Single eye analysis and contralateral eye comparison of tear proteins in normal and dry eye model rabbits by MALDI-ToF mass spectrometry using wax-coated target plates

Abstract

A study of rabbit tear protein expression in a dry eye rabbit model was performed to determine if a pattern in expressed proteins could be identified. The uniqueness of the model allows the comparison of normal (control) eye tear protein expression with surgically induced dry eye tear protein expression in individual animals. The sensitivity of the method allows for single eye analysis. One-dimensional mini-gel electrophoresis of the tear proteins did not show substantial differences between band patterns of the normal versus the dry eye, but was used to assess the molecular weight ranges of the major proteins. Specific assignments of some of the predominant proteins were obtained by tandem mass spectrometry (MS) which showed that the lower molecular weight lipid-binding proteins (approximately 10 kDa to 36 kDa) constitute a considerable amount of the observed protein, followed in lesser quantities by the transferrins which have higher molecular weights ranging from 70 kDa to 85 kDa. Enhancement of matrix-assisted laser desorption/ionization time-of-flight (MALDI-ToF) MS linear mode analysis of intact proteins in tear fluid was demonstrated through the use of wax-coated MALDI plates and spot washing. MALDI-ToF MS analysis of the expressed tear proteins illustrates that differences between normal eye tear and dry eye tear protein content are manifested in changes in the lower molecular weight lipid-binding proteins such as lipophilin which exhibits an increase in concentration in the dry eye, and beta-2 microglobulin which undergoes a decrease.

Fig. 1

1D SDS-PAGE (1-mm-thick gel with 12% acrylamide) results of protein separation in rabbit normal eye tear versus the dry eye model tear. Lanes 1 and 6 low molecular weight (LMW) standard; lanes 2 and 4 1:1 mixtures of sample and dye; lanes 3 and 5 1:3 mixtures of sample and dye

Fig. 2

MALDI-ToF mass spectra of a left aqueous 1 mg/mL lysozyme (14.4 kDa) standard yielding a S/N ratio of 71 acquired using a conventional, non-coated MALDI plate; right lysozyme protein standard spectrum acquired using a wax-coated plate giving a S/N of 154 and a decrease in dimerization at m/z 28.8 kDa; b left aqueous 1 mg/mL carbonic anhydrase (29.0 kDa) standard with a S/N of 12 acquired using a non-coated plate; right carbonic anhydrase protein standard acquired using a wax-coated plate giving a S/N of 135; c left 1 mg/mL BSA (33.2 kDa and 66.4 kDa) standard acquired using a non-coated plate. The S/N for the 33-kDa peak is 10, whereas that for the 66-kDa peak is 15; right BSA spectrum acquired using wax-coated plate (S/N for the 33-kDa peak is 39 and S/N for the 66-kDa peak is 158)

Fig. 3

MALDI-ToF mass spectral response for a normal eye rabbit tear protein sample: tear protein spectrum acquired using a conventional plate (a) and a wax-coated plate (b)

Fig. 4

MALDI-ToF mass spectra of a normal eye tear proteins and b dry eye tear proteins. The latter shows an increase in the 10.2-kDa lipophilin CL2 protein, and a decrease in both the 14.4-kDa cystatin/lysozyme protein(s) and the 16.5-kDa lipophilin protein

Fig. 5

PSD spectrum from an m/z 1,756 precursor peptide obtained from a tryptic digest of band 1 in the 1D SDS-PAGE gel, identified as the lipophilin CL2 protein at 10.5 kDa: a blow up of region below m/z 1,155; b full PSD spectrum

Fig. 6

MALDI-ToF mass spectra of a normal eye tear proteins and b dry eye tear proteins. The latter shows an increase in the 17.3-kDa lipophilin CL protein, and a decrease in both the 11.4-kDa β-2 microglobulin protein and the 30.4-kDa apoD protein

Fig. 7

a ES single stage mass spectrum of the normal eye peptides. b ES single stage mass spectrum of the dry eye peptides. c ES-MS/MS product ion spectrum of the m/z 661.3 (+3) peptide precursor (from normal eye tear)