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At least 163 records · Page 9Linked to original sources
PubMed2005

Proteomic profiling of recombinant Escherichia coli in high-cell-density fermentations for improved production of an antibody fragment biopharmaceutical.

By using two-dimensional polyacrylamide gel electrophoresis, a proteomic analysis over time was conducted with high-cell-density, industrial, phosphate-limited Escherichia coli fermentations at the 10-liter scale. During production, a recombinant, humanized antibody fragment was secreted and assembled in a soluble form in the periplasm. E. coli protein changes associated with culture conditions were distinguished from protein changes associated with heterologous protein expression. Protein spots were monitored quantitatively and qualitatively. Differentially expressed proteins were quantitatively assessed by using a t-test method with a 1% false discovery rate as a significance criterion. As determined by this criterion, 81 protein spots changed significantly between 14 and 72 h (final time) of the control fermentations (vector only). Qualitative (on-off) comparisons indicated that 20 more protein spots were present only at 14 or 72 h in the control fermentations. These changes reflected physiological responses to the culture conditions. In control and production fermentations at 72 h, 25 protein spots were significantly differentially expressed. In addition, 19 protein spots were present only in control or production fermentations at this time. The quantitative and qualitative changes were attributable to overexpression of recombinant protein. The physiological changes observed during the fermentations included the up-regulation of phosphate starvation proteins and the down-regulation of ribosomal proteins and nucleotide biosynthesis proteins. Synthesis of the stress protein phage shock protein A (PspA) was strongly correlated with synthesis of a recombinant product. This suggested that manipulation of PspA levels might improve the soluble recombinant protein yield in the periplasm for this bioprocess. Indeed, controlled coexpression of PspA during production led to a moderate, but statistically significant, improvement in the yield.

Bacterial Proteins↗
PubMed2004

An analysis of the proteomic profile for Thermoanaerobacter tengcongensis under optimal culture conditions.

The genome of Thermoanaerobacter tengcongensis is estimated to encode 2588 theoretical proteins. In this study, we have vitalized approximately 46% of the theoretical proteome experimentally using a proteomic strategy that combines three different methods, shotgun digestion plus high-performance liquid chromatography (HPLC) with ion-trap tandem mass spectrometry (shotgun-liquid chromatography (LC)/MS), one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) plus HPLC with ion-trap tandem mass spectrometry (one-dimensional electrophoresis (1DE)-LC/MS), and two-dimensional gel electrophoresis plus matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (2DE-MALDI-TOF-MS). Of the 1200 proteins identified, as few as 76 proteins were globally found by all three approaches, and notably, most of these proteins were in the soluble fraction. However, there were a number of unique proteins detected by one method only, suggesting that our strategy provides a means toward obtaining a comprehensive view of protein expression profile. Proteins from the major metabolic pathways are strongly represented on the map, and a number of these enzymes were identified by more than one proteomic method. Based upon the proteins identified in the present study, we are able to broaden the understanding of how T. tengcongensis survives under high temperature environment, whereas several of its properties can not be fully explained by genome data.

Amino Acid Sequence↗
PubMed2006

[Effect of some active Chinese herbal fraction on brain tissue proteomic profile of ischemic mouse].

OBJECTIVE: To study the effect of some active Chinese herbal fraction on protein expression of brain tissue in ischemic mouse with proteomic technique. METHODS: Ischemia-reperfusion mice were treated with baicalin, geniposide, cholic acid and concha margaritifera respectively for 3 hrs, and then their brain tissue were taken to extract the total protein. Protein expression in ischemic mouse brain was analyzed with surface-enhanced laser desorption/inionation-time of flight-mass spectra (SELDI-TOF-MS) protein-chip. RESULTS: The four components tested had effect on 3 target proteins at 5373Da, 5707Da and 15103Da, showing the nature of multi-target and with different action on protein expression. CONCLUSION: Protein-chip is an effective approach for exploring the pharmacological mechanism of Chinese herbal fraction.

Animals↗
PubMed2026

Quantitative proteomic profiling of neural cells-specific metabolic reprogramming in response to mitochondrial dysfunction using iMPAQT2.

Age-related mitochondrial dysfunction is increasingly recognized as a key contributor to neurodegenerative disease pathogenesis. In the central nervous system, neurons, oligodendrocytes, and astrocytes which derived from neural stem cells, fulfill distinct metabolic and functional roles. However, the specific vulnerabilities of these cell types to mitochondrial impairment remain unclear. In this study, we employed the iMPAQT2 proteomics platform to systematically compare the metabolic profiles of neurons, oligodendrocytes, and astrocytes, and to elucidate the molecular consequences of mitochondrial dysfunction induced by chloramphenicol and oligomycin. Our findings indicate that neurons and oligodendrocytes primarily rely on oxidative phosphorylation (OXPHOS) for ATP production, whereas astrocytes predominantly utilize glycolysis. It is noteworthy that oligodendrocytes exhibited enriched pathways for cholesterol synthesis, fatty acid degradation, and heme catabolism-processes that are critical for myelin maintenance. Treatment with the mitochondrial function inhibitors chloramphenicol or oligomycin reduced the expression of OXPHOS enzymes in all cell types. This reduction was particularly pronounced in oligodendrocytes for glycolysis, cholesterol synthesis, heme degradation, and fatty acid degradation. These results suggest that oligodendrocytes are particularly vulnerable to mitochondrial dysfunction, which may play a pivotal role in the pathogenesis of age-related neurodegenerative disorders.

Animals↗
PubMed2007

Quantitative proteomic profiling of membrane proteins from the mouse brain cortex, hippocampus, and cerebellum using the HysTag reagent: mapping of neurotransmitter receptors and ion channels.

Analysis of the brain proteome and studying brain diseases through clinical biopsies and animal disease models require methods of quantitative proteomics that are sensitive and allow identification and quantification of low abundant membrane proteins from minute amount of tissue. Taking advantage of recently developed methods for isolation of membrane proteins from 10-20 mg brain tissue [Nielsen, P.Aa., Olsen, J.V., Podtelejnokov, A.V., Andersen, J.R., Mann, M., Wiśniewski, J.R., 2005. Proteomic mapping of brain plasma membrane proteins. Mol. Cell. Proteomics 4, 402--408] and the HysTag-quantification method [Olsen, J.V., Andersen, J.R., Nielsen, P.Aa., Nielsen, M.L., Figeys, D., Mann, M., Wiśniewski, J.R., 2004. HysTag---A novel proteomic qualification tool applied to differential analysis of membrane proteins from distinct areas of mouse brain. Mol. Cell. Proteomics 3, 82--92] we performed quantitative proteomic analysis of three functionally distinct compartments of mouse brain: cortex, hippocampus, and cerebellum. In total, 976 unique peptides corresponding to 555 unique proteins were quantified. Up to 20-fold differences in the levels of some proteins between brain areas were measured. For many quantified proteins--as for glutamate receptors, calcium channel subunits, and ATP-ases--an excellent correlation between our proteomic data and previously published mRNA expression levels or intensity of immunostaining was found. Our results clearly demonstrate differences in levels of membrane proteins mapped in distinct brain compartments and offer a technology that allows in depth study of brain membrane proteomes, such as mouse models of neurological diseases.

Animals↗
PubMed2006

A multiplex tissue immunoblotting assay for proteomic profiling: a pilot study of the normal to tumor transition of esophageal squamous cell carcinoma.

Esophageal cancer remains a highly lethal malignancy for which the genetic and proteomic events are poorly understood. Studies have reported dysregulated proteins in esophageal carcinoma; however, the magnitude of these changes remains largely uncharacterized. Little is known about alterations early in the neoplastic pathway. Using multiplex tissue immunoblotting, we quantified the expression of seven proteins in esophageal carcinogenesis. Regions of normal, dysplasia, and invasive carcinoma of the squamous esophagus in six patients were characterized. Pan-cytokeratin (CK) was essentially unchanged across the transition (0.96 in dysplasia and 0.69 in tumor). Expression levels of annexin 1, CK-4, and CK-14 were all decreased in dysplasia and tumor compared with normal (reference, 1.00): annexin 1, 0.30 in dysplasia and 0.15 in tumor; CK-4, 0.20 in dysplasia and 0.16 in tumor; and CK-14, 0.54 in dysplasia and 0.40 in tumor. Expression of two proteins was increased in dysplasia and tumor versus normal: cyclooxygenase-2, 1.35 in dysplasia and 2.32 in tumor and p53, 1.29 in dysplasia and 2.37 in tumor. Secreted protein, acidic and rich in cysteine, which is expressed in the adjacent stroma, was 1.56-fold higher in stroma underlying dysplasia and 6.20-fold increased in dysplastic stroma surrounding invasive tumor. These findings suggest that changes in protein expression can be detected during the transition to dysplasia and may be useful biomarkers.

