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PubMed2006

Proteomics of the peroxisome.

Genomes provide us with a blue print for the potential of a cell. However, the activity of a cell is expressed in its proteome. Full understanding of the complexity of cells demands a comprehensive view of the proteome; its interactions, activity states and organization. Comprehensive proteomic approaches applied to peroxisomes have yielded new insights into the organelle and its dynamic interplay with other cellular structures. As technologies and methodologies improve, proteomics hold the promise for new discoveries of peroxisome function and a full description of this dynamic organelle.

Arabidopsis↗

PubMed2005

Preparation of postsynaptic density fraction from hippocampal slices and proteomic analysis.

Hippocampal slices offer an excellent experimental system for the study of activity-induced changes in the postsynaptic density (PSD). While studies have documented electrophysiological and structural changes at synapses in response to precise manipulations of hippocampal slices, parallel biochemical and proteomic analyses were hampered by the lack of subcellular fractionation techniques applicable to starting tissue about three orders of magnitude smaller than that used in conventional protocols. Here, we describe a simple and convenient method for the preparation of PSD fractions from hippocampal slices and the identification of its components by proteomic techniques. The "micro PSD fraction" obtained following two consecutive extractions of a synaptosomal fraction with Triton X-100 shows a significant enrichment in the marker protein PSD-95. Thin section electron microscopy shows PSDs similar to those observed in situ. However, other particulate material, especially myelin, and membrane vesicles are also present. The composition of the PSD fraction from hippocampal slices was analyzed by 2D LC/MS/MS. The proteomic approach which utilizes as little as 10microg total protein allowed the identification of >100 proteins. Many of the proteins detected in the fraction are the same as those identified in conventional PSD preparations including specialized PSD-scaffolding proteins, signaling molecules, cytoskeletal elements as well as certain contaminants. The results show the feasibility of the preparation of a PSD fraction from hippocampal slices of reasonable purity and of sufficient yield for proteomic analyses. In addition, we show that further purification of PSDs is possible using magnetic beads coated with a PSD-95 antibody.

Animals↗

PubMed2006

LC-MS/MS based proteomic analysis and functional inference of hypothetical proteins in Desulfovibrio vulgaris.

High efficiency capillary liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to examine the proteins extracted from Desulfovibrio vulgaris cells across six treatment conditions. While our previous study provided a proteomic overview of the cellular metabolism based on proteins with known functions [W. Zhang, M.A. Gritsenko, R.J. Moore, D.E. Culley, L. Nie, K. Petritis, E.F. Strittmatter, D.G. Camp II, R.D. Smith, F.J. Brockman, A proteomic view of the metabolism in Desulfovibrio vulgaris determined by liquid chromatography coupled with tandem mass spectrometry, Proteomics 6 (2006) 4286-4299], this study describes the global detection and functional inference for hypothetical D. vulgaris proteins. Using criteria that a given peptide of a protein is identified from at least two out of three independent LC-MS/MS measurements and that for any protein at least two different peptides are identified among the three measurements, 129 open reading frames (ORFs) originally annotated as hypothetical proteins were found to encode expressed proteins. Functional inference for the conserved hypothetical proteins was performed by a combination of several non-homology based methods: genomic context analysis, phylogenomic profiling, and analysis of a combination of experimental information, including peptide detection in cells grown under specific culture conditions and cellular location of the proteins. Using this approach we were able to assign possible functions to 20 conserved hypothetical proteins. This study demonstrated that a combination of proteomics and bioinformatics methodologies can provide verification of the expression of hypothetical proteins and improve genome annotation.

Amino Acid Sequence↗

PubMed2005

Investigation of cell cycle arrest effects of actinomycin D at G1 phase using proteomic methods in B104-1-1 cells.

