Single unit filter-aided method for fast proteomic analysis of tear fluid Cecilie Aass a,⇑ , Ingrid Norheim b , Erik Fink Eriksen b , Per M. Thorsby a , Milaim Pepaj a a Hormone Laboratory, Department of Medical Biochemistry, Oslo University Hospital, 0424 Oslo, Norway b Department of Endocrinology, Oslo University Hospital, 0424 Oslo, Norway article info Article history: Received 30 January 2015 Received in revised form 31 March 2015 Accepted 1 April 2015 Available online 8 April 2015 Keywords: Tear fluid Schirmer strips Liquid chromatography Mass spectrometry abstract Human tear fluid is a complex mixture containing high concentrations of proteins and is increasingly becoming an important source for studying protein biomarkers of eye-related diseases such as Graves’ ophthalmopathy. Today, the Schirmer tear test is the most widely used technique for tear collection. However, sample handling and protein extraction from these strips have been highly challenging. Cutting and removal of the Schirmer strips after extraction, which may lead to sample loss prior to down- stream analysis, are some of the challenges to consider. To address some of these limitations, we have developed a single-unit filter-aided method for both sample handling and protein extraction. In addition, we systematically investigated the most suitable conditions for protein extraction from these strips. Among the different extraction conditions applied, extraction with 100 mM ammonium bicarbonate con- taining 50 mM NaCl resulted in the highest number of identified proteins using one-dimensional liquid chromatography tandem mass spectrometry (LC–MS/MS). Moreover, 1526 proteins were identified when the optimized extraction method was combined with two-dimensional LC–MS/MS analysis, demonstrat- ing the applicability of this novel approach to the study of the tear proteome. This dataset of identified proteins represents a comprehensive catalogue of the tear proteome and may serve as a list for future biomarker research. Ó 2015 Elsevier Inc. All rights reserved. Human tear fluid is a complex biological mixture containing high concentrations of proteins [1,2] and is increasingly being used to detect protein biomarkers related to eye diseases such as dry eye [3], keratoconus [4], and Graves ophthalmopathy [5,6]. Lately, there has been an increase in proteomic analysis of tear fluid, and this has made it particularly interesting in diagnosis and pathogenesis in clinical medicine. Today, the commercially available Schirmer tear test is the most widely used collection technique of tears. However, sample handling and protein extraction from these strips remain a challenge in biochemical analysis. In general, extraction of proteins from Schirmer strips involves cutting and removal of the strip, which in turn results in multiple sample transfers, leading to considerable sample loss prior to downstream analysis [1,3,7– 12]. For instance, Lam and coworkers cut the strips into fine pieces and extracted proteins with ice-cold chloroform and methanol [7]. Others have used solely centrifugation force to extract the proteins from the strips [13,14]. Recently, Li and coworkers used ammonium bicarbonate to extract proteins before centrifugation using a cen- trifugal device [15]. Although a recent study evaluated protein loss post-collection due to interactions with the Schirmer strips [16], no study has systematically investigated the most suitable conditions for protein extraction from these strips. To reduce the inherent sample loss during cutting and transfer of the Schirmer strips, we have developed a single-unit filter-aided method for both sample handling and protein extraction that is compatible with down- stream analysis. In addition, we evaluated the effect of extraction solvent, time, temperature, and volume on the number of extracted proteins. Finally, the optimized single-unit filter-aided extraction method was combined with two-dimensional liquid chromatogra- phy tandem mass spectrometry (2D LC–MS/MS) 1 for deep sampling of the human tear proteome. Materials and methods Materials All chemicals, solvents, and columns were purchased from Sigma (St. Louis, MO, USA) unless stated otherwise. Type 1 water http://dx.doi.org/10.1016/j.ab.2015.04.002 0003-2697/Ó 2015 Elsevier Inc. All rights reserved. ⇑ Corresponding author. Fax: +47 22 84 40 77. E-mail address: cecilaas@medisin.uio.no (C. Aass). 1 Abbreviations used: 2D LC–MS/MS, two-dimensional liquid chromatography tandem mass spectrometry; CaCl 2 , calcium chloride; NaCl, sodium chloride; KH 2 PO 4 , potassium dihydrogen phosphate; HCl, hydrochloric acid; RG SF, RapiGest SF; PPS, PPS Silent Surfactant; FA, formic acid; SCX, strong cation exchange; ACN, acetonitrile; GO, Gene Ontology. Analytical Biochemistry 480 (2015) 1–5 Contents lists available at ScienceDirect Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio