Objective
With increasing age, skin is subject to alterations in its organization, which impact on its function as well as having clinical consequences. Proteomics is a useful tool for non‐targeted, ...semi‐quantitative simultaneous investigation of high numbers of proteins. In the current study, we utilize proteomics to characterize and contrast age‐associated differences in photoexposed and photoprotected skin, with a focus on the epidermis, dermal–epidermal junction and papillary dermis.
Methods
Skin biopsies from buttock (photoprotected) and forearm (photoexposed) of healthy volunteers (aged 18–30 or ≥65 years) were transversely sectioned from the stratum corneum to a depth of 250 μm. Following SDS‐PAGE, each sample lane was segmented prior to analysis by liquid chromatography‐mass spectrometry/mass spectrometry. Pathway analysis was carried out using Ingenuity IPA.
Results
Comparison of skin proteomes at buttock and forearm sites revealed differences in relative protein abundance. Ageing in skin on the photoexposed forearm resulted in 80% of the altered proteins being increased with age, in contrast to the photoprotected buttock where 74% of altered proteins with age were reduced. Functionally, age‐altered proteins in the photoexposed forearm were associated with conferring structure, energy and metabolism. In the photoprotected buttock, proteins associated with gene expression, free‐radical scavenging, protein synthesis and protein degradation were most frequently altered.
Conclusion
This study highlights the necessity of not considering photoageing as an accelerated intrinsic ageing, but as a distinct physiological process.
Résumé
Objectif
Avec l’âge, la peau est sujette à des altérations dans son organisation, et outre le fait d'avoir des conséquences cliniques cela a un impact sur sa fonction. La protéomique est un outil utile pour l’évaluation non ciblée, semi‐quantitative, simultanée d'un nombre élevé de protéines. Dans cette étude, nous utilisons la protéomique pour caractériser et comparer les différences associées à l’âge entre une peau photoexposée et une peau photoprotégée, avec une attention particulière sur l’épiderme, la jonction dermo–épidermique et le derme papillaire.
Méthodes
Des biopsies de peau de la fesse (photoprotégée) et de l'avant‐bras (photoexposée) de volontaires sains (âgés de 18 à 30 ans ou de ≥ 65 ans) ont été sectionnées transversalement depuis la couche cornée jusqu’à une profondeur de 250 μm. Suite à une électrophorèse SDS‐PAGE, chaque échantillon a été segmenté avant l'analyse par chromatographie en phase liquide couplée à la spectrométrie de masse/spectrométrie de masse. Une analyse des voies de signalisation a été réalisée à l'aide d'Ingenuity IPA.
Résultats
La comparaison des protéomes de la peau des sites des fesses et de l'avant‐bras a révélé des différences dans l'abondance relative de protéines. Le vieillissement de la peau de l'avant‐bras photoexposée montre une augmentation de 80% des protéines altérées avec l’âge, contrairement à la peau des fesses photoprotégée où une réduction de 74 % des protéines altérées avec l’âge a été mesurée. Sur le plan de la fonction, les protéines altérées par l’âge dans la peau de l'avant‐bras photoexposée étaient associées à une structure, une énergie et un métabolisme. Dans la peau des fesses photoprotégée, les protéines associées à l'expression génique, la neutralisation des radicaux‐libres, la synthèse des protéines et la dégradation des protéines étaient le plus fréquemment altérés.
Conclusion
Cette étude souligne la nécessité de ne pas considérer le photovieillissement comme un vieillissement accéléré intrinsèque, mais comme un processus physiologique distinct.
In this study, we investigated the impact of ageing and light on the proteomes of skin collected at photoexposed and photoprotected sites. Although most of the altered proteins in skin from aged individuals were higher in abundance than in skin from younger individuals, in the photoprotected buttock the majority of the altered proteins were reduced with age. Differences in the functions of altered proteins between photoprotected and photoexposed sites also suggest that the nature of photoageing in skin may be very different and perhaps distinct from non‐photoageing.
