We analyzed an ex vivo model of in situ aged human dermal fibroblasts, obtained from 15 adult healthy donors from three different age groups using an unbiased quantitative proteome-wide approach applying label-free mass spectrometry. Thereby, we identified 2409 proteins, including 43 proteins with an age-associated abundance change. Most of the differentially abundant proteins have not been described in the context of fibroblasts’ aging before, but the deduced biological processes confirmed known hallmarks of aging and led to a consistent picture of eight biological categories involved in fibroblast aging, namely proteostasis, cell cycle and proliferation, development and differentiation, cell death, cell organization and cytoskeleton, response to stress, cell communication and signal transduction, as well as RNA metabolism and translation. The exhaustive analysis of protein and mRNA data revealed that 77% of the age-associated proteins were not linked to expression changes of the corresponding transcripts. This is in line with an associated miRNA study and led us to the conclusion that most of the age-associated alterations detected at the proteome level are likely caused post-transcriptionally rather than by differential gene expression. In summary, our findings led to the characterization of novel proteins potentially associated with fibroblast aging and revealed that primary cultures of in situ aged fibroblasts are characterized by moderate age-related proteomic changes comprising the multifactorial process of aging.

The prediction of protein localization, such as in the extracellular space, from high-throughput data is essential for functional downstream inference. It is well accepted that some secreted proteins go through the classic endoplasmic reticulum-Golgi pathway with the guidance of a signal peptide. However, a large number of proteins have been found to reach the extracellular space by following unconventional secretory pathways. There remains a demand for reliable prediction of unconventional protein secretion (UPS). Here, we present OutCyte, a fast and accurate tool for the prediction of UPS, which for the first time has been built upon experimentally determined UPS proteins. OutCyte mediates the prediction of protein secretion in two steps: first, proteins with N-terminal signals are accurately filtered out; second, proteins without N-terminal signals are classified as UPS or intracellular proteins based on physicochemical features directly generated from their amino acid sequences. We are convinced that OutCyte will play a relevant role in the annotation of experimental data and will therefore contribute to further characterization of the extracellular nature of proteins by considering the commonly neglected UPS proteins.OutCyte has been implemented as a web server at www.outcyte.com.

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Stem cell transplantation is a promising therapeutic strategy to enhance axonal regeneration after spinal cord injury. Unrestricted somatic stem cells (USSC) isolated from human umbilical cord blood is an attractive stem cell population available at GMP grade without any ethical concerns. It has been shown that USSC transplantation into acute injured rat spinal cords leads to axonal regrowth and significant locomotor recovery, yet lacking cell replacement. Instead, USSC secrete trophic factors enhancing neurite growth of primary cortical neurons in vitro. Here, we applied a functional secretome approach characterizing proteins secreted by USSC for the first time and validated candidate neurite growth promoting factors using primary cortical neurons in vitro. By mass spectrometric analysis and exhaustive bioinformatic interrogation we identified 1156 proteins representing the secretome of USSC. Using Gene Ontology we revealed that USSC secretome contains proteins involved in a number of relevant biological processes of nerve regeneration such as cell adhesion, cell motion, blood vessel formation, cytoskeleton organization and extracellular matrix organization. We found for instance that 31 well-known neurite growth promoting factors like, e.g. Injury to the spinal cord leads to a multiple damaging process including axonal contusion and transection with subsequent degeneration, massive apoptosis of oligodendrocytes and break-down of the blood-spinal cord barrier accompanied by invasion of immune cells resulting in sustained motoric and sensory impairments. Glial-fibrotic scarring and the lack of growth promoting factors impair axonal regrowth, which is currently the main target for therapeutic interventions to treat spinal cord injury. In addition, modulation of neuronal survival, remyelination of axons, and the immune reaction could promote functional regeneration (1-3). The inhibition of axonal regeneration might be overcome by exogenous application of growth factors or by transplantation of stem cells directly into the lesion site, which locally release trophic factors and thus support axonal regrowth. For clinical applications, stem cells should be ideally available on a clinical scale without ethical concerns or invasive interventions. Human umbilical cord blood (hUCB) 1 is

