Indirect protein quantification of drug-transforming enzymes using peptide group-specific immunoaffinity enrichment and mass spectrometry

Immunoaffinity enrichment of proteotypic peptides, coupled with selected reaction monitoring, enables indirect protein quantification. However the lack of suitable antibodies limits its widespread application. We developed a method in which multi-specific antibodies are used to enrich groups of peptides, thus facilitating multiplexed quantitative protein assays. We tested this strategy in a pharmacokinetic experiment by targeting a group of homologous drug transforming proteins in human hepatocytes. Our results indicate the generic applicability of this method to any biological system

Indirect protein quantification of drug-transforming enzymes using peptide group-specific immunoaffinity enrichment and mass spectrometry

Immunoaffinity enrichment of proteotypic peptides, coupled with selected reaction monitoring, enables indirect protein quantification. However the lack of suitable antibodies limits its widespread application. We developed a method in which multi-specific antibodies are used to enrich groups of peptides, thus facilitating multiplexed quantitative protein assays. We tested this strategy in a pharmacokinetic experiment by targeting a group of homologous drug transforming proteins in human hepatocytes. Our results indicate the generic applicability of this method to any biological system

Human tear fluid proteome dataset for usage as a spectral library and for protein modeling

This article provides a detailed dataset of human tear fluid proteins. Samples were fractionated by sodium dodecyl sulfate (SDS) gel electrophoresis resulting in 48 fractions that were spiked with an indexed retention time (iRT) peptide standard. These data are based on a data-dependent acquisition (DDA) mass spectrometric approach and can be used for example as a spectral library for tear fluid proteome analysis by data-independent acquisition (DIA). Moreover, the provided data set can be used with optimized HPLC and mass spectrometric settings for proteins/peptides of interest. Besides these aspects, this dataset can serve as a protein overview for gene ontology enrichment analysis and for modeling and benchmarking of multiple signaling pathways associated with the ocular surface in healthy or disease stages. The mass spectrometry proteomics data from the described workflow have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD011075

Proteomic Analysis of Retinal Tissue in an S100B Autoimmune Glaucoma Model

Glaucoma is a neurodegenerative disease that leads to damage of retinal ganglion cells and the optic nerve. Patients display altered antibody profiles and increased antibody titer, e.g., against S100B. To identify the meaning of these antibodies, animals were immunized with S100B. Retinal ganglion cell loss, optic nerve degeneration, and increased glial cell activity were noted. Here, we aimed to gain more insights into the pathophysiology from a proteomic point of view. Hence, rats were immunized with S100B, while controls received sodium chloride. After 7 and 14 days, retinae were analyzed through mass spectrometry and immunohistology. Using data-independent acquisition-based mass spectrometry, we identified more than 1700 proteins on a high confidence level for both study groups, respectively. Of these 1700, 43 proteins were significantly altered in retinae after 7 days and 67 proteins revealed significant alterations at 14 days. For example, α2-macroglobulin was found significantly increased not only by mass spectrometry analysis, but also with immunohistological staining in S100B retinae at 7 and 14 days. All in all, the identified proteins are often associated with the immune system, such as heat shock protein 60. Once more, these data underline the important role of immunological factors in glaucoma pathogenesis

Quality control - a stepchild in quantitative proteomics

Proteomic studies using mass spectrometry (MS)-based quantification are a main approach to the discovery of new biomarkers. However, a number of analytical conditions in front and during MS data acquisition can affect the accuracy of the obtained outcome. Therefore, comprehensive quality assessment of the acquired data plays a central role in quantitative proteomics, though, due to the immense complexity of MS data, it is often neglected. Here, we address practically the quality assessment of quantitative MS data, describing key steps for the evaluation, including the levels of raw data, identification and quantification. With this, four independent datasets from cerebrospinal fluid, an important biofluid for neurodegenerative disease biomarker studies, were assessed, demonstrating that sample processing-based differences are already reflected at all three levels but with varying impacts on the quality of the quantitative data. Specifically, we provide guidance to critically interpret the quality of MS data for quantitative proteomics. Moreover, we provide the free and open source quality control tool $\it MaCProQC$, enabling systematic, rapid and uncomplicated data comparison of raw data, identification and feature detection levels through defined quality metrics and a step-by-step quality control workflow

Proteomic analysis of retinal tissue in an S100B autoimmune glaucoma model

Glaucoma is a neurodegenerative disease that leads to damage of retinal ganglion cells and the optic nerve. Patients display altered antibody profiles and increased antibody titer, e.g., against S100B. To identify the meaning of these antibodies, animals were immunized with S100B. Retinal ganglion cell loss, optic nerve degeneration, and increased glial cell activity were noted. Here, we aimed to gain more insights into the pathophysiology from a proteomic point of view. Hence, rats were immunized with S100B, while controls received sodium chloride. After 7 and 14 days, retinae were analyzed through mass spectrometry and immunohistology. Using data-independent acquisition-based mass spectrometry, we identified more than 1700 proteins on a high confidence level for both study groups, respectively. Of these 1700, 43 proteins were significantly altered in retinae after 7 days and 67 proteins revealed significant alterations at 14 days. For example, $\alpha$2-macroglobulin was found significantly increased not only by mass spectrometry analysis, but also with immunohistological staining in S100B retinae at 7 and 14 days. All in all, the identified proteins are often associated with the immune system, such as heat shock protein 60. Once more, these data underline the important role of immunological factors in glaucoma pathogenesis