title: Absolute quantification of human tear lactoferrin using multiple reaction monitoring technique with stable-isotopic labeling.
abstract: The mass spectrometry technique of multiple reaction monitoring (MRM) was used to quantify and compare the expression level of lactoferrin in tear films among control, prostate cancer (CaP), and benign prostate hyperplasia (BPH) groups. Tear samples from 14 men with CaP, 15 men with BPH, and 14 controls were analyzed in the study. Collected tears (2 μl) of each sample were digested with trypsin overnight at 37 °C without any pretreatment, and tear lactoferrin was quantified using a lactoferrin-specific peptide, VPSHAVVAR, both using natural/light and isotopic-labeled/heavy peptides with MRM. The average tear lactoferrin concentration was 1.01 ± 0.07 μg/μl in control samples, 0.96 ± 0.07 μg/μl in the BPH group, and 0.98 ± 0.07 μg/μl in the CaP group. Our study is the first to quantify tear proteins using a total of 43 individual (non-pooled) tear samples and showed that direct digestion of tear samples is suitable for MRM studies. The calculated average lactoferrin concentration in the control group matched that in the published range of human tear lactoferrin concentration measured by enzyme-linked immunosorbent assay (ELISA). Moreover, the lactoferrin was stably expressed across all of the samples, with no significant differences being observed among the control, BPH, and CaP groups.
authors.affiliation: St George and Sutherland Clinical School, Faculty of Medicine, UNSW Australia, Sydney, NSW 2052, Australia; Minomic International Ltd, Macquarie Park, NSW 2113, Australia; Save Sight Institute, Sydney Medical School, University of Sydney, NSW 2001, Australia.
authors.forename: Jingjing
authors.initials: J
authors.lastname: You
authors.affiliation: School of Optometry and Vision Science, UNSW Australia, Sydney, NSW 2052, Australia.
authors.forename: Mark
authors.initials: M
authors.lastname: Willcox
authors.affiliation: Minomic International Ltd, Macquarie Park, NSW 2113, Australia.
authors.forename: Anna
authors.initials: A
authors.lastname: Fitzgerald
authors.affiliation: Minomic International Ltd, Macquarie Park, NSW 2113, Australia.
authors.forename: Belinda
authors.initials: B
authors.lastname: Schiller
authors.affiliation: St George and Sutherland Clinical School, Faculty of Medicine, UNSW Australia, Sydney, NSW 2052, Australia; Department of Surgery, St George Hospital, Kogarah, NSW 2217, Australia.
authors.forename: Paul J
authors.initials: PJ
authors.lastname: Cozzi
authors.affiliation: Australian Prostate Cancer Research Centre-Queensland, Institute of Health and Biomedical Innovation, Queensland University of Technology, Translational Research Institute, Brisbane, QLD 4102, Australia.
authors.forename: Pamela J
authors.initials: PJ
authors.lastname: Russell
authors.affiliation: Minomic International Ltd, Macquarie Park, NSW 2113, Australia.
authors.forename: Bradley J
authors.initials: BJ
authors.lastname: Walsh
authors.affiliation: Bioanalytical Mass Spectrometry Facility, Mark Wainwright Analytical Centre, UNSW Australia, Sydney, NSW 2052, Australia; School of Medical Sciences, UNSW Australia, Sydney, NSW 2052, Australia.
authors.forename: Valerie C
authors.initials: VC
authors.lastname: Wasinger
authors.affiliation: St George and Sutherland Clinical School, Faculty of Medicine, UNSW Australia, Sydney, NSW 2052, Australia; Cancer Care Centre, St George Hospital, Kogarah, NSW 2217, Australia.
authors.forename: Peter H
authors.initials: PH
authors.lastname: Graham
authors.affiliation: St George and Sutherland Clinical School, Faculty of Medicine, UNSW Australia, Sydney, NSW 2052, Australia; Cancer Care Centre, St George Hospital, Kogarah, NSW 2217, Australia. Electronic address: y.li@unsw.edu.au.
authors.forename: Yong
authors.initials: Y
authors.lastname: Li
mesh_terms: D000368:Aged; D000369:Aged, 80 and over; D000595:Amino Acid Sequence; D002138:Calibration; D016022:Case-Control Studies; D006801:Humans; D007553:Isotope Labeling; D007781:Lactoferrin / Q000032:analysis* / Q000737:chemistry; D057230:Limit of Detection; D008297:Male; D008875:Middle Aged; D011470:Prostatic Hyperplasia / Q000378:metabolism; D015203:Reproducibility of Results; D013666:Tears / Q000737:chemistry*
chemical_list: D007781:Lactoferrin
keywords: Lactoferrin; Multiple reaction monitoring; Prostate cancer; Tears