Rheumatoid arthritis (RA) is a common autoimmune disease that causes significant disability and reduced life expectancy. The folate antagonist methotrexate (MTX) is first-line therapy for RA when ...used weekly at low doses (5-25 mg). However, the true rate of adherence to MTX is uncertain. This is in part due to the different methods of measurement of adherence employed with no biochemical test currently available to determine adherence to low dose MTX. Common methods of MTX measurement include immunoassays in patients with high dose therapy, but these assays cross-react with MTX metabolites and lack the sensitivity required to measure adherence to low dose MTX. HPLC-SRM-MS (selected reaction monitoring-mass spectrometry) has several theoretical advantages over immunoassays with improved specificity, minimal cross-reaction and higher sensitivity. The aim of this study was to develop an assay to measure MTX and its major metabolite 7-OH-MTX in urine as a tool to monitor adherence to low dose MTX in clinic. As a proof of concept, urine samples from 4 participants with RA were measured after directly observed therapy. The assay showed improved sensitivity compared to that reported by immunoassays, with low carryover and high within-run precision. In participant samples, MTX was measurable in the urine for up to 105 hours after administration and 7-OH-MTX was detectable up to 98 hours after administration, suggesting that this assay is suitable for the measurement of adherence to therapy. The assay requires minimal sample preparation and can be adopted by other laboratories with minimal study set up.
Functional genomic experiments frequently involve a comparison of the levels of gene expression between two or more genetic, developmental, or physiological states. Such comparisons can be carried ...out at either the RNA (transcriptome) or protein (proteome) level, but there is often a lack of congruence between parallel analyses using these two approaches. To fully interpret protein abundance data from proteomic experiments, it is necessary to understand the contributions made by the opposing processes of synthesis and degradation to the transition between the states compared. Thus, there is a need for reliable methods to determine the rates of turnover of individual proteins at amounts comparable to those obtained in proteomic experiments. Here, we show that stable isotope-labeled amino acids can be used to define the rate of breakdown of individual proteins by inspection of mass shifts in tryptic fragments. The approach has been applied to an analysis of abundant proteins in glucose-limited yeast cells grown in aerobic chemostat culture at steady state. The average rate of degradation of 50 proteins was 2.2%/h, although some proteins were turned over at imperceptible rates, and others had degradation rates of almost 10%/h. This range of values suggests that protein turnover is a significant missing dimension in proteomic experiments and needs to be considered when assessing protein abundance data and comparing it to the relative abundance of cognate mRNA species.
Saccharomyces cerevisiae activates general amino acid control (GCN) in response to amino acid starvation. Some aspects of this response are known to be conserved in other fungi including Candida ...albicans, the major systemic fungal pathogen of humans. Here, we describe a proteomic comparison of the GCN responses in S. cerevisiae and C. albicans. We have used high-resolution two-dimensional (2-D) gel electrophoresis and peptide mass fingerprinting to develop a 2-D protein map of C. albicans. A total of 391 protein spots, representing 316 open reading frames, were identified. Fifty-five C. albicans and 65 S. cerevisiae proteins were identified that responded reproducibly to 3-aminotriazole (3AT) in a Gcn4p-dependent fashion. The changes in the S. cerevisiae proteome correlated with the response in the S. cerevisiae transcript profile to 3AT treatment (rank correlation coefficient = 0.59; Natarajan et al., Molec. Cell. Biol. 2001, 21, 4347-4368). Significant aspects of the GCN response were conserved in C. albicans and S. cerevisiae. In both fungi, amino acid biosynthetic enzymes on multiple metabolic pathways were induced by 3AT in a Gcn4p-dependent fashion. Carbon metabolism functions were also induced. However, subtle differences were observed between these fungi. For example, purine biosynthetic enzymes were induced in S. cerevisiae, but were not significantly induced in C. albicans. These differences presumably reflect the contrasting niches of these relatively benign and pathogenic yeasts, respectively.
As data rates rise, there is a danger that informatics for high‐throughput LC‐MS becomes more opaque and inaccessible to practitioners. It is therefore critical that efficient visualisation tools are ...available to facilitate quality control, verification, validation, interpretation, and sharing of raw MS data and the results of MS analyses. Currently, MS data is stored as contiguous spectra. Recall of individual spectra is quick but panoramas, zooming and panning across whole datasets necessitates processing/memory overheads impractical for interactive use. Moreover, visualisation is challenging if significant quantification data is missing due to data‐dependent acquisition of MS/MS spectra. In order to tackle these issues, we leverage our seaMass technique for novel signal decomposition. LC‐MS data is modelled as a 2D surface through selection of a sparse set of weighted B‐spline basis functions from an over‐complete dictionary. By ordering and spatially partitioning the weights with an R‐tree data model, efficient streaming visualisations are achieved. In this paper, we describe the core MS1 visualisation engine and overlay of MS/MS annotations. This enables the mass spectrometrist to quickly inspect whole runs for ionisation/chromatographic issues, MS/MS precursors for coverage problems, or putative biomarkers for interferences, for example. The open‐source software is available from http://seamass.net/viz/.