Biomarkers, Tumor↗
PubMed2006

Proteomic profiling of differentiating osteoblasts.

The major event that triggers osteogenesis is the transition of mesenchymal stem cells into bone-forming, differentiating osteoblast cells. Osteoblast differentiation is the primary event of bone formation, exemplified by the synthesis, deposition and mineralization of extracellular matrix. Osteoblast differentiation is controlled tightly by sequential activation of diverse transcription factors that regulate the expression of specific genes. The spatial and temporal regulation of the differentiation process is not completely understood at the cellular or molecular level. Recent advances in mass spectrometry-based proteomics have allowed for the systematic qualitative and quantitative profiling of differentiating osteoblasts, enabling a better understanding of the multiple factors and signaling events that control the differentiation process at a molecular level. This review focuses on recent developments in the proteomic analysis of differentiating osteoblasts, including advances, challenges and future prospects of using mass spectrometry to investigate the local and systemic factors regulating bone formation and its homeostasis.

Animals↗
PubMed2003

Genomic and proteomic profiling of responses to toxic metals in human lung cells.

Examining global effects of toxic metals on gene expression can be useful for elucidating patterns of biological response, discovering underlying mechanisms of toxicity, and identifying candidate metal-specific genetic markers of exposure and response. Using a 1,200 gene nylon array, we examined changes in gene expression following low-dose, acute exposures of cadmium, chromium, arsenic, nickel, or mitomycin C (MMC) in BEAS-2B human bronchial epithelial cells. Total RNA was isolated from cells exposed to 3 M Cd(II) (as cadmium chloride), 10 M Cr(VI) (as sodium dichromate), 3 g/cm2 Ni(II) (as nickel subsulfide), 5 M or 50 M As(III) (as sodium arsenite), or 1 M MMC for 4 hr. Expression changes were verified at the protein level for several genes. Only a small subset of genes was differentially expressed in response to each agent: Cd, Cr, Ni, As (5 M), As (50 M), and MMC each differentially altered the expression of 25, 44, 31, 110, 65, and 16 individual genes, respectively. Few genes were commonly expressed among the various treatments. Only one gene was altered in response to all four metals (hsp90), and no gene overlapped among all five treatments. We also compared low-dose (5 M, noncytotoxic) and high-dose (50 M, cytotoxic) arsenic treatments, which surprisingly, affected expression of almost completely nonoverlapping subsets of genes, suggesting a threshold switch from a survival-based biological response at low doses to a death response at high doses.

Cell Culture Techniques↗
PubMed2005

Proteomic profiling of facial development in chick embryos.

Craniofacial disorders are associated with one-third of human birth defects but the underlying molecular and cellular causes remain poorly understood. Proteomics seems well-placed to benefit this medically important area but the scarcity of embryonic tissues poses a major challenge. In this study, we applied a microsample proteomics strategy to investigate the first branchial arch, an embryonic structure crucial for facial development, and found that proteome analysis is both practicable and informative despite the scarcity of tissue. Exploiting the embryonic chick as a tractable source of accurately staged tissue, we developed a sequential extraction procedure to interface with one-dimensional polyacrylamide gel electrophoresis (1-D PAGE) and 2-D PAGE. In 2-D gels, about 8% of the visible proteome changed between embryonic days 3 and 5, and the identities determined for 21 proteins accorded with the rapid growth during this period. These results led to the first molecular identification of chicken alpha-fetoprotein, and an unusual localisation of vimentin to endoderm. With over 470 protein spots accessible, this comparative proteomics approach has good prospects for providing new markers, functional hypotheses and genes to target in functional tests. A broader value of extending these approaches to facial development in other species and to other areas in embryology can be anticipated.

Albumins↗
PubMed2026

Plasma Proteomic Profiling of Comorbid and Noncomorbid COVID-19 Patients in ICU.

