Human tear film protein sampling using soft contact lenses.

BACKGROUND: Human tear protein biomarkers are useful for detecting ocular and systemic diseases. Unfortunately, existing tear film sampling methods (Schirmer strip; SS and microcapillary tube; MCT) have significant drawbacks, such as pain, risk of injury, sampling difficulty, and proteomic disparities between methods. Here, we present an alternative tear protein sampling method using soft contact lenses (SCLs). RESULTS: We optimized the SCL protein sampling in vitro and performed in vivo studies in 6 subjects. Using Etafilcon A SCLs and 4M guanidine-HCl for protein removal, we sampled an average of 60 ± 31 µg of protein per eye. We also performed objective and subjective assessments of all sampling methods. Signs of irritation post-sampling were observed with SS but not with MCT and SCLs. Proteomic analysis by mass spectrometry (MS) revealed that all sampling methods resulted in the detection of abundant tear proteins. However, smaller subsets of unique and shared proteins were identified, particularly for SS and MCT. Additionally, there was no significant intrasubject variation between MCT and SCL sampling. CONCLUSIONS: These experiments demonstrate that SCLs are an accessible tear-sampling method with the potential to surpass current methods in sampling basal tears.

Utilizing Nonequilibrium Isotope Enrichments to Dramatically Increase Turnover Measurement Ranges in Single Biopsy Samples from Humans.

The synthesis of new proteins and the degradation of old proteins in vivo can be quantified in serial samples using metabolic isotope labeling to measure turnover. Because serial biopsies in humans are impractical, we set out to develop a method to calculate the turnover rates of proteins from single human biopsies. This method involved a new metabolic labeling approach and adjustments to the calculations used in previous work to calculate protein turnover. We demonstrate that using a nonequilibrium isotope enrichment strategy avoids the time dependent bias caused by variable lag in label delivery to different tissues observed in traditional metabolic labeling methods. Turnover rates are consistent for the same subject in biopsies from different labeling periods, and turnover rates calculated in this study are consistent with previously reported values. We also demonstrate that by measuring protein turnover we can determine where proteins are synthesized. In human subjects a significant difference in turnover rates differentiated proteins synthesized in the salivary glands versus those imported from the serum. We also provide a data analysis tool, DeuteRater-H, to calculate protein turnover using this nonequilibrium metabolic 2H2O method.

Liquid Biopsy for Proliferative Diabetic Retinopathy: Single-Cell Transcriptomics of Human Vitreous Reveals Inflammatory T-Cell Signature.

Purpose: Current therapies for proliferative diabetic retinopathy (PDR) do not specifically target VEGF-independent, cell-type-specific processes that lead to vision loss, such as inflammatory pathways. This study aimed to identify targetable cell types and corresponding signaling pathways by elucidating the single-cell landscape of the vitreous of patients with PDR. Design: Case series. Subjects: Vitreous and peripheral blood obtained from 5 adult patients (6 eyes) undergoing pars plana vitrectomy for vision-threatening PDR. Methods: Single-cell RNA sequencing (scRNA-seq) was performed on vitreous cells obtained from diluted cassette washings during vitrectomy from 6 eyes and peripheral blood mononuclear cells (PBMCs, n = 5). Droplet-based scRNA-seq was performed using the Chromium 10x platform to obtain single-cell transcriptomes. Differences in tissue compartments were analyzed with gene ontology enrichment of differentially expressed genes and an unbiased ligand-receptor interaction analysis. Main Outcome Measures: Single-cell transcriptomic profiles of vitreous and peripheral blood. Results: Transcriptomes from 13 675 surgically harvested vitreous cells and 22 636 PBMCs were included. Clustering revealed 4 cell states consistently across all eyes with representative transcripts for T cells (CD2, CD3D, CD3E, and GZMA), B cells (CD79A, IGHM, MS4A1 (CD20), and HLA-DRA), myeloid cells (LYZ, CST3, AIF1, and IFI30), and neutrophils (BASP1, CXCR2, S100A8, and S100A9). Most vitreous cells were T cells (91.6%), unlike the peripheral blood (46.2%), whereas neutrophils in the vitreous were essentially absent. The full repertoire of adaptive T cells including CD4+, CD8+ and T regulatory cells (Treg) and innate immune system effectors (i.e., natural killer T cells) was present in the vitreous. Pathway analysis also demonstrated activation of CD4+ and CD8+ memory T cells and ligand-receptor interactions unique to the vitreous. Conclusions: In the first single-cell transcriptomic characterization of human vitreous in a disease state, we show PDR vitreous is primarily composed of T cells, a critical component of adaptive immunity, with activity and proportions distinct from T cells within the peripheral blood, and neutrophils are essentially absent. These results demonstrate the feasibility of liquid vitreous biopsies via collection of otherwise discarded, diluted cassette washings during vitrectomy to gain mechanistic and therapeutic insights into human vitreoretinal disease. Financial Disclosures: Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

