, mouse and guinea pig is driven by concerted evolution with extensive gene conversion events in mouse and guinea pig and by positive selection mainly acting on paralogous genes in horse and guinea pig, (3) positive selection was detected for amino acids involved in pheromone binding and amino acids putatively involved in olfactory receptor binding, (4) positive selection was also found for lineage, indicating a species-specific strategy for amino acid selection. CONCLUSIONS: This work provides new insights into the evolutionary history of SAL1 and its orthologs. On one hand, some genes are subject to concerted evolution and to an increase in dosage, suggesting the need for homogeneity of sequence and function in certain species. On the other hand, positive selection plays a role in the diversification of the functions of the family and in lineage, suggesting adaptive evolution, with possible consequences for speciation and for the reinforcement of prezygotic barriers.

Two newly identified cat allergens: the von Ebner gland protein Fel d 7 and the latherin-like protein Fel d 8.

INTRODUCTION: Characterization of the complete IgE binding spectrum of cat allergens is important for the development of improved diagnosis and effective immunotherapeutics. While Fel d 1 remains unchallenged as the major cat allergen, we now report the isolation of two new allergens capable of binding similar concentrations of IgE in the allergic sera of some individuals. MATERIALS AND METHODS: Cat tongue and submandibular salivary gland cDNA libraries were screened by DNA hybridisation and IgE immunoassay. The isolated DNA fragments were sub-cloned into an E. coli expression system and the IgE reactivity was examined with human cat-allergic sera using a DELFIA IgE quantitation assay. RESULTS: Fel d 7, an 18 kDa von Ebner gland protein Can f 1 homologue, was isolated from the tongue library. Fel d 8, a 24-kDa latherin-like protein with homology to Equ c 5, was isolated from the submandibular library. The frequency of IgE binding of cat-allergic sera to recombinant Fel d 1, 7 and 8 was 60.5, 37.6 and 19.3%, respectively. Inhibition studies indicated some IgE binding cross-reactivity between Fel d 7 and dog dander extracts. DISCUSSION: The study reports the isolation and characterization of two new cat allergens. The isolation of these allergens provides the opportunity to determine the role that IgE binding proteins other than Fel d 1 play in cat-allergic disease. For cat-allergic individuals with moderate to mild rhinoconjunctivitis these allergens may play a more important role in the manifestation of their allergic disease.

Tear lipocalin: structure and function.

Lipocalins are a family of diverse low molecular weight proteins that act extracellularly. They use multiple recognition properties that include 1) ligand binding to small hydrophobic molecules, 2) macromolecular complexation with other soluble macromolecules, and 3) binding to specific cell surface receptors to deliver cargo. Tear lipocalin (TLC) is a major protein in tears and has a large ligand-binding cavity that allows the lipocalin to bind an extensive and diverse set of lipophilic molecules. TLC can also bind to macromolecules, including the tear proteins lactoferin and lysozyme. The receptor to which TLC binds is termed tear lipocalin-interacting membrane receptor (LIMR). LIMR appears to work by endocytosis. TLC has a variety of suggested functions in tears, including regulation of tear viscosity, binding and release of lipids, endonuclease inactivation of viral DNA, binding of microbial siderophores (iron chelators used to deliver essential iron to bacteria), serving as a biomarker for dry eye, and possessing anti-inflammatory activity. Additional research is warranted to determine the actual functions of TLC in tears and the presence of its receptor on the ocular surface.

pH-Dependent conformational changes in tear lipocalin by site-directed tryptophan fluorescence.

