Analysis of rabbit tear proteins by high‐pressure liquid chromatography/electrospray ionization mass spectrometry

Abstract

The aim of this study was to develop a fast and reliable analytical procedure for the display of the protein components of tears that can be used to differentiate the status of the ocular surface. Using this new procedure, we analyzed the tear protein components following a corneal wound in the rabbit. Calibrated 10-microL glass, fire-polished capillary micropipettes were used to collect tears from New Zealand White rabbits prior to and daily for 9 days following a unilateral 6-mm diameter centrally placed anterior keratectomy. Tear proteins were eluted by a reversed-phase high-performance liquid chromatography (RP-HPLC) column and the tear protein profile was monitored by electrospray ionization (ESI) mass spectrometry positive total ion current (TIC) chromatography. Tear proteins were reliably separated into 17 peaks, each of which contained one or a number of protein components. The molecular weight of each protein component was determined by on-line ESI. Major tear protein components, lactoferrin, lysozyme (minimally detectable in rabbit tears), albumin, lipocalin, lipophilin and beta2-microglobulin, were tentatively identified by this method. Based on the mass spectrometric data, beta2-microglobulin was found to be glycosylated with N-acetylhexosamine. ESI-positive TIC chromatograms and mass spectra revealed comparative differences in the tear protein spectra after corneal wounding. One day after wounding, rabbit lysozyme with a molecular weight of 14,717 Da was found to be 8-fold higher in the tears of wounded eyes when compared with tears from unwounded eyes. It dropped back to normal 3 days after wounding. The expression of an unidentified tear protein with the molecular weight of 16,060 Da was also elevated after corneal wounding and returned to normal level by day 5. In this study, LC/ESI-MS was developed as a fast, reproducible and simple method for the identification and analysis of many of the protein components of the tears. Importantly, this technique also allows quantification of each component resolved in the chromatogram. This method is very suitable for mapping peptides and proteins (<80 kDa) in tears.

iTRAQ Quantitative Proteomics in the Analysis of Tears in Dry Eye Patients

“…13 Differential regulation of inflammatory proteins in the tear film has been evaluated using a variety of techniques in several ocular surface conditions, such as meibomian gland dysfunction, 14 DE, 15 Sjögren's syndrome, 13,[16][17][18] and wound healing processes of the ocular surface. 6,19 Versura et al, in a recent study, showed that the tear protein changes anticipate the onset of more extensive clinical signs in early stage DE disease. 20 Changes of tear protein profile also have been shown to correlate with DE severity, 13 and the levels of certain proteins have been correlated with the severity of meibomian gland disease.…”

“…[3][4][5][6] The major source of tear proteins is the secretory acinar cells of the lacrimal gland, including the primary proteins of the tear film: lysozyme, lactoferrin, and lipocalin. 7 Numerous proteins have been identified previously in human tears; however, there is an inconsistency in the number of proteins in tears and their specific functions in the existing literature.…”

Comparison of two methods of tear sampling for protein quantification by Bradford method

“…Previous comparisons between the Lowry and Bradford methods for the quantification of tear proteins showed significant differences between both and higher values attributed by the Bradford method, alerting to the need of a careful interpretation of the results (Lu et al 2010). Studies reported several techniques used for proteomic analysis of tears, like sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (Green-Churchet et al 2008), enzyme-linked immunosorbent assay (ELISA), high--performance liquid chromatography (HPLC), matrix assisted laser desorption ionization-time of flight (MALDI-TOF) (De Souza et al 2008), surface-enhanced laser desorption ionization-TOF (SELDI-TOF), liquid chromatography coupled with electrospray ionization (LC/MS) (Zhou et al 2003) and LTQ-Orbitrap mass spectrometer (De Souza et al 2008). However, a simple change in the method of obtaining the sample can affect the results (Li et al 2008), since the quality of the analysis depends on the sample preparation (Ananthi et al 2008).…”

“…In our study the samples were frozen at -80 o C (Zhou et al 2003, and stored for no longer than 2 months. It was proved that samples stored at -70°C (Sitaramamma et al 1998, Ham et al 2007, Yoon et al 2010) for up to 4 months preserved the tear proteins better than in 4°C or -20 o C in a shorter time, when it was observed a reduction in the proteins of the sample (Sitaramamma et al 1998).…”

Diabetic retinopathy: Proteomic approaches to help the differential diagnosis and to understand the underlying molecular mechanisms

“…The chemical barrier contains mostly constitutively expressed AMPs, while some of them show inducible feature upon pathogenic or other stimuli. The tear fluid contains several prototypic AMPs such as defensins, dermcidin and LL-37 cathelicidin [74,85,101,102]. Defensins are small cationic peptides produced by epithelial cells with cysteine rich sequences forming a very stable 3D structure [103].…”