New perspectives in human tear analysis?

Abbreviations: DTT-dithiothreitol IAA-iodoacetamide LC-MS/MS-liquid chromatography (nano HPLC) and mass spectrometry SDS-sodium dodecyl sulphate T he human tear has the primary function to lubricate the cornea and to be the first-line protective surface against pathogens. Due to difficulties with their collection, tears are rarely utilized in clinical medicine. Nevertheless, these exocrine secretions could be very valuable as a source of potential biomarkers. Direct and prompt regulations of the tears production via parasympa-thetic tract of nucleus lacrimalis (n. VII) in pons Varoli throughout n. maxillaris of n. V enables to reveal changes in tear composition as an indicative response to various neuropsychiatric entities. Plasma, urine, and cerebrospinal fluid have been studied extensively in clinical medicine, but tears attracted only marginal interest so far. However, due to major advances in proteomics technique we can readily identify and quantify hundreds to thousands of proteins in single experiment. This can contribute to our understanding of complex biological phenomena and diseases. In our previous studies, we found deviations of human saliva and sweat structures in patients suffering from panic disorder (Kukumberg et al. 2009). Currently, we aim to compare proteins of human tears of healthy subjects with tear proteome of patients affected by different neuropsychiatric disorders. In our pilot study we analysed 54 tear samples from 7 healthy individuals (5 males and 2 females w/ average age 30). Tear proteins were extracted with SDS-Tris-HCl (pH 6.8) containig 2% β-mercaptoethanol, purified via methanol-chlo-roform precipitation, reduced with DTT, alkylated with IAA, digested with sequencing grade trypsin and analyzed by LC-MS/MS on LTQ-Orbitrap Velos mass spectrometer (Thermo Scientific) (Takáč et al. 2014). We identified 300 proteins, out of which only 59 (19.6%) were detected previously (Zhou et al. 2012) while 241 (80.4%) proteins were novel, not reported in the literature yet. To our knowledge, we are first ones to discover the presence of pituitary adenylate cyclase-activating polypeptide (PACAP) in human tear. PACAP and its receptors are expressed in the hypothalamus, the gonado-trope cells of the anterior pituitary gland, and the