Proteome-wide analysis reveals an age-associated cellular phenotype of in situ aged human fibroblasts

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

Sparse Proteomics Analysis - A compressed sensing-based approach for feature selection and classification of high-dimensional proteomics mass spectrometry data

Background: High-throughput proteomics techniques, such as mass spectrometry (MS)-based approaches, produce very high-dimensional data-sets. In a clinical setting one is often interested in how mass spectra differ between patients of different classes, for example spectra from healthy patients vs. spectra from patients having a particular disease. Machine learning algorithms are needed to (a) identify these discriminating features and (b) classify unknown spectra based on this feature set. Since the acquired data is usually noisy, the algorithms should be robust against noise and outliers, while the identified feature set should be as small as possible. Results: We present a new algorithm, Sparse Proteomics Analysis (SPA), based on the theory of compressed sensing that allows us to identify a minimal discriminating set of features from mass spectrometry data-sets. We show (1) how our method performs on artificial and real-world data-sets, (2) that its performance is competitive with standard (and widely used) algorithms for analyzing proteomics data, and (3) that it is robust against random and systematic noise. We further demonstrate the applicability of our algorithm to two previously published clinical data-sets

A proteomics-based approach for the identification of biliary markers of cholangiocarcinoma

Prognosis for patients with cholangiocarcinoma (CCA) continues to be poor as a result of the difficulty in distinguishing malignant from benign bile duct disease, late stage diagnosis and a lack of sufficiently sensitive and specific diagnostic markers. These factors underlie the pressing clinical need for novel disease biomarkers. The utility of bile as a proximal fluid for biomarker discovery (compared to serum) was investigated using two dimensional difference gel electrophoresis (2D DIGE). Significant differences between the proteomes of bile and serum were identified, supporting the hypothesis that bile offers a potentially enriched microenvironment of proteins shed/secreted by tumour. However, as with serum, a few major abundant proteins dominate the bile proteome, therefore an albumin/IgG depletion technique was optimised to improve biomarker identification. The bile proteome was initially characterised in samples from patients with hilar CCA. A protein mastermap was generated by two dimensional polyacrylamide gel electrophoresis (2D PAGE) and a catalogue of proteins by liquid chromatography – tandem mass spectrometry (LC-MS/MS), which identified 80 and 813 unique proteins respectively. This represents one of the largest compendiums to date and forms a basis for future proteomic-based biomarker studies. A comparative analysis of biliary proteins in CCA and benign biliary disease was performed using a label-free proteomic approach to identify potential diagnostic biomarker(s). Comparative analysis of bile protein profile in 5 patients with CCA versus benign biliary disease identified 13 proteins which were at higher levels in malignant disease of which metalloproteinase-9 (MMP-9), Rho GDP-dissociation inhibitor 2 (also known as Ly-GDI), Annexin A3 and pre-B-cell colony-enhancing factor (PBEF) were taken forward in immunoblotting-based validation. MMP-9 was shown to be overexpressed in bile of CCA and represents a potential diagnostic marker. In addition analysis of bile samples showed lipocalin-2 and its complex with MMP-9 were present in greater amounts in CCA compared to benign biliary disease

Age-related changes in CNS and PNS neuronal structures within C57BL / 6J male mice that regulate continence

The prevalence of urinary incontinence (UI), faecal incontinence (FI) and chronic constipation increases with age. Sufferers tend to have reduced quality of life, with treatments being far from ideal. Furthermore, treatment costs place significant financial burden on the economy. The lumbosacral somatic dorsolateral nucleus (DLN) and spinal nucleus of the bulbospongiosus (SNB), and the sacral parasympathetic nucleus (SPN), exert control over the external urethral sphincter (EUS) external anal sphincter (EAS), and bladder detrusor / colorectal smooth muscle, respectively. Pontine nuclei, including the pontine micturition centre (PMC), locus coeruleus (LC) and laterodorsal tegmental nucleus (LDTg), and the hypothalamic paraventricular nucleus (PVN) share connection pathways and exert control over defaecation and micturition. In this work, lumbosacral spinal structures were immunolabelled alongside inhibitory methionine-enkephalin (met-ENK) and gamma aminobutyric acid (GABA) boutons; pontine structures were immunolabelled alongside inhibitory met-ENK boutons; and the PVN was immunolabelled alongside inhibitory GABA and excitatory glutamate. The density of GABA and met-ENK in the SPN significantly decreased with age; the density of glutamate significantly increased in the PVN periventricular region (PVNpv); and the number of GABA inputs onto OXY+ and VP+ parvocellular soma within the PVN medial parvocellular dorsal division (PVNmpd) significantly increased with age. In all other nuclei the density / number of inputs from immunolabelled boutons remained unchanged with age. Furthermore, soma size and cell number (observed in pontine and spinal nuclei) were maintained with age. The distal colon (DC) is also extensively controlled by the intrinsic enteric nervous system (ENS) which is known to be subject to age-related structural changes. Protein was extracted from the whole DC with the future aim of extracting proteins specifically from the myenteric plexus (MP). Subsequently, whole DC protein extract was subject to downstream protein analysis to determine expression changes with age. Forty-four proteins showed age-associated change in regulation. These findings indicate that age-associated changes occur at all levels of nervous and non-nervous structures that may contribute to age-related voiding dysfunctions