Tear lipocalin (TL), a major protein of human tears, binds a broad array of endogenous ligands. pH-dependent ligand binding in TL may have functional implications in tears. Previously, conformational selections of the AB and GH loops have been implicated in ligand binding by site-directed tryptophan fluorescence (SDTF). In this study, SDTF was applied to the AB and GH loops to investigate pH-driven conformational changes relevant to ligand binding. Both loops demonstrate significant but distinct conformational rearrangements over a wide pH range. In the low-pH transition, from 7.3 to 3.0, residues of the GH loop exhibit decreased solvent accessibilities. In acrylamide quenching experiments, the average quenching rate constant (k(q), accessibility parameter) of the residues in the GH loop is decreased approximately 38%, from 2.1 x 10(9) to 1.3 x 10(9) M(-1) s(-1). However, despite the significant changes in accessibilities for some residues in the AB loop, the average accessibility per residue remained unchanged (average k(q) = 1.2 M(-1) s(-1)). Accordingly, the low-pH transition induces conformational changes that reshuffle the accessibility profiles of the residues in the AB loop. A significant difference in the titration curves between the holo and apo forms of the W28 mutant suggests that the protonation states of the residues around position 28 modulate conformational switches of the AB loop relevant to ligand binding.

Tear lipocalin is the predominant phosphoprotein in human tear fluid.

Proteins are very important components in tears. Their phosphorylation is an important posttranslational modification affecting biological activity. Using proteomic techniques, this study was designed to analyze phosphoproteins found in open eye basal tears from normal human subjects. Proteins in tear samples were separated in 1-dimensional (1D) and 2-dimensional (2D) gels and phosphoproteins were selectively stained with Pro-Q diamond dye before visualization of all proteins using Sypro Ruby. Potential phosphoproteins in 2D gels were identified by liquid chromatography-mass spectrometry (LC-MS/MS) after trypsin digestion and phosphopeptide enrichment using titanium dioxide (TiO(2)) columns. The tryptic digests of the tear samples were also analyzed to identify phosphoproteins directly by LC-MS/MS after phosphopeptide enrichment. The major phosphoprotein stained by Pro-Q diamond in the gels and identified by LC-MS/MS from the spots was tear lipocalin. Tear lipocalin was separated into 3 different isoforms and one phosphorylation site (serine at position 24) was identified in one of the isoforms. Prolactin-induced protein, nucleobindin-2 and lipophilin C were also stained with Pro-Q diamond although no phosphorylated peptides from these proteins could be found using LC-MS/MS. Direct analysis of the tear tryptic digests by LC-MS/MS identified a further 12 potential phosphoproteins with tear lipocalin predominant. Four phosphorylation sites (position 24 (serine), 32 (serine), 34 (threonine) and 36 (tyrosine)) were identified for tear lipocalin using this method. These results indicate that tear lipocalin is the predominant phosphoprotein in normal human basal tears. Nucleobindin-2, prolactin-induced protein and lipophilin C also appear to be phosphorylated in basal tear samples.

Fluorescent aptasensor based on G-quadruplex-assisted structural transformation for the detection of biomarker lipocalin 1.

Diabetic retinopathy (DR) is the leading global cause of blindness in the working-age population. Early diagnosis and intervention can effectively reduce the risk for blindness. However, the current diagnostic methods in clinical practice remain constrained by nonquantitative examinations and individual ophthalmologists' experiences. Sensitive, specific and accurate detection of DR-specific biomarkers is an important approach to achieve its early and rapid diagnosis. In this study, a high-affinity aptamer APT12TM that specifically binds to the tear-derived DR biomarker lipocalin 1 was obtained. The aptamer APT12TM can be folded into a stable B-DNA structure, and its strong interaction with LCN 1, including hydrogen bonding and hydrophobic interactions, is an important factor for targeted recognition and high-affinity binding. A G-rich DNA fragment was further assembled at both ends of the aptamer APT12TM, and the B-DNA form was successfully converted into a parallel G-quadruplex. Most importantly, LCN 1 could induce further transformation of the G-quadruplex structure. Therefore, a fluorescent aptasensor based on G-quadruplex-assisted structural transformation was developed through the Thioflavin T mediator. The aptasensor exhibited a broad detection window from 0.25 to 1000 nM LCN 1, with a limit of detection of 0.2 nM. Furthermore, the aptasensor was applied to LCN 1 detection in artificial tear samples and displayed good reproducibility and stability. These results show that the developed aptasensor has significant potential for sensitive, specific and convenient detection of the DR-specific biomarker LCN 1.