November 2011
Volume 52, Issue 12
Free
Biochemistry and Molecular Biology  |   November 2011
Analysis and Comparison of Proteomic Profiles of Tear Fluid from Human, Cow, Sheep, and Camel Eyes
Author Affiliations & Notes
  • Farrukh A. Shamsi
    From the King Khaled Eye Specialist Hospital, Riyadh, Saudi Arabia;
  • Ziyan Chen
    the Zhongshan Ophthalmic Center, State Key Laboratory of Ophthalmology, Sun Yat-sen University, Guangzhou, P.R. China;
  • Jingwen Liang
    the Zhongshan Ophthalmic Center, State Key Laboratory of Ophthalmology, Sun Yat-sen University, Guangzhou, P.R. China;
  • Kaijun Li
    the Zhongshan Ophthalmic Center, State Key Laboratory of Ophthalmology, Sun Yat-sen University, Guangzhou, P.R. China;
  • Ali A. Al-Rajhi
    L. J. Memorial Hospital, Rasalgunj, Aligarh, Uttar Pradesh, India; and
  • Imtiaz A. Chaudhry
    L. J. Memorial Hospital, Rasalgunj, Aligarh, Uttar Pradesh, India; and
  • Mingtao Li
    the The Proteomics Lab, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, P.R. China.
  • Kaili Wu
    the Zhongshan Ophthalmic Center, State Key Laboratory of Ophthalmology, Sun Yat-sen University, Guangzhou, P.R. China;
    the The Proteomics Lab, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, P.R. China.
  • *Each of the following is a corresponding author: Kaili Wu, Zhongshan Ophthalmic Center, State Key Laboratory of Ophthalmology, Sun Yat-sen University, Guangzhou 510060, P.R. China; wukaili@mail.sysu.edu.cn. Farrukh A. Shamsi, L. J. Memorial Hospital, Yousuf Building, Yousuf Road, Rasalgunj, Aligarh 202 001, Uttar Pradesh, India; dr_farrukhshamsi@yahoo.com
  • Footnotes
    2  These authors contributed equally to the work presented here and should therefore be regarded as equivalent authors.
Investigative Ophthalmology & Visual Science November 2011, Vol.52, 9156-9165. doi:10.1167/iovs.11-8301
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      Farrukh A. Shamsi, Ziyan Chen, Jingwen Liang, Kaijun Li, Ali A. Al-Rajhi, Imtiaz A. Chaudhry, Mingtao Li, Kaili Wu; Analysis and Comparison of Proteomic Profiles of Tear Fluid from Human, Cow, Sheep, and Camel Eyes. Invest. Ophthalmol. Vis. Sci. 2011;52(12):9156-9165. doi: 10.1167/iovs.11-8301.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose.: To investigate the tear proteome profiles of human, cow, sheep, and camel comparatively and to explore the difference of tear protein profiles among different species.

Methods.: Tears were collected from both eyes of 25 clinically healthy volunteers, 50 cows, 25 sheep, and 50 camels. Pooled tear protein samples were separated by SDS-PAGE and two-dimensional electrophoresis. Protein spots of differential expression were excised and subjected to in-gel digestion and identification by matrix assisted laser desorption/ionization-time-of-flight/time-of-flight mass spectrum analysis. Because of the incomplete genomic data of cow, sheep, and camel, a combined strategy of de novo sequencing and BLAST (Best Local Alignment Search Tool) homology searching was also used for protein identification. The differentially expressed proteins were validated by Western blot analysis.

Results.: On comparison with human tears (182 ± 6 spots), 223 ± 8, 217 ± 11, and 241 ± 3 well-resolved protein spots were detected in triphenylmethane dye–stained gels of cow, sheep, and camel tears, respectively. Similar high-abundant proteins (lactoferrin, lysozyme, etc.) were found in all tear fluids. Tear lipocalins have been identified in cow and sheep tears. BLAST searching revealed a 21-kDa protein, identical with human vitelline membrane outer layer protein 1 (VMO1) homolog, in camel tears. The Western blot confirmed that VMO1 homolog was present in both camel and sheep tears but not in human and cow tears.

Conclusions.: The comparative proteomic analyses of tears from healthy humans, cows, sheep, and camels were first reported. Differential protein expression existed in the tear among species, offering useful information for further study on tear proteins and the related ocular diseases.

The preconjunctival and precorneal viscous liquid film, known as tears or tear film, forms an interface between the ocular tissues and the air. 1 The external surface of the eye is protected by the tear fluid and is therefore considered as a first resistance in the ocular defense system. 2 Tear film is a unique and complex fluid containing such factors as proteins, lipids, carbohydrates, and electrolytes. 3 6 Proteins in the tear film are believed to play an important role in the maintenance of ocular surface, such as protecting the external surface from potential pathogens and modulating the wound-healing process. 7 9 Changes in expression of tear proteins are associated with many systemic and ocular diseases. 10 15 This suggests that analysis of the tear fluid may provide a unique noninvasive tool for the discovery of molecular markers for disease diagnosis and prognosis. Several techniques have been used in studies on tear proteins including ELISA, 6 SDS-polyacrylamide gel electrophoresis (SDS-PAGE), two-dimensional electrophoresis (2-DE), 16 and high-performance liquid chromatography (HPLC). 17 More recently, mass spectrometry (MS)–based proteomic methods applied for detailed analysis of tear proteins with high sensitivity and resolution include SELDI-TOF MS (surface-enhanced laser desorption/ionization time-of-flight mass spectrometry), 12,18 LC-MS/MS (liquid chromatography coupled with tandem mass spectrometry), 19,20 LTQ-FT (linear ion trap–Fourier transform mass spectrometry), and LTQ-Orbitrap (linear ion trap with Orbitrap, a new high-resolution mass spectrometer). 21 To date, >509 proteins have been reported in the human tear fluid using multiple proteomic technologies. 20 22  
Several high-abundant proteins have been reported in the tear fluid of animals. Lysozyme has been found in the tear fluids of several domestic animals (sheep, goats, llamas, cattle, horses, dogs, and rabbits). 23,24 Homologous tear lipocalins have been detected in rats, pigs, dogs, hamsters, and rabbits. 25,26 Lipophilins have been reported only in tears of humans and rabbits. 26 Previous studies were limited to analysis of one or a few tear proteins of interest or comparison of different animal tear proteins using SDS-PAGE, immunologic, or enzymatic assays. 24,27 Using SDS-PAGE and Western blot, Gionfriddo et al. 27 demonstrated that llama tears had a distinct 13.6-kDa band that was confirmed as a lysozyme, although the same was not detected in cattle. They also detected lactoferrin, IgA, transferrin, ceruloplasmin, α1-antitrypsin, α1-amylase, α2-macroglobulin, and proteases in both species using anti-human or anti-mouse antibodies in the Western blot. 27 Pinard et al. 23 showed that lactoferrin was detectable via SDS-PAGE and Western blot in bison tears, which did not reveal lysozyme. 
Few studies focus on a comprehensive and comparative analysis of the tear proteins from different species using proteomics techniques. Hemsley and colleagues 28 established species-specific fractionation profiles of normal koala, mouse, dog, rat, and cat tears using SE-HPLC (size exclusion–high performance liquid chromatography), whereas they did not characterize the proteins represented by peaks in tear elution profiles of different animals. Campos et al. 29 reported the first 2-DE reference map of the proteome of pooled normal dog tears. 29 Identification by MALDI-TOF-MS (matrix-assisted laser desorption/ionization–time-of-flight–mass spectrometry) suggested that the major canine allergen protein, analogous to the lipocalin in human tears, was present in dog tears but there was little detectable lactoferrin or lysozyme in dog tears compared with human tears. Moreover, in the associated area of the human tear protein map, zinc-α-2-glycoprotein seemed to be absent or substantially reduced in dog tears. 29 In the studies of Zhou et al., 8,9 the expression of proteins in rabbit tears during corneal wound healing was analyzed using different technologies (liquid chromatography electrospray ionization mass spectrometry [LC-ESI-MS], SELDI ProteinChip, LC-ESI-MS/MS, and isobaric tags for relative and absolute quantitation [iTRAQ]). The data revealed that besides major tear protein components identified in rabbit tears, the levels of rabbit defensins (NP-1 and NP-2), which were elevated after wounding, returned to normal levels by the time the corneal abrasion healed. To our knowledge, the comparative protein profiles of normal human, cow, sheep, and camel tears have not been described. Previous studies suggested that llamas and cattle have different susceptibilities to ocular diseases. 30,31 For example, llamas appear to be resistant to infectious keratoconjunctivitis, whereas cattle seem to be much more susceptible to ocular or periocular squamous cell carcinoma compared with llamas. 27,30 Reasons for the apparently different susceptibilities to ocular disease between llamas and cattle are still not clear. The differences of the tear components among humans and different species are worthy of further investigation. 
Proteomic identification by MS is mainly dependent on genomic data. One potential obstacle to comprehensive assessments of proteins of nonmodel animals is the lack of available genomic data for the species. As of December 2010, the National Center for Biotechnology Information (NCBI) listed 553,545 human protein annotations, whereas only 89,596, 7056, and 1260 protein annotations were listed for Bos taurus, Ovis aries, and Camelus, respectively. However, researches on numerous nonmodel animals are essential for expanding our knowledge about the structure and function of proteins and the related diseases in humans. With the development of accurate MS, the interested proteins can be characterized using a de novo sequencing strategy, in which partial or complete peptide sequences are directly derived from tandem MS/MS signals. The derived sequence can then be homology matched to existing databases using BLAST (Best Local Alignment Search Tool)–related algorithms to perform putative functional classification. 32 The alternative approach makes it possible to identify proteins with high throughput in species that lack genomic and proteomic databases. 33  
Our previous study showed that the seasonal variation in camel tears collected during the summer and winter seasons was found in the composition of proteins, including lactoferrin (LF) and vitelline membrane outer layer protein 1 (VMO1) homolog. 34 In the present study, using 2-DE and MALDI-TOF/TOF-MS with a combined strategy of de novo sequencing and BLAST homology searching, we made the comparative proteomic analysis of tears from healthy humans, cows, sheep, and camels, as a basis for further studies on biochemical characterization of tear proteins and the related ocular disorders. 
Materials and Methods
Tear Sampling
This study was approved by the Institutional Review Board of the King Khaled Eye Specialist Hospital, Riyadh, Saudi Arabia, in accordance with the tenets of the Declaration of Helsinki and the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 
The normal young (mean age, 35 ± 5 years), healthy volunteers (n = 25; 10 females, 15 males) with no ocular disease, using no current eye medications, with no eye-related symptoms were recruited for the collection of human tears after obtaining informed consent. Tears were collected using small-volume (1–5 μL) glass microcapillary tubes (Drummond Scientific Co., Broomall, PA) under ×16 slit-lamp magnification. Nonreflex tears were collected from the inferior tear prism without contact with the lower lid until a total of 5 μL had been collected. Human tears were collected several times from each individual and pooled for analysis. 
Camel and sheep owners outside Riyadh, Saudi Arabia were contacted and approached for collecting camel and sheep tears. Two commercial farms (Al-Marai and Al-Safi farms, Al-Kharj, Saudi Arabia) were approached for the collection of tears from cows. Tears were collected from both eyes of 50 clinically normal camels, 50 clinically normal cows, and 25 clinically normal sheep. All animals had no signs of disease of the external ocular structures. No agents to induce lacrimation or anesthetic were used for the collection of tears. Tears were collected with a 50-μL sterile plastic pipette by placing it in the lower conjunctival fornix. Care was taken to cause as little conjunctival trauma as possible during collection. The animal samples were immediately stored on ice and brought back to the laboratory for further processing. Unless otherwise stated, all the tear samples were centrifuged at 10,000g for 5 minutes at 4°C to remove gross debris and mucus and pooled. The concentration of protein in the sample was measured by the bicinchoninic acid method using BSA as a protein standard. The tear samples were stored at −80°C and thawed only once before analysis. 
SDS-PAGE
SDS-PAGE was performed on a minivertical electrophoresis system (Bio-Rad Mini-PROTEAN 3 Cell; Bio-Rad Laboratories, Hercules, CA). Each tear sample with an equal amount of total proteins was separated on a 12% acrylamide resolving gel (0.1% SDS, 1.5 M Tris-HCl, pH 8.8) with a 5% acrylamide stacking gel (0.1% SDS, 0.5 M Tris-HCl, pH 6.8). Electrophoresis was performed in electrode buffer (0.1% SDS, 0.25 M glycine, 0.025 M Tris-HCl, pH 8.3) at 60 V for 10 minutes, and then at 120 V for 120 minutes. Each experiment was repeated three times in different gels and running buffers. 
2-DE
The sample preparation and 2-DE were conducted according to our previously reported protocols. 34,35 Briefly, prechilled acetone was added to tear samples at fourfold volume that of the sample to be precipitated. The tube was vortexed and incubated at −20°C for 120 minutes. The precipitated proteins were pelleted by centrifuging at 4°C for 10 minutes at 13,000g. The acetone was discarded and the protein pellet in the tube was air dried. 
2-DE was performed using commercial reagents and instruments (GE Health Care Bio-Sciences AB, Uppsala, Sweden). First-dimensional isoelectric focusing (IEF) was performed using a commercial unit (Ettan IPGphor II unit). Protein samples (100 μg per gel) were diluted to 250 μL in a rehydration buffer (7 M urea, 2 M thiourea, 2% CHAPS, 2.8 mg/mL dithiothreitol, 0.002% bromophenol blue, 0.5% pH 3–10 immobilized pH gradient buffer). Immobilized pH gradient (IPG) strips (13 cm, pH 3–10 linear gradient) were loaded with protein samples and rehydrated using the passive rehydration method for 1 hour and the active rehydration method at 50 V for 11 hours. Isoelectric focusing was run at 20°C with the voltage settings of 500 V for 1 hour (step and hold), 1000 V for 1 hour (gradient), 8000 V for 3 hours and 30 minutes (gradient), and finally, 8000 V for 30 minutes (step and hold). The IEF strips were subjected to the standard equilibration steps before second-dimensional electrophoresis. The IEF strips were soaked for 15 minutes in the equilibration buffer (6 M urea, 50 mM, pH 8.8, Tris-HCl, 2% SDS, 29.3% glycerol, 0.002% bromophenol blue, 1% DTT). They were then soaked for an additional 15 minutes in the same solution, except that 1% DTT was replaced with 2.5% idoacetaminde. The IEF strips were applied onto 13% SDS-PAGE. The second-dimensional SDS-PAGE was performed with a vertical electrophoresis system (Amersham Pharmacia Biotechnology, Uppsala, Sweden) at 60 V for 15 minutes, 150 V for 2 hours, and 300 V for 3 hours. The 2-DE of each group was run three times. 
Staining and Image Analysis
Gels were stained with hot triphenylmethane dye (Coomassie blue R-350; Imperial Chemical Industries, London, UK) in accordance with the protocol we used in the previous study. 34 Briefly, gels were fixed in 40% (v/v) methanol and 10% (v/v) acetic acid for 1 hour and then stained in a staining solution (0.025% Coomassie blue R-350 in 10% acetic acid) heated to 80–90°C. The gels were destained in 10% acetic acid. All gel images were recorded immediately after destaining to minimize any possibility of fading. Images were acquired with a commercial imaging program (Kodak Image Station 4000MM; Kodak, Rochester, NY). For 1-DE images, the molecular imaging program (Kodak Image Station 4000MM) was used to examine the lane profiles; 2-DE images were analyzed with a commercial software program (Melanie Ver. 4.0; GeneBio, Geneva, Switzerland). According to the method introduced by Westermeier and Naven, 36 experimental M r values were calculated by mobility comparisons with protein standard markers (Tiangen Biotech Co. Ltd, Beijing, China) run in a separate marker lane on the SDS-PAGE gel, whereas pI was determined by using a 3–10 linear scale over the total dimension of the IPG strip. 
In-Gel Digestion and MALDI-TOF/TOF-MS Analysis
The protein identification was performed using the methods reported previously. 34 Briefly, the protein spots selected for identification were manually excised and subjected to in-gel digestion. Excised gel spots were destained at 37°C with 25 mM ammonium bicarbonate/50% (v/v) acetonitrile (ACN) and then dehydrated with ACN. For digestion, the gel pieces were rehydrated in 25 mM ammonium bicarbonate solution containing 12.5 ng/μL trypsin (sequencing grade; Promega, Madison, WI) and incubated at 4°C for 30 minutes. The supernatant was discarded; gels were incubated at 37°C for 8 hours in 25 mM ammonium bicarbonate. Finally, peptides were eluted and dissolved with 25 mM ammonium bicarbonate for MALDI-TOF/TOF-MS analysis. 
For mass spectral analysis, the matrix solution was prepared by dissolving R-cyano-4-hydroxycinnamic acid (CHCA) in an ethanol/acetone mixture (2:1, v/v) to a final concentration of 1 μg/μL 2 μL sample followed by 0.1 μL matrix was applied to a chip (Anchor Chip; Bruker Daltonics, Bremen, Germany). Crystallization occurred at room temperature. MALDI-TOF and MALDI-TOF/TOF spectra were obtained using a mass spectrometer (Ultraflex III TOF/TOF mass spectrometer, controlled by FlexControl Ver. 2.4 software; Bruker Daltonics). For MS and MS/MS experiments, the mass spectrometer was calibrated externally with a mixture of bradykinin, angiotensin I and II, substance P, bombesin, renin, ACTH, somatostatin, and oxidized insulin β-chain. Peptide mass fingerprint (PMF) and MS/MS spectra were processed with commercial software (FlexAnalysis Ver. 2.4 software; Bruker Daltonics) for mass (m/z) annotation, another software package (BioTools Ver. 3.2; MASCOT MS; Matrix Science, London, UK) to search mass spectrometry databases and MS/MS ion searching, and another software package (RapiDeNovo; Bruker, Bremen) to obtain de novo sequence information. 
Database Searches
First, the PMF data combined with the corresponding MS/MS spectra data of the tryptic peptides derived from the gel spots were searched against protein sequences from the NCBI nonredundant (nr) database or International Protein Index (IPI) database (using the local MASCOT search program; http://www.matrixscience.com/). Only Homo sapiens and Bos taurus databases have been established. The data from humans and cows were searched against NCBInr Homo sapiens database and IPI_bovin database, respectively. With respect to sheep and camels, the protein annotations for Ovis aries and Camelus in the NCBInr database were loaded onto the search program (MASCOT). The data from sheep and camels were searched against the loaded protein annotations of related species. Second, the cross-species identification of the unidentified proteins from cows, sheep, and camels was performed (using MASCOT) to search against the NCBInr mammalian database. The search parameters were set as followed. One missed cleavage for tryptic peptides was allowed. Carbamidomethylation of cysteine as fixed modifications and oxidation of methionine as variable modifications were selected. The mass tolerance adjustments for the precursor ions and the resulting fragments were set to 50–100 ppm and 0.2–0.7 Da, respectively. 
Due to the poor protein and DNA sequence database coverage for Ovis aries and Camelus, proteins were identified by de novo sequencing and an MS-driven homology search (BLAST) by following a procedure outlined by Liska and Shevchenko. 32 De novo peptide sequences were deduced from the annotated fragment spectra of precursor ions (RapiDeNovo software; precursor mass tolerance, 100 ppm; fragment mass tolerance, 0.3 Da; variable modification: oxidation of methionine; fixed modification: carbamidomethylation of cysteine). Homology searches were executed with the commercial search engine (MS BLAST; http://genetics.bwh.harvard.edu/msblast, performed with the following settings: Program, blast2p; Database, NCBInr_95 and Swiss-Prot; Matrix, PAM30MS; Expect, 100; other advanced options, nogap-hspmax100-sort_by_totalscore-span1). Protein identification significance (P < 0.05) was judged using the scoring from a commercial search engine (MS BLAST). A deduced sequence should be more than seven amino acids. Most proteins included in the results list were matched by at least two unique peptide sequences. 
Western Blot Analysis
Rabbit anti-human VMO1 antibody (GeneTex Inc., San Antonio, TX) was used for Western blot analysis to validate the 2-DE and mass spectrum results. Equal amounts of total tear proteins were separated by 12% acrylamide SDS-PAGE, and then blotted onto the polyvinylidene fluoride membrane using the mini trans-blot system (Bio-Rad, Hercules, CA). The membrane was blocked with 5% fat-free milk in TBST (Tris-buffered saline including 0.1% Tween) and incubated with the blocking solution containing 1:800 dilution of the primary antibody at 4°C overnight. The membrane was subsequently incubated with the blocking solution containing a 1:4000 dilution of goat anti-rabbit IgG, horseradish peroxidase (HRP)-linked secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA). Detection was performed with a commercial detection system (Phototope-HRP Western Blot Detection System; Cell Signaling Technology, Inc., Danvers, MA). Western blot analyses were scanned and analyzed (Kodak Image Station 4000MM). The results were repeated and confirmed in three independent tests. 
Results
SDS-PAGE Patterns and 2-DE Proteome Profiles of Tears from Different Species
To investigate the difference in protein composition of tears among different species, samples were initially analyzed by SDS-PAGE after loading equal amounts of total tear proteins. The distribution of protein bands after SDS-PAGE was reproducible and species specific (Fig. 1). Approximately 12 well-resolved bands were observed in human tears, 13 bands in cow tears, 12 bands in sheep tears, and 21 bands in camel tears. Since differences in the tear protein profiles of the species were noted, we attempted to identify proteins contained in each tear sample using 2-DE and MALDI-TOF/TOF-MS analysis. 
Figure 1.
 