Type 2 Diabetes (T2D) and hypertension (HTN) are common comorbidities in severe COVID-19, yet their specific impact on proteomic recovery remains unclear. This study analyzed plasma protein signatures of critical COVID-19 patients with and without these comorbidities (COVID-only group [COG] and COVID comorbid group [CTHG]) on the first and last days of ICU stay. Proteomic analysis revealed a systemic shift characterized by upregulated immune responses and downregulated metabolic processes at admission across all patients. Survival was fundamentally defined by the restoration of homeostasis; liver-derived proteins─including LPA, TTR, and AHSG─were initially suppressed but rebounded significantly in survivors. This homeostatic recovery was impaired in CTHG compared to COG, with CTHG survivors showing attenuated recovery of metabolic markers. Distinct mortality-associated signatures also emerged between groups. COG nonsurvivors exhibited liver failure and severe hemolysis marked by persistent suppression of haptoglobin (HP). In contrast, CTHG mortality was driven by lipid metabolism dysregulation, with CD5L and APOA2 levels dropping specifically in comorbid nonsurvivors, often accompanied by a paradoxical elevation in APOA4─likely reflecting impaired renal clearance rather than restored lipid homeostasis. These findings indicate that preexisting T2D and HTN hinder physiological resolution of metabolic and lipid dysregulation, providing proteomic evidence for distinct mortality risks associated with failure to restore metabolic homeostasis in comorbid COVID-19 patients.

Humans↗
PubMed2003

Comparative proteomic profiling of murine skin.

Mammalian skin is regularly exposed to different environmental stresses, each of which results in specific compensatory changes in protein expression that can be assessed by proteomic analysis. We have established a reference proteome map of BALB/c murine skin allowing the resolution of greater than 500 protein spots in a single two-dimensional polyacrylamide gel. Forty-four protein spots, corresponding to 28 different cutaneous proteins, were identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and the Mascot online database searching algorithm. Twenty-five proteins were expressed at higher levels in the epidermis, whereas only nine were found predominantly in the subepidermal tissues. A subset of protein spots exhibited strain-specific expression. Proteins of diverse function were identified, including those involved in stress response, apoptosis, growth inhibition, the maintenance of structural integrity, translational control, energy metabolism, calcium binding, cholesterol transport, and the scavenging of free radicals. Prohibitin expression was detected cutaneously, with more abundant protein and mRNA levels in the epidermis. Five molecular chaperones including protein di-sulfide isomerase, 78 kDa glucose-regulated protein precursor, heat shock protein 60 (HSP60), HSP70, and HSP27 were also identified. Of these, HSP27 expression was confined mainly to the epidermis, and expression of protein disulfide isomerase was found primarily in the subepidermal tissues. Proteomic analysis of skin following heat or cold shock resulted in increased levels of HSP27, HSP60, and HSP70 suggesting involvement of these chaperones in the cutaneous response mechanism to temperature stress. These data establish numerous reference markers within the proteome map of murine skin and provide an important framework for future efforts aimed at characterization of the epidermal and subepidermal responses to environmental changes.

Abdomen↗
PubMed2026

Integrative Transcriptomic and Proteomic Profiling Identifies S100P as a Potential Functional Biomarker for Sessile Serrated Lesions.

BACKGROUND: Sessile serrated lesions (SSLs) account for 15% of colorectal cancers (CRCs) but detection remains difficult due to flat morphology, mucinous features, and subtle histology. AIMS: This study aimed to identify novel and functionally relevant biomarkers of SSLs using transcriptomic screening and multi-omics validation. METHODS: Paired SSL and normal mucosa specimens (n = 6) underwent RNA sequencing. Differentially expressed genes (DEGs) were filtered for membrane or secretory proteins and validated across TCGA and adenoma transcriptomes. Functional significance was assessed using CRISPR dependency profiling, proteotranscriptomic concordance, pharmacogenomic sensitivity, and connectivity map analysis. RESULTS: We identified 216 upregulated genes in SSLs, including 68 encoding secretory/membrane proteins that better discriminated SSLs from controls and were enriched for adhesion and neuronal signaling while suppressing TNFα-NFκB inflammatory pathways. Cross-cohort comparison revealed five overlapping candidates between SSLs and TCGA CMS1 tumors. Among them, S100P emerged as the primary biomarker candidate, showing consistent upregulation in SSLs and CMS1 tumors while remaining low in normal mucosa and conventional adenomas. TFF1 also showed RNA-level upregulation but appeared more context-dependent. S100P demonstrated strong RNA-protein concordance in CRC cell-line profiling, supporting its detectability as a biomarker candidate. Pharmacogenomic profiling of LS411N cells revealed marked sensitivity to SN-38 and fluoropyrimidines, consistent with serrated CRC vulnerabilities. Connectivity map analysis identified perturbations, including MAPK1 and histone acetyltransferase suppression, that may reverse parts of the SSL transcriptional program. CONCLUSION: These findings prioritize S100P as a promising biomarker candidate for SSLs that warrants further validation in larger cohorts and clinically applicable platforms.