The key enzyme for C4 photosynthesis, Phosphoenolpyruvate Carboxylase (PEPC), evolved from nonphotosynthetic PEPC found in C3 ancestors. In all plants, PEPC is phosphorylated by Phosphoenolpyruvate Carboxylase Protein Kinase (PPCK). However, differences in the phosphorylation pattern exist among plants with these photosynthetic types, and it is still not clear if they are due to interspecies differences or depend on photosynthetic type. The genus Flaveria contains closely related C3, C3-C4 intermediate, and C4 species, which are evolutionarily young and thus well suited for comparative analysis. To characterize the evolutionary differences in PPCK between plants with C3 and C4 photosynthesis, transcriptome libraries from nine Flaveria spp. were used, and a two-member PPCK family (PPCKA and PPCKB) was identified. Sequence analysis identified a number of C3- and C4-specific residues with various occurrences in the intermediates. Quantitative analysis of transcriptome data revealed that PPCKA and PPCKB exhibit inverse diel expression patterns and that C3 and C4  Flaveria spp. differ in the expression levels of these genes. PPCKA has maximal expression levels during the day, whereas PPCKB has maximal expression during the night. Phosphorylation patterns of PEPC varied among C3 and C4  Flaveria spp. too, with PEPC from the C4 species being predominantly phosphorylated throughout the day, while in the C3 species the phosphorylation level was maintained during the entire 24 h. Since C4  Flaveria spp. evolved from C3 ancestors, this work links the evolutionary changes in sequence, PPCK expression, and phosphorylation pattern to an evolutionary phase shift of kinase activity from a C3 to a C4 mode.

A label-free solution basing on a highly reproducible and stable LC-MS/MS system allows quantitative proteome analyses. Due to nonlabeling approach, the label-free method has the potential to measure samples from clinical specimen monitoring and comparing thousands of proteins. The presented label-free workflow includes in-solution digest, LC-MS analyses, data evaluation by the means of Progenesis™ software, and validation of the differential proteins. We successfully applied this workflow in a proteomics study analyzing the human lung carcinoma cell line A549 treated with transforming growth factor beta 1, a cell culture model of lung fibrosis. The differential analysis of only 1 μg protein per sample led to 202 significantly regulated proteins.

Primary central nervous system lymphomas (PCNSLs) are mature B-cell lymphomas confined to the central nervous system (CNS). Blood-brain barrier (BBB) dysfunction drastically alters the cerebrospinal fluid (CSF) proteome in PCNSL patients. To reveal the interaction of PCNSL tumors with CNS structures and the vasculature, we conducted a whole-proteome analysis of CSF from PCNSL patients (n = 17 at initial diagnosis) and tumor-free controls (n = 10) using label-free quantitative mass spectrometry. We identified 601 proteins in the CSF proteome using a one-step approach without further prefractionation, and quantified 438 proteins in detail using the Hi-N method. An immunoassay revealed that 70% of the patients in our unselected PCNSL patient cohort had BBB dysfunction. Correlation analysis indicated that 127 (30%) of the quantified proteins were likely increased in PCSNL patients due to BBB dysfunction. After the exclusion of these proteins, 66 were found to differ in abundance (fold-change > 2.0, p < 0.05) between PCNSL and control CSF proteomes, and most of those were associated with the CNS. These data also provide the first evidence that proteomic changes in CSF from PCNSL patients are mainly associated with protein ectodomain shedding, and that shedding of human leukocyte antigen class 2 proteins is a mechanism of tumor-cell immune evasion.

For production of different monoclonal antibodies (mAbs), biopharmaceutical companies often use related upstream and downstream manufacturing processes. Such platforms are typically characterized regarding influence of upstream and downstream process (DSP) parameters on critical quality attributes (CQAs). CQAs must be monitored strictly by an adequate control strategy. One such process-related CQA is the content of host cell protein (HCP) which is typically analyzed by immunoassay methods (e.g., HCP-ELISA). The capacity of the immunoassay to detect a broad range of HCPs, relevant for the individual mAb-production process should be proven by orthogonal proteomic methods such as 2D gel electrophoresis or mass spectrometry (MS). In particular MS has become a valuable tool to identify and quantify HCP in complex mixtures. We evaluate up-and DSP parameters of four different biopharmaceutical products, two different process variants, and one mock fermentation on the HCP pattern by shotgun MS analysis and ELISA. We obtained a similar HCP pattern in different cell culture fluid harvests compared to the starting material from the downstream process. During the downstream purification process of the mAbs, the HCP level and the number of HCP species significantly decreased, accompanied by an increase in diversity of the residual HCP pattern. Based on this knowledge, we suggest a control strategy that combines multi product ELISA for in-process control and release analytics, and MS testing for orthogonal HCP characterization, to attain knowledge on the HCP level, clusters and species. This combination supports a control strategy for HCPs addressing safety and efficacy of biopharmaceutical products. K E Y W O R D S biopharmaceuticals, ELISA, host cell protein, mass spectrometry, proteomics 1 | INTRODUCTION Recombinant monoclonal antibodies (mAbs) produced in Chinese Hamster Ovarian cells are among the most important biopharmaceutical drugs. Different sub-populations of the Chinese hamster ovary (CHO) cell line are commonly used in biopharmaceutical processes for mAbs 1 . mAbs are steadily secreted into the cell culture fluid (CCF) during the fermentation process. During different steps of the DSP mAbs are purified and concentrated from CCF by separating DNA, RNA, lipids, other cell and process derived components,