Multicenter Collaborative Study to Optimize Mass Spectrometry Workflows of Clinical Specimens.

The foundation for integrating mass spectrometry (MS)-based proteomics into systems medicine is the development of standardized start-to-finish and fit-for-purpose workflows for clinical specimens. An essential step in this pursuit is to highlight the common ground in a diverse landscape of different sample preparation techniques and liquid chromatography-mass spectrometry (LC-MS) setups. With the aim to benchmark and improve the current best practices among the proteomics MS laboratories of the CLINSPECT-M consortium, we performed two consecutive round-robin studies with full freedom to operate in terms of sample preparation and MS measurements. The six study partners were provided with two clinically relevant sample matrices: plasma and cerebrospinal fluid (CSF). In the first round, each laboratory applied their current best practice protocol for the respective matrix. Based on the achieved results and following a transparent exchange of all lab-specific protocols within the consortium, each laboratory could advance their methods before measuring the same samples in the second acquisition round. Both time points are compared with respect to identifications (IDs), data completeness, and precision, as well as reproducibility. As a result, the individual performances of participating study centers were improved in the second measurement, emphasizing the effect and importance of the expert-driven exchange of best practices for direct practical improvements.

Phenotypic map of porcine retinal ganglion cells.

PURPOSE: Porcine retina is an excellent model for studying diverse retinal processes and diseases. The morphologies of porcine retinal ganglion cells (RGCs) have, however, not yet been described comprehensively. The aim of the present study was to créate a classification of the RGCs using the 1, 1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) tracing method. METHODS: About 170 RGCs were retrogradely labeled by injecting DiI into the optic nerve of postmortem eyes and statistically analyzed by two different clustering methods: Ward's algorithm and the K-means clustering. Major axis length of the soma, soma area size, and dendritic field area size were selected as main parameters for cluster classification. RESULTS: RGC distribution in clusters was achieved according to their morphological parameters. It was feasible to combine both statistical methods, thereby obtaining a robust clustering distribution. Morphological analysis resulted in a classification of RGCs in three groups according to the soma size and dendritic field: A (large somas and large dendritic fields), B (medium to large somas and medium to large dendritic fields), C (medium to small somas and medium to small dendritic fields). Within groups, fine clustering defined several subgroups according to dendritic arborization and level of stratification. Additionally, cells stratifying in two different levels of the inner plexiform layer were observed within the clusters. CONCLUSIONS: This comprehensive study of RGC morphologies in the porcine retina provides fundamental knowledge about RGC cell types and provides a basis for functional studies toward selective RGC cell degeneration in retinal disorders.

Apoe, Mbl2, and Psp plasma protein levels correlate with diabetic phenotype in NZO mice--an optimized rapid workflow for SRM-based quantification.

Male New Zealand Obese (NZO) mice progress through pathophysiological stages similar to humans developing obesity-associated type 2 diabetes (T2D). The current challenge is to establish quantitative proteomics from small plasma sample amounts. We established an analytical workflow that facilitates a reproducible depletion of high-abundance proteins, has high throughput applicability, and allows absolute quantification of proteins from mouse plasma samples by LC-SRM-MS. The ProteoMiner equalizing technology was adjusted to the small sample amount, and reproducibility of the identifications was monitored by spike proteins. Based on the label-free relative quantification of proteins in depleted plasma of a test set of NZO mice, assays for potential candidates were designed for the setup of a targeted selected reaction monitoring (SRM) approach and absolute quantification. We could demonstrate that apolipoprotein E (Apoe), mannose-binding lectin 2 (Mbl2), and parotid secretory protein (Psp) are present at significantly different quantities in depleted plasma of diabetic NZO mice compared to non-diabetic controls using AQUA peptides. Quantification was validated for Mbl2 using the ELISA technology on non-depleted plasma. We conclude that the depletion technique is applicable to restricted sample amounts and suitable for the identification of T2D signatures in plasma.

Label-free LC-MSMS analysis of vitreous from autoimmune uveitis reveals a significant decrease in secreted Wnt signalling inhibitors DKK3 and SFRP2.

Equine recurrent uveitis is a severe and frequent blinding disease in horses which presents with auto-reactive invading T-cells, resulting in the destruction of the inner eye. Infiltration of inflammatory cells into the retina and vitreous is driven by currently unknown guidance cues, however surgical removal of the vitreous (vitrectomy) has proven therapeutically successful. Therefore, proteomic analyses of vitrectomy samples are likely to result in detection of proteins contributing to disease pathogenesis. Vitreous from healthy and ERU diseased horses were directly compared by quantitative mass spectrometry based on label-free quantification of peak intensities across samples. We found a significant upregulation of complement and coagulation cascades and downregulation of negative paracrine regulators of canonical Wnt signalling including the Wnt signalling inhibitors DKK3 and SFRP2. Based on immunohistochemistry, both proteins are expressed in equine retina and suggest localisation to retinal Müller glial cells (RMG), which may be the source cells for these proteins. Furthermore, retinal expression levels and patterns of DKK3 change in response to ERU. Since many other regulated proteins identified here are associated with RMG cells, these cells qualify as the prime responders to autoimmune triggers.