Actinomycin D was previously reported as an inhibitor of Shc/Grb2 interaction in B104-1-1 cells. Actinomycin D arrested the cell cycle at the G1 phase at 1nM, which is about 10 times lower than the inhibition of Shc/Grb2 interactions in B104-1-1 cells. To evaluate other mechanisms of actinomycin D affected suppression of tumors and cell growth, except inhibition of Shc/Grb2 interactions, we examined the proteomic expression profile by proteomic technology. We found up-regulation of MEKK3 and down-regulation of Hsp70 expression from proteomic analysis, which is a very interesting observation because MEKK3 is strongly related with G1 arrest of cell cycle and Hsp70 is also involved in cell cycle regulation. These results indicate that the anti-tumor effects of actinomycin D is due to synergic effects of various proteins regulated by the compound including inhibition of the Shc/Grb2 interaction and other signaling pathways in the cytoplasm. Here we provide a mechanism-based explanation for growth inhibition by actinomycin D using proteomic technology. Thus, this approach may be a potentially useful method to reveal new mechanisms of active compounds or drugs with unknown cellular function.

Animals↗

PubMed2006

Effects of the incubation in vitro with sorbents on serum proteomic pattern and cytokine concentration in cancer patients during chemotherapy--preliminary results.

INTRODUCTION: Cancer and treatment by chemotherapy often produce abnormalities in endogenous cytokine, chemokine, and inflammatory mediator production. Sorbent-based adsorption therapies have been used to remove cytokines in diverse human diseases. AIM: The aim of this study was to evaluate the effects of chemotherapy on serum proteomic pattern and cytokine concentration, and to evaluate the ex vivo feasibility of using sorbents to remove cytokines, chemokines and other proteins in adult cancer patients undergoing chemotherapy with fluorouracil or carboplatin-taxane combinations. PATIENTS AND METHODS: Serum samples of three female adult patients (one affected by rectal cancer and two by ovarian cancer) were examined before and on the fourth day of the first cycle of chemotherapy with fluorouracil (rectal cancer patient) or carboplatin-taxane combination (ovarian cancer patients). The analysis was performed, by means of luminex technology and with a proteomic approach, on native serum samples, and on the same sera after 2 hours of in vitro incubation with a synthetic based styrenic divinylbenzene resin. RESULTS: Chemotherapy determined variable effects on serum concentration of cytokines, while the incubation in vitro of patients serum samples with the resin induced a significant decrease (>80%) in serum concentration of different chemokines, cytokines, growth factors and proteins. The proteomic approach, using SDS PAGE and 2-DE highlighted differences in protein expression between sera from healthy controls and cancer patients. Proteomic analysis demonstrated also variations in the expression of proteins, particularly those with low-molecular weight, due to chemotherapy. Finally, the incubation in vitro of serum samples with sorbents induced a general reduction of protein expression. Within the cancer patients maps, 10 spots were chosen for identification with MALDI-TOF analysis. CONCLUSION: The incubation in vitro with sorbents normalized the over-expression of different proteins and cytokines induced by chemotherapy, suggesting further evaluation as a possible adjuvant treatment.

Adsorption↗

PubMed2006

Inner ear proteomics of mouse models for deafness, a discovery strategy.

Inner ear dysfunction is often associated with defective hair cells. Therefore, hair cells are the focus of study in many of the mouse mutants showing auditory and vestibular deficits. However, harvesting sufficient numbers of hair cells from the tiny bony mouse inner ear for proteomic analysis is challenging. New approaches that would take advantage of mouse mutants and avoid processing steps, such as decalcification or microdissetion, would be more suitable for proteomic analysis. Here, we propose a novel approach called SSUMM-Subtractive Strategy Using Mouse Mutants. SSUMM takes advantage of the differences between control and affected or mutant samples. We predict that SSUMM would be a useful method in proteomics, especially in those cases in which the investigator must work with small numbers of diverse cell types from a tiny organ. Here, we discuss the potential utility of SSUMM to unravel the protein expression profiles of hair cells using the Pou4f3 mouse mutant as an example. Pou4f3 mutant mice exhibit a total loss of inner and outer hair cells, but supporting cells remain relatively intact in the cochlea, thus providing an excellent model for identifying proteins and transcripts that are specific to the hair cell at all life stages. SSUMM would maximize the sensitivity of the analyses while obviating the need for tedious sessions of microdissection and collection of hair cells. By comparing the mutant to control ears at specific time points, it is possible to identify direct targets of a gene product of interest. Further, SSUMM could be used to identify and analyze inner ear development markers and other known genes/proteins that are coexpressed in the ear. In this short technical report, we also discuss protein-profiling approaches suitable for SSUMM and briefly discuss other approaches used in the field of proteomics.