One-dimensional SDS-PAGE electrophoretic (1-DE) patterns of human, sheep, cow, and camel tears with equal amounts of total proteins. MW, molecular weight standards. The distribution of tear protein bands is species specific.
Figure 1.
 
One-dimensional SDS-PAGE electrophoretic (1-DE) patterns of human, sheep, cow, and camel tears with equal amounts of total proteins. MW, molecular weight standards. The distribution of tear protein bands is species specific.
In the present study, the 2-DE protein map of human tears was generally similar to those that have been reported previously. 16 We present here the comparative report of the 2-DE protein reference maps of human, cow, sheep, and camel tears (Fig. 2), as a basis for subsequent differential expression proteomic studies on tears of various species. The 2-DE maps of tears were reproducible and species specific. Approximately 182 ± 6, 223 ± 8, 217 ± 11, and 241 ± 3 well-resolved protein spots were detected in triphenylmethane (Coomassie)-stained 2-DE gels of human, cow, sheep, and camel tears, respectively. Several regions in the 2-DE analysis showed significant differences in protein patterns that were similar to those observed with the SDS gel band patterns. Triphenylmethane-stained 2-DE gels revealed that most of the spots were concentrated in the pH 5 to pH 8 range and 10- to 100-kDa M r ranges. 
Figure 2.
 
2-DE triphenylmethane dye (Coomassie)–stained protein profiles of tears from humans (A), sheep (B), cows (C), and camels (D). Protein (100 μg) was separated on first-dimensional pH 3–10 linear IPG gels (13 cm) and second-dimensional 13% vertical slab gels. The relative MW is given in the middle, whereas the pI is given at the top of the figure; 182 ± 6, 223 ± 8, 217 ± 11, and 241 ± 3 well-resolved protein spots were detected in gels of human, cow, sheep, and camel tears, respectively. The numbered spots were selected for protein identification (H, Homo sapiens; B, Bos taurus; S: Ovis aries; and C, Camelus dromedarius). *Spots were identified by de novo sequencing/MS BLAST; #spots failed to be identified.
Figure 2.
 
2-DE triphenylmethane dye (Coomassie)–stained protein profiles of tears from humans (A), sheep (B), cows (C), and camels (D). Protein (100 μg) was separated on first-dimensional pH 3–10 linear IPG gels (13 cm) and second-dimensional 13% vertical slab gels. The relative MW is given in the middle, whereas the pI is given at the top of the figure; 182 ± 6, 223 ± 8, 217 ± 11, and 241 ± 3 well-resolved protein spots were detected in gels of human, cow, sheep, and camel tears, respectively. The numbered spots were selected for protein identification (H, Homo sapiens; B, Bos taurus; S: Ovis aries; and C, Camelus dromedarius). *Spots were identified by de novo sequencing/MS BLAST; #spots failed to be identified.
Identification for Tear Proteins
After image analysis, we selected and excised 22, 26, 20, and 29 spots in the correspondent or varied regions from 2-DE gels of human (Fig. 2A, spots named H), cow (Fig. 2C, spots named B), sheep (Fig. 2B, spots named S), and camel (Fig. 2D, spots named C) tears, respectively, present in all replicates, to determine and identify these tear proteins. For most spots, we observed good PMFs suitable for de novo MALDI MS/MS analysis. Five of the most intense peaks in the PMF of each spot were selected for MS/MS analysis. The PMF data combined with MS/MS spectrum of each peptide were loaded (into the MASCOT program). In most spots, two or more peptides in the MS/MS analysis were matched to determine the identification of proteins. The resulting first-round of searching databases showed that 21 (95.45%), 21 (80.77%), 5 (25%), and 13 (44.83%) spots from the gels of human, cow, sheep, and camel tears respectively were successfully identified (Table 1). 
Table 1.
 