Purpose The esthetic and functional rehabilitation of oncologic patients subjected to major resection surgery constitutes one of the greatest challenges for the head and neck surgeon. Immediate bone ...reconstruction with microsurgical free tissue transfer and dental implants has constituted a genuine revolution in the management of such patients. Materials and Methods We present a series of 111 oncologic patients, involving a total of 706 implants, who underwent reconstruction with pedicled or free microsurgical flaps. Results The osseointegration success rate was 92.9%, with a global failure rate (malpositioning or failed osseointegration or loading) of 15%. Failure particularly affected the group of irradiated patients and those subjected to lateral osseomyocutaneous trapezial pedicled flap reconstruction. Excellent results were obtained with the fibular and iliac crest free flaps and osseointegrated dental implants. Conclusions The difficulties of prosthetic rehabilitation are discussed, along with the individualized solutions applied, the repercussions on the temporomandibular joint, and the management protocol adopted by our service.
Tandem mass spectrometry (MS/MS) of peptides plays a key role in the field of proteomics, and an understanding of the fragmentation mechanisms involved is vital for data interpretation. Not all the ...fragment ions observed by low-energy collision-induced dissociation of protonated peptides are readily explained by the generally accepted structures for a- and b-ions. The possibility of a macrocyclic structure for b-type ions has been recently proposed. In this study, we have undertaken investigations of linear protonated YAGFL-NH
2, N-acetylated-YAGFL-NH
2, and cyclo-(YAGFL) peptides and their fragments using a combination of ion mobility (IM) separation and mass spectrometry. The use of IM in this work both gives insight into relative structural forms of the ion species and crucial separation of isobaric species. Our study provides compelling evidence for the formation of a stable macrocyclic structure for the b
5 ion generated by fragmentation of protonated linear YAGFL-NH
2. Additionally we demonstrate that the a
4 ion fragment of protonated YAGFL-NH
2 has at least two structures; one of which is attributable to a macrocyclic structure on the basis of its subsequent fragmentation. More generally, this work emphasizes the value of combined IM-MS/MS in probing the detailed fragmentation mechanisms of peptide ions, and illustrates the use of combined ion mobility/collisional activation/mass spectrometry analysis in achieving an effective enhancement of the resolution of the mobility separator.
Both the generation and the analysis of proteome data are becoming increasingly widespread, and the field of proteomics is moving incrementally toward high-throughput approaches. Techniques are also ...increasing in complexity as the relevant technologies evolve. A standard representation of both the methods used and the data generated in proteomics experiments, analogous to that of the MIAME (minimum information about a microarray experiment) guidelines for transcriptomics, and the associated MAGE (microarray gene expression) object model and XML (extensible markup language) implementation, has yet to emerge. This hinders the handling, exchange, and dissemination of proteomics data. Here, we present a UML (unified modeling language) approach to proteomics experimental data, describe XML and SQL (structured query language) implementations of that model, and discuss capture, storage, and dissemination strategies. These make explicit what data might be most usefully captured about proteomics experiments and provide complementary routes toward the implementation of a proteome repository.
Age-related macular degeneration (AMD) is a leading cause of visual loss. It has a strong genetic basis, and common haplotypes on chromosome (Chr) 1 (CFH Y402H variant) and on Chr10 (near ...HTRA1/ARMS2) contribute the most risk. Little is known about the early molecular and cellular processes in AMD, and we hypothesized that analyzing submacular tissue from older donors with genetic risk but without clinical features of AMD would provide biological insights. Therefore, we used mass spectrometry–based quantitative proteomics to compare the proteins in human submacular stromal tissue punches from donors who were homozygous for high-risk alleles at either Chr1 or Chr10 with those from donors who had protective haplotypes at these loci, all without clinical features of AMD. Additional comparisons were made with tissue from donors who were homozygous for high-risk Chr1 alleles and had early AMD. The Chr1 and Chr10 risk groups shared common changes compared with the low-risk group, particularly increased levels of mast cell–specific proteases, including tryptase, chymase, and carboxypeptidase A3. Histological analyses of submacular tissue from donors with genetic risk of AMD but without clinical features of AMD and from donors with Chr1 risk and AMD demonstrated increased mast cells, particularly the tryptase-positive/chymase-negative cells variety, along with increased levels of denatured collagen compared with tissue from low–genetic risk donors. We conclude that increased mast cell infiltration of the inner choroid, degranulation, and subsequent extracellular matrix remodeling are early events in AMD pathogenesis and represent a unifying mechanistic link between Chr1- and Chr10-mediated AMD.
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