Animals↗

PubMed2026

Adaptive proteomic remodeling and eNOS upregulation in luminal endothelium and perivascular adipose tissue of patent saphenous vein grafts after CABG.

OBJECTIVE: Long-term patency of saphenous vein grafts (SVGs) remains a significant challenge in coronary artery bypass grafting (CABG). The biological factors underlying successful human grafts are poorly understood. We aimed to characterize the structural and molecular features associated with successful graft function. METHODS: Patent and occluded SVG and internal thoracic artery (ITA) grafts were obtained from explanted hearts of CABG patients undergoing heart transplantation for end-stage heart failure not attributable to graft failure, along with freshly harvested ITA and SVG controls. Samples underwent histomorphological analysis, immunohistochemistry (IHC), and liquid chromatography-tandem mass spectrometry (LC-MS/MS) proteomics. RESULTS: Patent ITA (ITA-P) showed minimal intimal hyperplasia with medial reinforcement, whereas patent SVGs (SVG-P) had organized, α-smooth muscle actin (αSMA)-positive myofibroblast-rich neointima. Endothelial nitric oxide synthase (eNOS) was markedly upregulated in patent grafts at two sites-the luminal endothelium and adventitial microvessels within perivascular adipose tissue (PVAT)-and lost at both sites in occluded SVG (SVG-O). Adventitial CD31-positive microvessels were significantly increased in patent grafts. Proteomically, ITA-P and SVG-P shared a largely common adaptive proteome enriched in translation, RNA processing, and extracellular matrix (ECM) organization, with shared upstream activation of NR4A3, EGFR, and STAT1, and conduit-specific signatures (IGF-1/RUNX2 in ITA-P; RETN/SRC/PTGES in SVG-P). PTGES was strongly expressed in the adventitia of SVG-P. CONCLUSIONS: Patent arterial and venous bypass grafts exhibited a shared adaptive phenotype characterized by dual-site upregulation of eNOS in both the luminal endothelium and the perivascular microvessels/PVAT. In SVG-P, PTGES was co-upregulated alongside eNOS, indicating a mechanistic link between the proteomic and IHC findings. These findings highlight the perivascular compartment as a site of adaptive, eNOS-associated changes in patent vein grafts.

Humans↗

PubMed2005

Computational analysis of shotgun proteomics data.

Proteomics technology is progressing at an incredible rate. The latest generation of tandem mass spectrometers can now acquire tens of thousands of fragmentation spectra in a matter of hours. Furthermore, quantitative proteomics methods have been developed that incorporate a stable isotope-labeled internal standard for every peptide within a complex protein mixture for the measurement of relative protein abundances. These developments have opened the doors for 'shotgun' proteomics, yet have also placed a burden on the computational approaches that manage the data. With each new method that is developed, the quantity of data that can be derived from a single experiment increases. To deal with this increase, new computational approaches are being developed to manage the data and assess false positives. This review discusses current approaches for analyzing proteomics data by mass spectrometry and identifies present computational limitations and bottlenecks.

Algorithms↗

PubMed2006

Tagging and detection strategies for activity-based proteomics.

The field of activity-based proteomics is a relatively new discipline that makes use of small molecules, termed activity-based probes (ABPs), to tag and monitor distinct sets of proteins within a complex proteome. These activity-dependant labels facilitate analysis of systems-wide changes at the level of enzyme activity rather than simple protein abundance. While the use of small molecule inhibitors to label enzyme targets is not a new concept, the past ten years have seen a rapid expansion in the diversity of probe families that have been developed. In addition to increasing the number and types of enzymes that can be targeted by this method, there has also been an increase in the number of methods used to visualize probes once they are bound to target enzymes. In particular, the use of small organic fluorophores has created a wealth of applications for ABPs that range from biochemical profiling of diverse proteomes to direct imaging of active enzymes in live cells and even whole animals. In addition, the advent of new bioorthogonal coupling chemistries now enables a diverse array of tags to be added after targets are labeled with an ABP. This strategy has opened the door to new in vivo applications for activity-based proteomic methods.