List of Proteins Identified by Searching Mass Spectrometry Databases*
Table 1.
 
List of Proteins Identified by Searching Mass Spectrometry Databases*
Spot No. Accession No.† Protein Name Obs. M r (kDa)/pI Theo. M r (kDa)/pI Matched Peptides Score Expect Sequence Coverage (%)‡
H1 gi 58372399 Lactoferrin 77.95/5.15 80.15/8.47 26 533 1.1e-048 42
H2 gi 58372399 Lactoferrin 72.14/7.14 80.15/8.47 29 573 1.1e-052 50
H3 gi 58372399 Lactoferrin 76.52/7.43 80.15/8.47 28 562 1.4e-051 45
H4 gi 58372399 Lactoferrin 77.09/8.78 80.15/8.47 18 574 8.8e-053 28
H5 gi 23307793 Serum albumin 67.27/6.02 71.34/6.05 28 584 8.8e-054 51
H6 gi 21619010 IGHA1 protein 61.80/5.95 54.48/6.26 10 328 3.5e-028 29
H18 gi 149673887 Ig light chain 27.38/7.66 23.67/6.97 11 376 5.6e-033 61
H19 gi 8569405 IGKC protein 27.78/8.42 26.32/6.71 9 323 1.1e-027 43
H24 gi 4504963 Lipocalin 1 precursor 15.30/5.17 19.41/5.39 9 482 1.4e-043 55
H25 gi 4504963 Lipocalin 1 precursor 15.87/5.40 19.41/5.39 6 228 3.5e-018 46
H27 gi 4504963 Lipocalin 1 precursor 14.11/5.10 19.41/5.39 9 560 2.2e-051 48
H53 gi 4504963 Lipocalin 1 precursor 14.32/4.93 19.41/5.39 11 423 1.1e-037 47
H26 gi 4505821 Prolactin-induced protein 14.42/4.76 16.85/8.26 7 333 1.1e-028 64
H51 gi 4505821 Prolactin-induced protein 14.47/4.46 16.85/8.26 8 385 7.0e-034 63
H36 gi 307141 Lysozyme precursor (EC 3.2.1.17) 12.91/9.89 17.00/9.38 13 359 2.8e-031 70
H37 gi 235948 Cystatin SA-III 11.39/4.80 14.41/4.74 11 435 7.0e-039 85
H54 gi 235948 Cystatin SA-III 11.65/4.58 14.41/4.74 9 466 5.6e-042 74
H38 gi 19882251 Cystatin-SN precursor 12.13/7.67 16.61/6.73 4 274 8.8e-023 48
H39 gi 265222 Beta 2-microglobulin 9.41/6.85 11.48/5.86 2 104 8.8e-006 27
H40 gi 4505171 Secretoglobin, family 2A, member 1 6.48/4.98 11.10/5.48 3 240 2.2e-019 30
H52 gi 38026 Zn-alpha2-glycoprotein 48.11/4.98 34.94/5.71 14 314 8.8e-027 53
B1 IPI00696714 IL of polymeric Ig receptor 74.28/7.19 83.70/7.07 25 488 5.0e-045 40
B2 IPI00696714 IL of polymeric Ig receptor 74.28/7.43 83.70/7.07 25 572 2.0e-053 38
B3 IPI00696714 IL of polymeric Ig receptor 74.28/7.66 83.70/7.07 27 620 3.2e-058 40
B5 IPI00708398 Serum albumin 69.01/5.81 71.52/5.80 8 436 7.9e-040 16
B6 IPI00852509 Putative uncharacterized protein 62.24/5.52 52.84/6.24 6 112 2.0e-007 38
B7 IPI00852509 Putative uncharacterized protein 61.56/5.70 52.84/6.24 11 385 1.0e-034 38
B8 IPI00852509 Putative uncharacterized protein 61.33/5.93 52.84/6.24 11 385 1.0e-034 38
B9 IPI00852509 Putative uncharacterized protein 61.56/7.43 52.84/6.24 11 385 1.0e-034 38
B10 IPI00852509 Putative uncharacterized protein 62.94/8.10 52.84/6.24 11 385 1.0e-034 38
B18 IPI00691861 IGL @ protein 30.12/7.83 24.92/7.53 7 167 6.3e-013 37
B20 IPI00701295 Ig J chain 26.87/4.91 18.36/5.10 6 247 6.3e-021 26
B21 IPI00702243 Similar to Equ c1 isoform 1 25.33/4.07 20.93/4.55 8 137 6.3e-010 54
B22 IPI00702243 Similar to Equ c1 isoform 1 21.31/4.89 20.93/4.55 8 144 1.3e-010 56
B25 IPI00688717 OBP (partial) 17.66/4.98 20.10/5.20 1 39 0.011 4
B36 IPI00717612 Lysozyme C, milk isozyme 12.96/9.97 19.15/9.97 2 86 7.4e-005 10
B41 IPI00716455 Hg subunit beta 11.18/7.67 16.00/7.01 6 63 0.016 42
B42 IPI00716455 Hg subunit beta 11.31/8.09 16.00/7.01 10 309 4.0e-027 64
B43 IPI00716455 Hg subunit beta 10.58/8.32 16.00/7.01 4 332 4.0e-029 43
B44 gi 122272 Hg subunit alpha-1 10.62/9.27 15.04/8.18 2 265 2.6e-012 26
B45 IPI00713229 Protein S100-A12 8.27/6.65 10.70/5.92 7 488 5.0e-045 68
B55 gi 193299659 Lactoferrin 78.80/6.05 80.00/8.69 20 136 2.0e-007 36
S2 gi 56544486 Lactoferrin 81.77/5.36 79.24/8.40 16 193 7.0e-016 21
S3 gi 56544486 Lactoferrin 80.82/7.16 79.24/8.40 30 625 4.4e-059 45
S5 gi 57164373 Pre-pro serum albumin 74.44/5.73 71.14/5.80 15 289 1.8e-025 29
S7 gi 2582411 Ig alpha HC 67.50/5.22 51.29/5.19 4 190 1.4e-015 9
S36 gi 841226 Lysozyme 11.96/9.53 3.70/8.42 2 168 2.2e-013 66
C3 gi 3431954 Lactoferrin 66.90/7.48 79.16/8.66 23 397 3.0e-037 42
C4 gi 3431954 Lactoferrin 65.80/7.52 79.16/8.66 20 287 2.1e-023 35
C6 gi 38092968 Ig HC VHDJ region 60.79/5.70 12.99/4.99 3 111 1.2e-008 42
C7 gi 38092968 Ig HC VHDJ region 61.01/6.97 12.99/4.99 1 84 6.3e-006 16
C9 gi 167473171 Ig HC variable region 52.26/6.68 15.18/8.39 1 13 0.055 10
C13 gi 21213823 Ig HC constant region 46.97/7.45 37.14/6.43 6 266 3.8e-024 23
C14 gi 21213823 Ig HC constant region 46.97/7.86 37.14/6.43 6 266 3.8e-024 23
C15 gi 21213823 Ig HC constant region 43.31/7.48 37.14/6.43 6 266 3.8e-024 23
C16 gi 21213823 Ig heavy chain constant region 42.99/7.86 37.14/6.43 6 266 3.8e-024 23
C33 gi 585437 Lysozyme C 13.23/6.05 15.24/5.91 11 415 4.8e-039 84
C34 gi 585437 Lysozyme C 12.89/6.59 15.24/5.91 7 273 7.6e-025 70
C13 gi 585437 Lysozyme C 11.16/9.94 15.24/5.91 4 76 3.4e-005 23
C50 gi 122440 Hg subunit alpha 3.60/6.21 15.30/8.07 1 39 0.00013 10
Most of the proteins from sheep and camel failed to be identified because neither genomes nor proteomes of these species have so far been extensively characterized. Under these conditions, through de novo sequencing software (RapiDeNovo software), amino acid sequences were derived from MS/MS spectra by measuring the mass differences between adjacent fragment ion peaks of the b- and y-ion series. The sequence similarity searches have been used to identify proteins via their known homologs in other species. 32,37 Therefore, 3 spots from cows, 5 spots from sheep, and 5 spots from camels, were identified as similar proteins from other organisms (Table 2). Most spots from sheep and camels matched protein sequences from cows (Bos taurus), whereas others matched sequences from pigs (Sus scrofa), rabbits (Oryctolagus cuniculus), and mice (Mus musculus). In all, 21 (95.45%), 24 (92.31%), 10 (50.0%), and 18 (62.07%) spots from gels of human, cow, sheep, and camel tears, respectively, were successfully identified using MALDI-TOF/TOF-MS analysis with a combined strategy of de novo sequencing and BLAST homology searching. 
Table 2.
 
