title: Quantitation of 47 human tear proteins using high resolution multiple reaction monitoring (HR-MRM) based-mass spectrometry.
abstract: Tear proteins are intimately related to the pathophysiology of the ocular surface. Many recent studies have demonstrated that the tear is an accessible fluid for studying eye diseases and biomarker discovery. This study describes a high resolution multiple reaction monitoring (HR-MRM) approach for developing assays for quantification of biologically important tear proteins. Human tear samples were collected from 1000 subjects with no eye complaints (411 male, 589 female, average age: 55.5±14.5years) after obtaining informed consent. Tear samples were collected using Schirmer's strips and pooled into a single global control sample. Quantification of proteins was carried out by selecting "signature" peptides derived by trypsin digestion. A 1-h nanoLC-MS/MS run was used to quantify the tear proteins in HR-MRM mode. Good reproducibility of signal intensity (using peak areas) was demonstrated for all 47 HR-MRM assays with an average coefficient of variation (CV%) of 4.82% (range: 1.52-10.30%). All assays showed consistent retention time with a CV of less than 0.80% (average: 0.57%). HR-MRM absolute quantitation of eight tear proteins was demonstrated using stable isotope-labeled peptides.
  
  
  CONCLUSIONS
  In this study, we demonstrated for the first time the technique to quantify 47 human tear proteins in HR-MRM mode using approximately 1μl of human tear sample. These multiplexed HR-MRM-based assays show great promise of further development for biomarker validation in human tear samples. Both discovery-based and targeted quantitative proteomics can be achieved in a single quadrupole time-of-flight mass spectrometer platform (TripleTOF 5600 system).
authors.affiliation: Singapore Eye Research Institute, Singapore, Singapore; Department of Ophthalmology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore; Singapore National Eye Center, Singapore, Singapore; Office of Clinical Academic and Faculty Affairs, Duke-NUS Graduate Medical School, Singapore, Singapore.
authors.forename: Louis
authors.initials: L
authors.lastname: Tong
authors.affiliation: Department of Ophthalmology, Second Affiliated Hospital, Chongqing Medical University, Chongqing, China.
authors.forename: Xi Yuan
authors.initials: XY
authors.lastname: Zhou
authors.affiliation: Department of Ophthalmology, University of Tampere Medical School, Tampere, Finland.
authors.forename: Antti
authors.initials: A
authors.lastname: Jylha
authors.affiliation: Department of Ophthalmology, University of Tampere Medical School, Tampere, Finland.
authors.forename: Ulla
authors.initials: U
authors.lastname: Aapola
authors.affiliation: Department of Ophthalmology, Second Affiliated Hospital, Chongqing Medical University, Chongqing, China.
authors.forename: Dan Ning
authors.initials: DN
authors.lastname: Liu
authors.affiliation: Singapore Eye Research Institute, Singapore, Singapore.
authors.forename: Siew Kwan
authors.initials: SK
authors.lastname: Koh
authors.affiliation: Department of Statistics and Applied Probability, National University of Singapore, Singapore, Singapore.
authors.forename: Dechao
authors.initials: D
authors.lastname: Tian
authors.affiliation: Singhealth Polyclinics, Singapore, Singapore.
authors.forename: Joanne
authors.initials: J
authors.lastname: Quah
authors.affiliation: Department of Ophthalmology, University of Tampere Medical School, Tampere, Finland.
authors.forename: Hannu
authors.initials: H
authors.lastname: Uusitalo
authors.affiliation: Singapore Eye Research Institute, Singapore, Singapore; Department of Ophthalmology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore; Department of Ophthalmology, University of Tampere Medical School, Tampere, Finland; SRP Neuroscience and Behavioral Disorder, Duke-NUS Graduate Medical School, Singapore, Singapore.
authors.forename: Roger W
authors.initials: RW
authors.lastname: Beuerman
authors.affiliation: Singapore Eye Research Institute, Singapore, Singapore; Department of Ophthalmology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore; SRP Neuroscience and Behavioral Disorder, Duke-NUS Graduate Medical School, Singapore, Singapore. Electronic address: zhou.lei@seri.com.sg.
authors.forename: Lei
authors.initials: L
authors.lastname: Zhou
mesh_terms: D005136:Eye Proteins / Q000032:analysis* / Q000378:metabolism*; D005260:Female; D006801:Humans; D008297:Male; D013058:Mass Spectrometry / Q000379:methods*; D008875:Middle Aged; D013666:Tears / Q000378:metabolism*
chemical_list: D005136:Eye Proteins; C042434:tear proteins
keywords: High resolution MRM; Human tear proteins; Quantitative proteomics; Targeted proteomics; Tear proteomics