Affinity Labels↗

PubMed2005

Proteome analysis of separated male and female gametocytes reveals novel sex-specific Plasmodium biology.

Gametocytes, the precursor cells of malaria-parasite gametes, circulate in the blood and are responsible for transmission from host to mosquito vector. The individual proteomes of male and female gametocytes were analyzed using mass spectrometry, following separation by flow sorting of transgenic parasites expressing green fluorescent protein, in a sex-specific manner. Promoter tagging in transgenic parasites confirmed the designation of stage and sex specificity of the proteins. The male proteome contained 36% (236 of 650) male-specific and the female proteome 19% (101 of 541) female-specific proteins, but they share only 69 proteins, emphasizing the diverged features of the sexes. Of all the malaria life-cycle stages analyzed, the male gametocyte has the most distinct proteome, containing many proteins involved in flagellar-based motility and rapid genome replication. By identification of gender-specific protein kinases and phosphatases and using targeted gene disruption of two kinases, new sex-specific regulatory pathways were defined.

Animals↗

PubMed2004

Bioinformatics strategies for proteomic profiling.

Clinical proteomics is an emerging field that involves the analysis of protein expression profiles of clinical samples for de novo discovery of disease-associated biomarkers and for gaining insight into the biology of disease processes. Mass spectrometry represents an important set of technologies for protein expression measurement. Among them, surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI TOF-MS), because of its high throughput and on-chip sample processing capability, has become a popular tool for clinical proteomics. Bioinformatics plays a critical role in the analysis of SELDI data, and therefore, it is important to understand the issues associated with the analysis of clinical proteomic data. In this review, we discuss such issues and the bioinformatics strategies used for proteomic profiling.

Computational Biology↗

PubMed2026

Proteomics as a theranostic compass in BCR::ABL1-negative myeloproliferative neoplasms: Integrating biomarker discovery with therapeutic stratification.

Classic BCR::ABL1-negative myeloproliferative neoplasms (MPNs)-polycythaemia vera, essential thrombocythaemia, and primary myelofibrosis-are clonal haematopoietic stem cell disorders with marked heterogeneity in clinical phenotype, disease trajectory, and therapeutic response. Genomic stratification by driver and cooperating mutations only partially accounts for this variability, leaving gaps in predicting thrombotic risk, fibrotic progression, leukaemic transformation, and treatment benefit. Proteomics bridges this gap by providing function-proximal readouts of protein abundance, post-translational modifications, pathway activity, and intercellular signalling that genomics and transcriptomics cannot capture, positioning it as a theranostic platform in which the same molecular readouts simultaneously inform diagnostic stratification and therapeutic decision-making. We propose a five-stage translational framework spanning from discovery-scale mass spectrometry and affinity-based plasma profiling to targeted validation, multicentre standardisation, and machine learning-integrated clinical panels. Proteomic evidence is synthesised across the following four disease axes: clonal fitness in haematopoietic stem and progenitor cells; bone marrow microenvironmental remodelling and fibrosis; chronic inflammation and thrombosis; and leukaemic transformation. We further describe how phosphoproteomics reveals resistance mechanisms to JAK inhibitors, including AXL-MAPK bypass and PP2A-autophagy-mediated tolerance, and how protein-level biomarkers (BCL2-BCL-XL, RAS-ERK, CAMK2G, and ROCK1/2) can guide individualised therapeutic selection. Affinity-based platforms (Olink PEA and SomaScan) and spatially resolved technologies (CODEX and single-cell proteomics) complement discovery proteomics. At present, however, this evidence base is constrained by small and heterogeneous cohorts, limited cross-platform reproducibility, and a scarcity of independent external validation for candidate protein panels. Realising this vision will require multicentre standardisation, analytically validated panel assays, and prospective clinical studies that translate molecular findings into decision-grade tools for patients with MPNs.

Humans↗

PubMed2026

Normoalbuminuric and albuminuric diabetic kidney disease exhibit divergent renal proteomic characteristics: implications for management.