List of Proteins Identified by Sequencing and MS BLAST*
Table 2.
 
List of Proteins Identified by Sequencing and MS BLAST*
Spot No. Precursor Ion (m/z) De Novo Sequence Homology-Matched Peptide Sequence Score Peptide Assigned to Protein Species
B17 1697.9434 ZEHTZSPSVSGSLGZR TQPPSVSGSLGQR 71 Ig LC variable region Bos taurus
B23 1398.6583 ADGVCLESSFTGR KADGVCIESSFTGR 133 Similar to OBP Bos taurus
1720.8996 ZLZTMDWTDNAGANR DAGANR
B29 871.4666 HVAYLLR KHVAYIIR 250 Lipocalin-1 Homo sapiens
1185.6364 GLSTESLLLPR RGLSTESILIPR
2341.0740 NCVVATEEELZDVSGTW ASDEEIQDVSGTW
2571.1942 HAVGSDHYLFWMMLYHMNFR DHYIF
S1 1514.8015 YGETAAVYVGLESR RYGETAAVYVAVESR 92 Polymeric Ig receptor Bos taurus
S10 1850.8954 VFGVVVSWFVDNVEVR SWFVDNVEVR 81 Ig gamma-1 chain Ovis aries
S21 1730.9158 LAGEWYSLLLASDHR KIAGEWYSILLASD 94 Salivary lipocalin Sus scrofa
S25 1366.6460 TGENGPMNVYLR KTSETGPLNVYL 118 Similar to OBP Bos taurus
1984.9393 HSEEAPHPHSELSGEWR EEAQPSLSELSGQWR
S31 1851.9117 ZEADRYTEZMRRLR EADRYAEQMR 70 MHC class I antigen Macaca mulatta
C1 1019.4382 WEETZNGR WEEAQNGR 196 Polymeric Ig receptor Bos taurus
1230.5420 CFYPSTSVNR KCYYPPTSVNR
1835.8640 ZLVESNGLVDEZYEGR LVESRGLIKEQYEGR
C5 1523.7522 AVEYGFZNDLLVR EYGFQNALIVR 227 Serum albumin Bos taurus
1597.7457 DVFLGMFLHEYAR KDAFLGSFLYEYSR
1665.9304 RLPZVSTFGNVEYR RKVPQVST
1684.7667 ZPHSEDYLSLLLGGR RMPCTEDYLSLIL
C11 1249.6455 WLHGNZELPR RWLQGNQELPR 71 IgA HC constant region Bos taurus
C23 1137.6026 ARDGYTSVLR RNGYTAVI 280 Vitelline membrane outer layer protein 1 homolog Homo sapiens
1164.5236 FGGWSEPCPZ FGDWSDHCPK
1584.7865 NAALDTHVVESESGR NTHVVESQSG
1822.9460 NNPPZGALGDDTALNVAR PPQGIPGDDTALN
1897.0345 ZTADSRTMDDTALNDAR DDTALNDAR
C49 1677.8134 ZLEMYZAPAEAVEAZ QIEIFNAPAEAVEAK 75 Lipophilin AL2 Oryctolagus cuniculus
Tear samples from humans, cows, sheep, and camels showed similar distribution of major tear proteins in the 2-DE maps, but some proteins appeared altered in the concentration. The majority of the tear proteins appear as large protein spots reflecting the high level of their expression. Many of these protein groups display charge heterogeneity, most likely due to posttranslational processing such as glycosylation. 29 Spots H1–H4, B55, S2, S3, C3, and C4 in the correspondent areas were positively identified as lactoferrin. The spots H5, B5, and S5 were verified as serum albumin, whereas the protein of C5 was analogous to serum albumin from Bos taurus. Spots B21 and B22 were identified as similar to Equ c1 isoform 1 and spots B25 and B23 were identified as odorant-binding protein (partial) (OBP) and similar to OBP, which were analogous to the lipocalin found in human tears (spots H24, H25, H27, and H53). Spot B29 homologically matched lipocalin 1 from Homo sapiens. Spots S21 and S25 were identified as salivary lipocalin from Sus scrofa and similar to OBP from Bos taurus, respectively. Lysozyme was found in all the tear samples, presented as H36, B36, S36, and C36. Moreover, in the camel tear samples, C33 and C34 were also identified as lysozyme. Based on the intensity of staining (with Coomassie blue), the amount of lysozyme in cow and sheep tears may be less than that in human and camel tears. Spot H40 was identified as secretoglobin, family 2A, member 1, which was also described as lipophilin C. Although matched by a single peptide, spot C49 may possibly have matched lipophilin AL found in rabbit tears. 26 According to its location at the low molecular weight area, we guess that lipophilin homologs may exist in camel tears. Besides major tear proteins mentioned earlier, protein S100-A12 was detected in cow tears (spot B45). Five peptides of protein spot C23 were elicited from de novo sequencing: ARDGYTSVLR, FGGWSEPCPK, NAALDTHVVESESGR, NNPPQGALGDDTALNVAR, and KTADSRTMDDTALNDAR. The protein corresponding to these peptides was annotated as vitelline membrane outer layer protein 1 homolog from human by MS BLAST (Fig. 3). The same spots from three duplicate gels were cut and analyzed by three independent MS/MS analyses. To validate this result, Western blot analysis for VMO1 homolog protein in tear fluids was conducted. 
Figure 3.
 
De novo analysis of the MALDI-TOF/TOF spectrum. (A) The PMF signals of spot C23; *indicates the parent ions further analyzed by MS/MS. (BF) The MS/MS spectra corresponding to the parent ions 1137.603, 1164.524, 1584.787, 1822.946, and 1897.036. Five peptides were elicited from de novo sequencing: ARDGYTSVLR, FGGWSEPCPK, NAALDTHVVESESGR, NNPPQGALGDDTALNVAR, and KTADSRTMDDTALNDAR. The protein corresponding to these peptides was annotated as vitelline membrane outer layer protein 1 homolog from human by MS BLAST.
Figure 3.
 
De novo analysis of the MALDI-TOF/TOF spectrum. (A) The PMF signals of spot C23; *indicates the parent ions further analyzed by MS/MS. (BF) The MS/MS spectra corresponding to the parent ions 1137.603, 1164.524, 1584.787, 1822.946, and 1897.036. Five peptides were elicited from de novo sequencing: ARDGYTSVLR, FGGWSEPCPK, NAALDTHVVESESGR, NNPPQGALGDDTALNVAR, and KTADSRTMDDTALNDAR. The protein corresponding to these peptides was annotated as vitelline membrane outer layer protein 1 homolog from human by MS BLAST.
VMO1 Homolog Detection by Western Blot Analysis
Identification of VMO1 homolog in tear fluids was further confirmed by Western blot analysis. An approximate 20-kDa protein band was recognized by rabbit anti-human VMO1 antibody in camel and sheep tears, whereas binding was not apparent in human and cow tears (Fig. 4). To the best of our knowledge a similar study has never been previously reported in the literature. 
Figure 4.
 
The Western blot immunoassay of human, sheep, cow, and camel tears. MW, molecular weight standards. In the area of approximately 20-kDa protein band, binding of anti-human VMO1 antibody indicates detection of VMO1 homolog in camel and sheep tears, whereas binding is not apparent in human and cow tears.
Figure 4.
 