Humans↗
PubMed2004

Activity-based probes for the proteomic profiling of metalloproteases.

Metalloproteases (MPs) are a large and diverse class of enzymes implicated in numerous physiological and pathological processes, including tissue remodeling, peptide hormone processing, and cancer. MPs are tightly regulated by multiple posttranslational mechanisms in vivo, hindering their functional analysis by conventional genomic and proteomic methods. Here we describe a general strategy for creating activity-based proteomic probes for MPs by coupling a zinc-chelating hydroxamate to a benzophenone photocrosslinker, which promote selective binding and modification of MP active sites, respectively. These probes labeled active MPs but not their zymogen or inhibitor-bound counterparts and were used to identify members of this enzyme class up-regulated in invasive cancer cells and to evaluate the selectivity of MP inhibitors in whole proteomes. Interestingly, the matrix metalloproteinase inhibitor GM6001 (ilomastat), which is currently in clinical development, was found to also target the neprilysin, aminopeptidase, and dipeptidylpeptidase clans of MPs. These results demonstrate that MPs can display overlapping inhibitor sensitivities despite lacking sequence homology and stress the need to evaluate MP inhibitors broadly across this enzyme class to develop agents with suitable target selectivities in vivo. Activity-based profiling offers a powerful means for conducting such screens, as this approach can be carried out directly in whole proteomes, thereby facilitating the discovery of disease-associated MPs concurrently with inhibitors that selectively target these proteins.

Dipeptides↗
PubMed2005

Changes in the proteomic profile during differentiation and maturation of human monocyte-derived dendritic cells stimulated with granulocyte macrophage colony stimulating factor/interleukin-4 and lipopolysaccharide.

Dendritic cells (DCs) are highly specialized antigen-presenting cells that play an essential role in the immune response. We used the proteomic approach based on two-dimensional gel electrophoresis and mass spectrometry to identify the protein changes that occur during differentiation of DCs from monocytes (Mo) stimulated with granulocyte macrophage colony stimulating factor/interleukin-4 (GM-CSF/IL-4) and during the maturation of immature DCs stimulated with lipopolysaccharide. Sixty-three differentially expressed proteins (+/- two-fold) were unambiguously identified with sequence coverage greater than 20%. They corresponded to only 36 different proteins, because 11 were present as 38 electrophoretic forms. Some proteins such as tropomyosin 4 and heat shock protein 71 presented differentially expressed electrophoretic forms, suggesting that many of the changes in protein expression that accompany differentiation and maturation of DCs occur in post-translationally modified proteins. The largest differences in expression were observed for actin (21-fold in Mo), Rho GDP-dissociation inhibitor 2 (20-fold in Mo), vimentin (eight-fold in immature DCs), lymphocyte-specific protein 1 (12-fold in mature DCs) and thioredoxin (14-fold in mature DCs). Several proteins are directly related to functional and morphological characteristics of DCs, such as cytoskeletal proteins (cytoskeleton rearrangement) and chaperones (antigen processing and presentation), but other proteins have not been assigned specific functions in DCs. Only a few proteins identified here were the same as those reported in proteomic studies of DCs, which used different stimuli to produce the cells (GM-CSF/IL-4 and tumor necrosis factor-alpha). These data suggest that the DC protein profile depends on the stimuli used for differentiation and especially for maturation.

Animals↗
PubMed2007

Proteomics profiling of urine with surface enhanced laser desorption/ionization time of flight mass spectrometry.

BACKGROUND: Urine consists of a complex mixture of peptides and proteins and therefore is an interesting source of biomarkers. Because of its high throughput capacity SELDI-TOF-MS is a proteomics technology frequently used in biomarker studies. We compared the performance of seven SELDI protein chip types for profiling of urine using standard chip protocols. RESULTS: Performance was assessed by determining the number of detectable peaks and spot to spot variation for the seven array types and two different matrices: SPA and CHCA. A urine sample taken from one healthy volunteer was applied in eight-fold for each chip type/matrix combination. Data were analyzed for total number of detected peaks (S/N > 5). Spot to spot variation was determined by calculating the average CV of peak intensities. In addition, an inventory was made of detectable peaks with each chip and matrix type. Also the redundancy in peaks detected with the different chip/matrix combinations was determined. A total of 425 peaks (136 non-redundant peaks) could be detected when combining the data from the seven chip types and the two matrices. Most peaks were detected with the CM10 chip with CHCA (57 peaks). The Q10 with CHCA (51 peaks), SEND (48 peaks) and CM10 with SPA (48 peaks) also performed well. The CM10 chip with CHCA also has the best reproducibility with an average CV for peak intensity of 13%. CONCLUSION: The combination of SEND, CM10 with CHCA, CM10 with SPA, IMAC-Cu with SPA and H50 with CHCA provides the optimal information from the urine sample with good reproducibility. With this combination a total of 217 peaks (71 non-redundant peaks) can be detected with CV's ranging from 13 to 26%, depending on the chip and matrix type. Overall, CM10 with CHCA is the best performing chip type.