BACKGROUND: The pathogenesis of diabetic kidney disease (DKD) is complex. Normoalbuminuric diabetic kidney disease (NADKD) is a special subtype of DKD that often progresses insidiously without detectable albuminuria, posing diagnostic and therapeutic challenges. Its pathogenesis remains unclear. Proteomic analysis of renal tissues may offer insights into its pathogenesis and identify biomarkers. METHODS: Clinicopathological data from 295 biopsy-proven DKD patients were collected and classified into normoalbuminuric (UACR < 30 mg/g, n = 25), microalbuminuric (UACR 30-300 mg/g, n = 26), and macroalbuminuric (UACR > 300 mg/g, n = 244) groups. Laser microdissection combined with mass spectrometry (LMD/MS) was used to analyze glomerular and proximal tubule proteomics in 5 patients per DKD subgroup and 5 control subjects. Associations with clinical features were examined. RESULTS: Glomerular proteomic analysis revealed that oxidative stress and metabolic pathways (UQCRC1) were upregulated in NADKD group, whereas the complement and coagulation cascades (C3, C5, C6, C9, CFH, CFHR1) were significantly upregulated in the microalbuminuric and macroalbuminuric DKD groups. The proximal tubule proteomics analysis showed that oxidative phosphorylation-related proteins (SDHA, CYCS, UQCRQ) were upregulated in NADKD, and collagen I related proteins (COL1A1, COL1A2) were significantly upregulated. CONCLUSION: Oxidative stress and mitochondrial dysfunction are involved in the progression of NADKD, lesions predominantly located in the tubulointerstitium. The complement pathway participates in the pathogenesis and progression of albuminuric DKD (ADKD). These divergent molecular profiles suggest that NADKD and ADKD may reflect different pathophysiological mechanisms and have important implications for therapeutic strategies in diabetes management.

Humans↗

PubMed2026

Early proteomic and metabolic signatures of liver and eye in OAT-deficient mice.

Ornithine aminotransferase (OAT) deficiency causes hyperornithinemia and gyrate atrophy (GA) of the choroid and retina, a rare inherited retinal degeneration. To understand the early molecular changes that make the eye susceptible to damage, we performed quantitative proteomic and metabolomic profiling of liver, retina, and retinal pigment epithelium and choroid (RPE/Cho) from OAT-deficient (Oatrhg) mice prior to detectable vision impairment. In addition to reduced OAT expression and elevated ornithine, methylation-related metabolites such as N(6)-methyl-lysine were altered in all examined tissues of Oatrhg mice. In the liver, excess ornithine was directed into urea cycle metabolism, together with altered expression of detoxification enzymes and histone H2B proteins. In contrast, the retina showed minimal proteomic changes but pronounced alterations in amino acid pathways that support glutamate homeostasis. The RPE/Cho demonstrated the most extensive proteomic changes, particularly in mitochondrial metabolism, cytoskeleton, and extracellular matrix, along with changes in metabolites involved in lysine metabolism, energy metabolism, and antioxidant capacity. Incubation with 13C lysine demonstrated that lysine was primarily degraded in RPE/Cho but not the retina, and ornithine enhanced lysine degradation in an OAT-dependent manner. Together, these findings highlight common and tissue-specific impacts of OAT on the liver and ocular tissues and provide insight into early molecular changes that contribute to the selective vulnerability of the eye in GA. Proteomics data are available via ProteomeXchange (PXD063614) and metabolomics data via MassIVE repository (MSV000101103).

Animals↗

PubMed2004

Proteomic analysis of post-mitochondrial fractions of young and old rat kidney.