The Western blot immunoassay of human, sheep, cow, and camel tears. MW, molecular weight standards. In the area of approximately 20-kDa protein band, binding of anti-human VMO1 antibody indicates detection of VMO1 homolog in camel and sheep tears, whereas binding is not apparent in human and cow tears.
Discussion
To our best knowledge, this is the first study that reports the comparative proteome profile of tears from nonmodel animals, including camels, in comparison with human tears by 2-DE and MALDI-TOF/TOF-MS analysis, with a combined strategy of de novo sequencing and BLAST homology searching. Camels, cows, and sheep have their own respective physical features. Camels can exist in arid and semiarid regions. Their ability to withstand long periods without water is due to a series of physiological adaptations. With respect to their immune system, for example, camels have antibody molecules that have only two heavy chains, which makes them smaller and more durable. 38 In addition, camels' eyes play an important role in their survival. Camels' eyes are protected from blowing sand and dust by a double row of eyelashes and three eyelids on each eye. The extra eyelid also helps protect against the blazing sun and stops them from going blind. Camels have been reported to have a weak dilator and a relatively strong constrictor iridial muscle that is a prerequisite of a strong miosis. 39 This character seems to be suitable for the bright environment of camels' habitat. 39 In terms of the categories of species, camel belongs to Tylopoda, whereas sheep and cow both belong to Ruminantia. Our results show that the spot distributions in 2-DE gels of cow and sheep tear fluids seem similar to each other but appear a bit different from the 2-DE map of camel tear fluids, which shows special low molecular weight protein spots (e.g., C49) with high content. 
The comparison of the tears from humans, cows, sheep, and camels showed the identities and differences in the tear proteome profiles of humans and other species. Similar to human tears, lysozyme, lipocalin, lactoferrin, serum albumin, and immunoglobulin were also the predominant constituents of cow, sheep, and camel tears, suggesting their important roles in the tear fluids. Meanwhile, animal tear profiles varied according to the species and did not correspond in all respects to human tear profiles. The interspecies variation of tear fluids has been found in the protein compositions; for example, homologous VMO1 was present in camel and sheep tears but not in human and cow tears. We speculated that the alteration of tear protein profiles among species might be the outcome of ocular surface structural and physiologic variation induced by a number of endogenous and exogenous elements (e.g., environmental effect) through the evolutionary mechanism. The specific presence of certain proteins (e.g., VMO1 homolog) in animals (e.g., camels) suggests that they may have special functions in the tear film, perhaps helping camels to keep the ocular surface healthy and withstand the ocular discomfort associated with its desert environment. Since multiple environmental risk factors (e.g., dry weather, windy condition, long hours of sunlight, and ultraviolet radiation) are associated with dry eye syndrome, 40 42 investigation into the composition of the tear film in camels, which live in the extremely hot, dry, and dusty desert conditions, will contribute to the evaluation of the pathophysiology and treatment of dry eye syndrome in humans. 
Proteomic techniques provide us with useful tools in analysis of tear proteins, although there are limitations in the protein separation and identification. Previous reports showed that the use of 2-DE would improve the resolution and the success rate of protein identification. 6,13,20 Results reported by Gustavo et al. 21 showed that in-gel digestion identified about fivefold more proteins than in-solution digestion in tear fluids. 21 The advantage of 2-DE analysis is that it gives the visible pattern of the expressed protein, including the MW and pI of the isolated proteins. However, the gel-based approach has certain intrinsic limitations. For example, very high molecular weight or low molecular weight proteins and strong acidic or basic proteins may not be separated in 2D gel electrophoresis. 6,20 Moreover, the incompletion of the genomic or proteomic data of nonmodel animals was a hindrance in our analysis of tear proteins from other species directly and widely. The limitations of protein separation and identification method used in this study unavoidably led to failure in the identification of certain tear proteins. Also, since we selected partial tear protein spots for further identification, it is thus highly likely that the unidentified proteins in the tears of some species possibly exist but may have possibly been missed, especially less-abundant tear proteins. 
The variation of protein spots in the 2-DE maps of tear fluids can be observed among humans and different animals, such as lysozyme. Human tear lysozyme, which is manufactured in acinar cells of the lacrimal glands and accounts for 20–40% of tear total proteins, has an antibacterial action with hydrolyzing glycosidic bonds, particularly those of certain Gram-positive bacteria, in the bacterial cell walls. 43,44 Previous studies has reported lysozyme in the tear fluids of llama, bovine, and sheep using different assays. 23,24 Gionfriddo et al. 24 found high concentrations in llama tears but lower concentrations in sheep and cattle tears by use of the microtiter plate colorometric assay. However, they failed to detect a 13-kDa band of lysozyme in cattle tears that was detected in llama and sheep tears by Western blot analysis. 24 In the study by Pinard et al., 23 Western blots of bison tears also did not reveal lysozyme. This may possibly be attributed to molecular differences in lysozymes among different species, which induce sufficient variability, preventing the antibody from binding. 23,24,45 Our study has detected lysozyme in all the tear samples through 2-DE and MS analysis. According to the intensity of spots (H36, B36, S36, and C36) in the similar regions of the gels, we inferred that cow and sheep tears might contain lower concentrations of lysozyme compared with that of human and camel tears. 
Moreover, differences in the molecular weight (MW)/isoelectric point (pI) of individual proteins (e.g., lysozyme) were present in 2-DE profiles of tear fluids. In camel tears, lysozyme was not only observed in spot C36 at approximately 11.0 kDa and pI 9.0, where it corresponded with other species, but was also observed in spots C33 and C34 at approximately 13.0 kDa and pI 6.0. Earlier studies have reported that proteins can be observed multiple times at different MW or pI regions of the 2-DE gel. 16 This may likely be due to posttranslational modifications, the formation of protein homopolymers (dimers, trimers, and multimers of a protein) of low MW proteins, proteolytic degradation, different isoforms derived from different genes of a multigene family, or chemical modifications of proteins, such as carbamoylation, during sample preparation. 16,46,47 In the animals, three major distinct lysozyme types have been identified, the c-type (chicken or conventional type), the g-type (goose-type), and the i-type (invertebrate type) lysozyme. The c-type lysozyme has been found in mammals, including primates (human) and artiodactylia (cow and sheep). In humans, c-type lysozyme is found in various body fluids (e.g., tears). Lysozyme functions according to its type and biochemical properties. Taking pH optimum for instance, defensive lysozymes typically have a neutral pH optimum and pI values of 8.0 or higher, whereas digestive lysozymes tend to have a low pH optimum and low pI value, and a higher resistance to proteases. 48  
Human tear lipocalin, previously known as tear prealbumin, comprises approximately 33% of the protein content 49 and possesses the functions with high promiscuity and binding characteristics for relative insoluble lipids, inhibitory activity on cysteine proteinases similar to cystatins, and scavenger of potentially harmful substances. 25,26,49 Tear lipocalin has been reported to consist of several isoforms with MWs between 17 and 18 kDa and pI values varying from 4.9 to 5.2, in accordance with our study. These isoforms, which show in 2D gels of human tear fluids, were speculated to be due to genetic polymorphism–heterogeneity of the N-terminus. 25 Redl 25 has reported that homologous tear lipocalins are present in various species; and OBP II of rat and Equ c 1 from horse are highly similar to tear lipocalins. Our findings, similar to Equ c1 isoform 1 and OBP (partial) (spots B21, B22, and B25) were identified in cow tears, and homology searching suggested that homologous tear lipocalins (spots S21 and S25) also existed in sheep tears. Due to the poor sequence coverage, spots C21 and C22 failed to be identified. 
Using anti-human VMO1 antibody for Western blot analysis, we demonstrated that homologous VMO1 was present in camel and sheep tears but not in human and cow tears. Although we found it only in the 2-DE gels of camel tears, the VMO1 homolog could also be detected in sheep tears by Western blot analysis. We speculate that it may be one of multiple proteins in unidentified spot S23 or spot S25 that appears as the large protein spot containing several kinds of proteins in sheep tears. The result that the homologous VMO1 was not detected in cow tears using Western blot analysis does not mean that it was absent in cow tears, given that there may be sufficient variability with the sequence of VMO1 homolog from cow that may prevent binding to anti-human VMO1 antibody. 
The analysis of the differentially expressed proteins in tear fluids can be helpful in understanding the mechanism of ocular surface maintenance and further exploring potential application for the treatment of ocular surface disorders. Take lacritin, a novel tear glycoprotein for example. It was observed in tears only from primates such as monkeys and humans, but not from dog, rabbit, or rat tears. 50 Laurie's group has applied it topically in rabbits and found lacritin appeared to increase the volume of basal tear secretion. 51 Our previous study has first identified VMO1 in camel tears and found its increasing level in the summer compared with the winter. 34 VMO1 is one of the proteins found in the outer layer of the vitelline membrane of poultry eggs. VMO1, lysozyme, and VMO2 are tightly bound to ovomucin to form a protein complex that structures the backbone of the outer layer. 52,53 Shimizu et al. 54 have analyzed the crystal structure of VMO1 and spectulated that VMO1 might have an enzymatic activity related to saccharides. So far VMO1 has not been found in human tears but has been reported in human urine and amniotic fluid. 55,56 The origin and the exact function of VMO1 in tear fluids are still unknown. The differential expression of VMO1 in tears between humans and camels gives us a clue of its important role in ocular surface maintenance, such as keeping the tear film stable and resistant against infection under the harsh environment. Thus, further insights into the function of the novel proteins (e.g., VMO1) could help to elucidate how the stable tear film is maintained. It would be interesting to know whether applying VMO1 to the therapy of dry eye or other ocular surface diseases is beneficial for the tear film and ocular surface. 
Conclusions
This is the first study that extensively compares human tear proteome with the proteomic profiles of cow, sheep, and camel tears. Differential protein expression (e.g., VMO1) existed in the tear fluids among various species. Although further studies are required to completely investigate the reason for the interspecies variation of the tear proteomic composition, especially its relationship with the environmental effect, our finding can expand the knowledge about roles of tear proteins in the maintenance of ocular surface. Our study provides an initial platform for further evaluation of modalities at the protein level that may successfully be used as an option in the diagnosis and treatment of human ocular surface disorders (e.g., dry eye syndrome). 
Footnotes
 Supported in part by National Natural Science Foundation of China Grant 81170827 (KW), Fundamental Research Funds of State Key Lab (KW), and King Abdul Aziz City for Science and Technology, Riyadh, Saudi Arabia Grant AT26-11 (FAS).
Footnotes
 Disclosure: F.A. Shamsi, None; Z. Chen, None; J. Liang, None; K. Li, None; A.A. Al-Rajhi, None; I.A. Chaudhry, None; M. Li, None; K. Wu, None
The authors thank personnel from Al-Marai and Al-Safi farms in Al-Kharj, Saudi Arabia for help in the collection of cow tears; Abulrahman Al-Hommadi, Manahi Al-Gahtani, Abdulwahab Al-theeb, and Edith Pineda for assisting with the collection and processing of tears in this study; and Kunhua Hu, The Proteomics Laboratory, Zhongshan School of Medicine, Sun Yat-sen University, for assistance in the mass spectrometric analysis. 
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Figure 1.
 