Combining offline high performance liquid chromatography fractionation of peptides and intact proteins to enhance proteome coverage in bottom-up proteomics.

Offline peptide separation (PS) using high-performance liquid chromatography (HPLC) is currently used to enhance liquid chromatography-tandem mass spectrometry (LC-MS/MS) detection of proteins. In search of more effective methods for enhancing MS proteome coverage, we developed a robust method for intact protein separation (IPS), an alternative first-dimension separation technique, and explored additional benefits that it offers. Comparing IPS to the traditional PS method, we found that both enhance detection of unique protein IDs to a similar magnitude, though in diverse ways. IPS was especially effective in serum, which has a small number of extremely high abundance proteins. PS was more effective in tissues with fewer dominating high-abundance proteins and was more effective in enhancing detection of post-translational modifications (PTMs). Combining the IPS and PS methods together (IPS+PS) was especially beneficial, enhancing proteome detection more than either method could independently. The comparison of IPS+PS versus six PS fractionation pools increased total number of proteins IDs by nearly double, while also significantly increasing number of unique peptides detected per protein, percent peptide sequence coverage of each protein, and detection of PTMs. This IPS+PS combined method requires fewer LC-MS/MS runs than current PS methods would need to obtain similar improvements in proteome detection, and it is robust, time- and cost-effective, and generally applicable to various tissue and sample types.

Modification of the structural stability of human serum albumin in rheumatoid arthritis.

Differential scanning calorimetry (DSC) can indicate changes in structure and/or concentration of the most abundant proteins in a biological sample via heat denaturation curves (HDCs). In blood serum for example, HDC changes result from either concentration changes or altered thermal stabilities for 7-10 proteins and has previously been shown capable of differentiating between sick and healthy human subjects. Here, we compare HDCs and proteomic profiles of 50 patients experiencing joint-inflammatory symptoms, 27 of which were clinically diagnosed with rheumatoid arthritis (RA). The HDC of all 50 subjects appeared significantly different from expected healthy curves, but comparison of additional differences between the RA and the non-RA subjects allowed more specific understanding of RA samples. We used mass spectrometry (MS) to investigate the reasons behind the additional HDC changes observed in RA patients. The HDC differences do not appear to be directly related to differences in the concentrations of abundant serum proteins. Rather, the differences can be attributed to modified thermal stability of some fraction of the human serum albumin (HSA) proteins in the sample. By quantifying differences in the frequency of artificially induced post translational modifications (PTMs), we found that HSA in RA subjects had a much lower surface accessibility, indicating potential ligand or protein binding partners in certain regions that could explain the shift in HSA melting temperature in the RA HDCs. Several low abundance proteins were found to have significant changes in concentration in RA subjects and could be involved in or related to binding of HSA. Certain amino acid sites clusters were found to be less accessible in RA subjects, suggesting changes in HSA structure that may be related to changes in protein-protein interactions. These results all support a change in behavior of HSA which may give insight into mechanisms of RA pathology.