Proteomic analysis is defined as the characterization of the entire set of proteins encoded by a genome. Two-dimensional (2D) electrophoresis and matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) are key technologies used in proteomic analysis to gain information about protein expression profiles and post-translational modifications. Knowledge about aging processes can be gained by recognizing changes in protein expression. Thus, to better understand the aging process through protein profiling, post-mitochondrial (PM) fractions of young (13-month) and old (31-month) male Fischer 344 rat kidney were differentially analyzed by 2D. We detected a total number of 380 spots on 2D gel images. Among them, 167 spots showed 2-fold significant alterations (p<0.05) between young and old PM fractions. Further, 103 proteins were identified by MALDI-TOF MS. The PM fraction of aged rat kidney showed increases in antioxidative and proteolytic proteins and decreases in cytoskeletal proteins. In addition, we found age-related changes in transport and homeostasis proteins. Thus, our results demonstrated that proteomic analysis can be effectively applied to the assessment of the age status of protein expression, and thereby provide valuable information on age-related changes of proteome.

Aging↗

PubMed2003

The potential for proteomic definition of stem cell populations.

Embryonic and adult stem cell populations have great potential value in medicine, and hematopoietic stem cells are already being used in transplantation. Definition of these populations to increase our understanding of the programs that control differentiation, self-renewal, and possibly plasticity would be of great interest. The relative quantitation of transcriptional activity in stem cells and other populations has defined a profile of gene expression activity in stem cells. Confirmation that these differences have an impact on protein levels within stem cells via their complete protein complement and protein interactions will enable further understanding of regulatory processes in these cells. The recent developments in proteomics and their potential application to the definition of the stem cell proteome are discussed, and examples are given. Advances in mass spectrometry, subcellular prefractionation protocols, and electrophoresis that make stem cell proteomics a tractable problem are discussed. Beyond the proteome per se, advances in post-translational modification profiling mean that comparative analysis of phosphorylation patterns between stem cells and other populations can be approached.

Animals↗

PubMed2006

The heterogeneity of human mesenchymal stem cell preparations--evidence from simultaneous analysis of proteomes and transcriptomes.

OBJECTIVE: Mesenchymal stem cells (MSC) raise high hopes in clinical applications. However, the lack of common standards and a precise definition of MSC preparations remains a major obstacle in research and application of MSC. Whereas surface antigen markers have failed to precisely define this population, a combination of proteomic data and microarray data provides a new dimension for the definition of MSC preparations. METHODS: In our continuing effort to characterize MSC, we have analyzed the differential transcriptome and proteome expression profiles of MSC preparations isolated from human bone marrow under two different expansion media (BM-MSC-M1 and BM-MSC-M2). RESULTS: In proteomics, 136 protein spots were unambiguously identified by MALDI-TOF-MS and corresponding cDNA spots were selected on our "Human Transcriptome cDNA Microarray." Combination of datasets revealed a correlation in differential gene expression and protein expression of BM-MSC-M1 vs BM-MSC-M2. Genes involved in metabolism were more highly expressed in BM-MSC-M1, whereas genes involved in development, morphogenesis, extracellular matrix, and differentiation were more highly expressed in BM-MSC-M2. Interchanging culture conditions for 8 days revealed that differential expression was retained in several genes whereas it was altered in others. CONCLUSION: Our results have provided evidence that homogeneous BM-MSC preparations can reproducibly be isolated under standardized conditions, whereas culture conditions exert a prominent impact on transcriptome, proteome, and cellular organization of BM-MSC.

Cell Culture Techniques↗

PubMed2005

Multidimensional separation of peptides for effective proteomic analysis.

Current solution based proteomic analysis methods are generally based on enzymatic digestion of a protein mixture followed by separation using multidimensional liquid chromatography and/or electrophoresis where peptide identification is typically accomplished by tandem mass spectrometry (MS/MS). It is generally accepted that no single chromatographic or electrophoretic procedure is capable of resolving the complex mixture of peptides that results from a global proteolytic digest of a proteome. Therefore, combining two or more orthogonal (multimodal) separation procedures dramatically improves the overall resolution and results in a larger number of peptides being identified from complex proteome digests. Separation of a proteome digest is a particularly challenging analytical problem due to the large number of peptides and the wide concentration dynamic range. While it has been demonstrated that increasing the number of dimensions of separation prior to MS analysis increases the number of peptides that may be identified, a balance between the time invested and the overall results obtained must be carefully considered. This manuscript provides a review of two- and three-dimensional peptide separation strategies combined with MS for the analysis of complex peptide mixtures.

Chromatography, Liquid↗