One-dimensional SDS-PAGE electrophoretic (1-DE) patterns of human, sheep, cow, and camel tears with equal amounts of total proteins. MW, molecular weight standards. The distribution of tear protein bands is species specific.
Figure 1.
 
One-dimensional SDS-PAGE electrophoretic (1-DE) patterns of human, sheep, cow, and camel tears with equal amounts of total proteins. MW, molecular weight standards. The distribution of tear protein bands is species specific.
Figure 2.
 
2-DE triphenylmethane dye (Coomassie)–stained protein profiles of tears from humans (A), sheep (B), cows (C), and camels (D). Protein (100 μg) was separated on first-dimensional pH 3–10 linear IPG gels (13 cm) and second-dimensional 13% vertical slab gels. The relative MW is given in the middle, whereas the pI is given at the top of the figure; 182 ± 6, 223 ± 8, 217 ± 11, and 241 ± 3 well-resolved protein spots were detected in gels of human, cow, sheep, and camel tears, respectively. The numbered spots were selected for protein identification (H, Homo sapiens; B, Bos taurus; S: Ovis aries; and C, Camelus dromedarius). *Spots were identified by de novo sequencing/MS BLAST; #spots failed to be identified.
Figure 2.
 
2-DE triphenylmethane dye (Coomassie)–stained protein profiles of tears from humans (A), sheep (B), cows (C), and camels (D). Protein (100 μg) was separated on first-dimensional pH 3–10 linear IPG gels (13 cm) and second-dimensional 13% vertical slab gels. The relative MW is given in the middle, whereas the pI is given at the top of the figure; 182 ± 6, 223 ± 8, 217 ± 11, and 241 ± 3 well-resolved protein spots were detected in gels of human, cow, sheep, and camel tears, respectively. The numbered spots were selected for protein identification (H, Homo sapiens; B, Bos taurus; S: Ovis aries; and C, Camelus dromedarius). *Spots were identified by de novo sequencing/MS BLAST; #spots failed to be identified.
Figure 3.
 
De novo analysis of the MALDI-TOF/TOF spectrum. (A) The PMF signals of spot C23; *indicates the parent ions further analyzed by MS/MS. (BF) The MS/MS spectra corresponding to the parent ions 1137.603, 1164.524, 1584.787, 1822.946, and 1897.036. Five peptides were elicited from de novo sequencing: ARDGYTSVLR, FGGWSEPCPK, NAALDTHVVESESGR, NNPPQGALGDDTALNVAR, and KTADSRTMDDTALNDAR. The protein corresponding to these peptides was annotated as vitelline membrane outer layer protein 1 homolog from human by MS BLAST.
Figure 3.
 
De novo analysis of the MALDI-TOF/TOF spectrum. (A) The PMF signals of spot C23; *indicates the parent ions further analyzed by MS/MS. (BF) The MS/MS spectra corresponding to the parent ions 1137.603, 1164.524, 1584.787, 1822.946, and 1897.036. Five peptides were elicited from de novo sequencing: ARDGYTSVLR, FGGWSEPCPK, NAALDTHVVESESGR, NNPPQGALGDDTALNVAR, and KTADSRTMDDTALNDAR. The protein corresponding to these peptides was annotated as vitelline membrane outer layer protein 1 homolog from human by MS BLAST.
Figure 4.
 
The Western blot immunoassay of human, sheep, cow, and camel tears. MW, molecular weight standards. In the area of approximately 20-kDa protein band, binding of anti-human VMO1 antibody indicates detection of VMO1 homolog in camel and sheep tears, whereas binding is not apparent in human and cow tears.
Figure 4.
 
The Western blot immunoassay of human, sheep, cow, and camel tears. MW, molecular weight standards. In the area of approximately 20-kDa protein band, binding of anti-human VMO1 antibody indicates detection of VMO1 homolog in camel and sheep tears, whereas binding is not apparent in human and cow tears.
Table 1.
 
List of Proteins Identified by Searching Mass Spectrometry Databases*
Table 1.
 
List of Proteins Identified by Searching Mass Spectrometry Databases*
Spot No. Accession No.† Protein Name Obs. M r (kDa)/pI Theo. M r (kDa)/pI Matched Peptides Score Expect Sequence Coverage (%)‡
H1 gi 58372399 Lactoferrin 77.95/5.15 80.15/8.47 26 533 1.1e-048 42
H2 gi 58372399 Lactoferrin 72.14/7.14 80.15/8.47 29 573 1.1e-052 50
H3 gi 58372399 Lactoferrin 76.52/7.43 80.15/8.47 28 562 1.4e-051 45
H4 gi 58372399 Lactoferrin 77.09/8.78 80.15/8.47 18 574 8.8e-053 28
H5 gi 23307793 Serum albumin 67.27/6.02 71.34/6.05 28 584 8.8e-054 51
H6 gi 21619010 IGHA1 protein 61.80/5.95 54.48/6.26 10 328 3.5e-028 29
H18 gi 149673887 Ig light chain 27.38/7.66 23.67/6.97 11 376 5.6e-033 61
H19 gi 8569405 IGKC protein 27.78/8.42 26.32/6.71 9 323 1.1e-027 43
H24 gi 4504963 Lipocalin 1 precursor 15.30/5.17 19.41/5.39 9 482 1.4e-043 55
H25 gi 4504963 Lipocalin 1 precursor 15.87/5.40 19.41/5.39 6 228 3.5e-018 46
H27 gi 4504963 Lipocalin 1 precursor 14.11/5.10 19.41/5.39 9 560 2.2e-051 48
H53 gi 4504963 Lipocalin 1 precursor 14.32/4.93 19.41/5.39 11 423 1.1e-037 47
H26 gi 4505821 Prolactin-induced protein 14.42/4.76 16.85/8.26 7 333 1.1e-028 64
H51 gi 4505821 Prolactin-induced protein 14.47/4.46 16.85/8.26 8 385 7.0e-034 63
H36 gi 307141 Lysozyme precursor (EC 3.2.1.17) 12.91/9.89 17.00/9.38 13 359 2.8e-031 70
H37 gi 235948 Cystatin SA-III 11.39/4.80 14.41/4.74 11 435 7.0e-039 85
H54 gi 235948 Cystatin SA-III 11.65/4.58 14.41/4.74 9 466 5.6e-042 74
H38 gi 19882251 Cystatin-SN precursor 12.13/7.67 16.61/6.73 4 274 8.8e-023 48
H39 gi 265222 Beta 2-microglobulin 9.41/6.85 11.48/5.86 2 104 8.8e-006 27
H40 gi 4505171 Secretoglobin, family 2A, member 1 6.48/4.98 11.10/5.48 3 240 2.2e-019 30
H52 gi 38026 Zn-alpha2-glycoprotein 48.11/4.98 34.94/5.71 14 314 8.8e-027 53
B1 IPI00696714 IL of polymeric Ig receptor 74.28/7.19 83.70/7.07 25 488 5.0e-045 40
B2 IPI00696714 IL of polymeric Ig receptor 74.28/7.43 83.70/7.07 25 572 2.0e-053 38
B3 IPI00696714 IL of polymeric Ig receptor 74.28/7.66 83.70/7.07 27 620 3.2e-058 40
B5 IPI00708398 Serum albumin 69.01/5.81 71.52/5.80 8 436 7.9e-040 16
B6 IPI00852509 Putative uncharacterized protein 62.24/5.52 52.84/6.24 6 112 2.0e-007 38
B7 IPI00852509 Putative uncharacterized protein 61.56/5.70 52.84/6.24 11 385 1.0e-034 38
B8 IPI00852509 Putative uncharacterized protein 61.33/5.93 52.84/6.24 11 385 1.0e-034 38
B9 IPI00852509 Putative uncharacterized protein 61.56/7.43 52.84/6.24 11 385 1.0e-034 38
B10 IPI00852509 Putative uncharacterized protein 62.94/8.10 52.84/6.24 11 385 1.0e-034 38
B18 IPI00691861 IGL @ protein 30.12/7.83 24.92/7.53 7 167 6.3e-013 37
B20 IPI00701295 Ig J chain 26.87/4.91 18.36/5.10 6 247 6.3e-021 26
B21 IPI00702243 Similar to Equ c1 isoform 1 25.33/4.07 20.93/4.55 8 137 6.3e-010 54
B22 IPI00702243 Similar to Equ c1 isoform 1 21.31/4.89 20.93/4.55 8 144 1.3e-010 56
B25 IPI00688717 OBP (partial) 17.66/4.98 20.10/5.20 1 39 0.011 4
B36 IPI00717612 Lysozyme C, milk isozyme 12.96/9.97 19.15/9.97 2 86 7.4e-005 10
B41 IPI00716455 Hg subunit beta 11.18/7.67 16.00/7.01 6 63 0.016 42
B42 IPI00716455 Hg subunit beta 11.31/8.09 16.00/7.01 10 309 4.0e-027 64
B43 IPI00716455 Hg subunit beta 10.58/8.32 16.00/7.01 4 332 4.0e-029 43
B44 gi 122272 Hg subunit alpha-1 10.62/9.27 15.04/8.18 2 265 2.6e-012 26
B45 IPI00713229 Protein S100-A12 8.27/6.65 10.70/5.92 7 488 5.0e-045 68
B55 gi 193299659 Lactoferrin 78.80/6.05 80.00/8.69 20 136 2.0e-007 36
S2 gi 56544486 Lactoferrin 81.77/5.36 79.24/8.40 16 193 7.0e-016 21
S3 gi 56544486 Lactoferrin 80.82/7.16 79.24/8.40 30 625 4.4e-059 45
S5 gi 57164373 Pre-pro serum albumin 74.44/5.73 71.14/5.80 15 289 1.8e-025 29
S7 gi 2582411 Ig alpha HC 67.50/5.22 51.29/5.19 4 190 1.4e-015 9
S36 gi 841226 Lysozyme 11.96/9.53 3.70/8.42 2 168 2.2e-013 66
C3 gi 3431954 Lactoferrin 66.90/7.48 79.16/8.66 23 397 3.0e-037 42
C4 gi 3431954 Lactoferrin 65.80/7.52 79.16/8.66 20 287 2.1e-023 35
C6 gi 38092968 Ig HC VHDJ region 60.79/5.70 12.99/4.99 3 111 1.2e-008 42
C7 gi 38092968 Ig HC VHDJ region 61.01/6.97 12.99/4.99 1 84 6.3e-006 16
C9 gi 167473171 Ig HC variable region 52.26/6.68 15.18/8.39 1 13 0.055 10
C13 gi 21213823 Ig HC constant region 46.97/7.45 37.14/6.43 6 266 3.8e-024 23
C14 gi 21213823 Ig HC constant region 46.97/7.86 37.14/6.43 6 266 3.8e-024 23
C15 gi 21213823 Ig HC constant region 43.31/7.48 37.14/6.43 6 266 3.8e-024 23
C16 gi 21213823 Ig heavy chain constant region 42.99/7.86 37.14/6.43 6 266 3.8e-024 23
C33 gi 585437 Lysozyme C 13.23/6.05 15.24/5.91 11 415 4.8e-039 84
C34 gi 585437 Lysozyme C 12.89/6.59 15.24/5.91 7 273 7.6e-025 70
C13 gi 585437 Lysozyme C 11.16/9.94 15.24/5.91 4 76 3.4e-005 23
C50 gi 122440 Hg subunit alpha 3.60/6.21 15.30/8.07 1 39 0.00013 10
Table 2.
 
List of Proteins Identified by Sequencing and MS BLAST*
Table 2.
 
List of Proteins Identified by Sequencing and MS BLAST*
Spot No. Precursor Ion (m/z) De Novo Sequence Homology-Matched Peptide Sequence Score Peptide Assigned to Protein Species
B17 1697.9434 ZEHTZSPSVSGSLGZR TQPPSVSGSLGQR 71 Ig LC variable region Bos taurus
B23 1398.6583 ADGVCLESSFTGR KADGVCIESSFTGR 133 Similar to OBP Bos taurus
1720.8996 ZLZTMDWTDNAGANR DAGANR
B29 871.4666 HVAYLLR KHVAYIIR 250 Lipocalin-1 Homo sapiens
1185.6364 GLSTESLLLPR RGLSTESILIPR
2341.0740 NCVVATEEELZDVSGTW ASDEEIQDVSGTW
2571.1942 HAVGSDHYLFWMMLYHMNFR DHYIF
S1 1514.8015 YGETAAVYVGLESR RYGETAAVYVAVESR 92 Polymeric Ig receptor Bos taurus
S10 1850.8954 VFGVVVSWFVDNVEVR SWFVDNVEVR 81 Ig gamma-1 chain Ovis aries
S21 1730.9158 LAGEWYSLLLASDHR KIAGEWYSILLASD 94 Salivary lipocalin Sus scrofa
S25 1366.6460 TGENGPMNVYLR KTSETGPLNVYL 118 Similar to OBP Bos taurus
1984.9393 HSEEAPHPHSELSGEWR EEAQPSLSELSGQWR
S31 1851.9117 ZEADRYTEZMRRLR EADRYAEQMR 70 MHC class I antigen Macaca mulatta
C1 1019.4382 WEETZNGR WEEAQNGR 196 Polymeric Ig receptor Bos taurus
1230.5420 CFYPSTSVNR KCYYPPTSVNR
1835.8640 ZLVESNGLVDEZYEGR LVESRGLIKEQYEGR
C5 1523.7522 AVEYGFZNDLLVR EYGFQNALIVR 227 Serum albumin Bos taurus
1597.7457 DVFLGMFLHEYAR KDAFLGSFLYEYSR
1665.9304 RLPZVSTFGNVEYR RKVPQVST
1684.7667 ZPHSEDYLSLLLGGR RMPCTEDYLSLIL
C11 1249.6455 WLHGNZELPR RWLQGNQELPR 71 IgA HC constant region Bos taurus
C23 1137.6026 ARDGYTSVLR RNGYTAVI 280 Vitelline membrane outer layer protein 1 homolog Homo sapiens
1164.5236 FGGWSEPCPZ FGDWSDHCPK
1584.7865 NAALDTHVVESESGR NTHVVESQSG
1822.9460 NNPPZGALGDDTALNVAR PPQGIPGDDTALN
1897.0345 ZTADSRTMDDTALNDAR DDTALNDAR
C49 1677.8134 ZLEMYZAPAEAVEAZ QIEIFNAPAEAVEAK 75 Lipophilin AL2 Oryctolagus cuniculus
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