Bottom–up protein identifications from microliter quantities of individual human tear samples. Important steps towards clinical relevance.

•Workflow for replicate bottom–up proteome analyses from microliter amounts of tears.•Samples obtained in a manner well compatible with current clinical practice.•Confident IDs of a few hundred proteins from analyses of individual patient.•This is a valuable advancement over previous work using pooled samples.•Triplicate analyses of dry eye patient at different treatment stages illustrate this.

A relatively simple combination of Schirmer strip sampling with straightforward sensitive nanoLC quadrupole-Orbitrap tandem mass spectrometry after a minimum of sample processing steps allows for replicate proteomic analysis of single human tears, i.e., without the requirement for sample pooling. This opens the way to clinical applications of the analytical workflow, e.g., to monitor disease progression or treatment efficacy within individual patients. Proof of concept is provided by triplicate analyses of a singular sampling of tears of a dry eye patient, before and one and two months after minor salivary gland transplantation. To facilitate comparison with the outcome of previously reported analytical protocols, we also include the data from a typical healthy young adult tear sample as obtained by our streamlined method.With 375 confidently identified proteins in the healthy adult tear, the obtained results are comprehensive and in large agreement with previously published observations on pooled samples of multiple patients. We conclude that, to a limited extent, bottom–up tear protein identifications from individual patients may have clinical relevance.

Graphical abstractFigure optionsDownload full-size imageDownload as PowerPoint slide