Evaluation of inter-day and inter-individual variability of tear peptide/protein profiles by MALDI-TOF MS analyses.
OBJECTIVE
To characterize the tear film peptidome and low molecular weight protein profiles of healthy control individuals, and to evaluate changes due to day-to-day and individual variation and tear collection methods, by using solid phase extraction coupled to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) profiling.


METHODS
The tear protein profiles of six healthy volunteers were analyzed over seven days and inter-day and inter-individual variability was evaluated. The bilaterality of tear film and the effect of tear collection methods on protein profiles were also analyzed in some of these patients. MALDI-TOF MS analyses were performed on tear samples purified by using a solid phase extraction (SPE) method based on C18 functionalized magnetic beads for peptide and low molecular weight protein enrichment, focusing spectra acquisition on the 1 to 20 kDa range. Spectra were analyzed using principal component analysis (PCA) with MultiExperiment Viewer (TMeV) software. Volunteers were examined in terms of tear production status (Schirmer I test), clinical assessment of palpebral lids and meibomian glands, and a subjective OSD questionnaire before tear collection by a glass micro-capillary.


RESULTS
Analysis of peptides and proteins in the 1-20 kDa range showed no significant inter-day differences in tear samples collected from six healthy individuals during seven days of monitoring, but revealed subtle intrinsic inter-individual differences. Profile analyses of tears collected from the right and left eyes confirmed tear bilaterality in four healthy patients. The addition of physiologic serum for tear sample collection did not affect the peptide and small protein profiles with respect to the number of resolved peaks, but it did reduce the signal intensity of the peaks, and increased variability. Magnetic beads were found to be a suitable method for tear film purification for the profiling study.


CONCLUSIONS
No significant variability in tear peptide and protein profiles below 20 kDa was found in healthy controls over a seven day period, nor in right versus left eye profiles from the same individual. Subtle inter-individual differences can be observed upon tear profiling analysis and confirm intrinsic variability between control subjects. Addition of physiologic serum for tear collection affects the proteome and peptidome in terms of peak intensities, but not in the composition of the profiles themselves. This work shows that MALDI-TOF MS coupled with C18 magnetic beads is an effective and reproducible methodology for tear profiling studies in the clinical monitoring of patients.
Bioftalmik, Parque Tecnológico de Vizcaya, Derio, Spain.
Nerea
N
González
Ibon
I
Iloro
Juan A
JA
Durán
Félix
F
Elortza
Tatiana
T
Suárez
D000328:Adult; D003228:Conjunctiva; D005136:Eye Proteins / Q000032:analysis*; D005260:Female; D006801:Humans; D008297:Male; D008537:Meibomian Glands; D008970:Molecular Weight; D015588:Observer Variation; D010455:Peptides / Q000032:analysis*; D025341:Principal Component Analysis; D020543:Proteome / Q000032:analysis*; D044967:Serum / Q000737:chemistry; D052616:Solid Phase Extraction / Q000379:methods*; D013048:Specimen Handling; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D011795:Surveys and Questionnaires; D013666:Tears / Q000737:chemistry*
D005136:Eye Proteins; D010455:Peptides; D020543:Proteome; C042434:tear proteins