Journal Article↗
PubMed2006

Molecular phenotype of zebrafish ovarian follicle by serial analysis of gene expression and proteomic profiling, and comparison with the transcriptomes of other animals.

BACKGROUND: The ability of an oocyte to develop into a viable embryo depends on the accumulation of specific maternal information and molecules, such as RNAs and proteins. A serial analysis of gene expression (SAGE) was carried out in parallel with proteomic analysis on fully-grown ovarian follicles from zebrafish (Danio rerio). The data obtained were compared with ovary/follicle/egg molecular phenotypes of other animals, published or available in public sequence databases. RESULTS: Sequencing of 27,486 SAGE tags identified 11,399 different ones, including 3,329 tags with an occurrence superior to one. Fifty-eight genes were expressed at over 0.15% of the total population and represented 17.34% of the mRNA population identified. The three most expressed transcripts were a rhamnose-binding lectin, beta-actin 2, and a transcribed locus similar to the H2B histone family. Comparison with the large-scale expressed sequence tags sequencing approach revealed highly expressed transcripts that were not previously known to be expressed at high levels in fish ovaries, like the short-sized polarized metallothionein 2 transcript. A higher sensitivity for the detection of transcripts with a characterized maternal genetic contribution was also demonstrated compared to large-scale sequencing of cDNA libraries. Ferritin heavy polypeptide 1, heat shock protein 90-beta, lactate dehydrogenase B4, beta-actin isoforms, tubulin beta 2, ATP synthase subunit 9, together with 40 S ribosomal protein S27a, were common highly-expressed transcripts of vertebrate ovary/unfertilized egg. Comparison of transcriptome and proteome data revealed that transcript levels provide little predictive value with respect to the extent of protein abundance. All the proteins identified by proteomic analysis of fully-grown zebrafish follicles had at least one transcript counterpart, with two exceptions: eosinophil chemotactic cytokine and nothepsin. CONCLUSION: This study provides a complete sequence data set of maternal mRNA stored in zebrafish germ cells at the end of oogenesis. This catalogue contains highly-expressed transcripts that are part of a vertebrate ovarian expressed gene signature. Comparison of transcriptome and proteome data identified downregulated transcripts or proteins potentially incorporated in the oocyte by endocytosis. The molecular phenotype described provides groundwork for future experimental approaches aimed at identifying functionally important stored maternal transcripts and proteins involved in oogenesis and early stages of embryo development.

Animals↗
PubMed2006

Proteomic profiling of ClpXP substrates after DNA damage reveals extensive instability within SOS regulon.

ClpXP, a bacterial AAA+ protease, controls intracellular levels of many stress-response proteins. To investigate substrate profile changes caused by a specific environmental stress, quantitative mass spectrometry (SILAC) was used to analyze proteins trapped by ClpXP(trap) before and after DNA damage. The abundance of half of the trapped proteins changed more than 3-fold after damage. Overrepresented substrates included the DNA-repair proteins RecN and UvrA. Among SOS-response proteins, 25% were ClpXP substrates and, importantly, nearly all of the highly induced regulon members were rapidly degraded. Other proteins, including the stress regulator sigma(S), were underrepresented in ClpXP(trap) after DNA damage; overproduction experiments suggest that simple substrate competition does not account for this reduced recognition. We conclude that damage-response proteins are an unusually rapidly degraded family and that ClpXP has substantial capacity to process the influx of newly synthesized substrates while maintaining the ability to degrade its other substrates in an environmentally responsive manner.

Adenosine Triphosphatases↗
PubMed1997

Proteome profiling of bladder squamous cell carcinomas: identification of markers that define their degree of differentiation.

One hundred fifty fresh bladder tumors were analyzed blindly by two-dimensional PAGE in combination with proteome identification techniques (microsequencing and mass spectrometry) and immunofluorescence of cryostat sections. Of these, six showed protein expression patterns corresponding to squamous cell carcinomas (SCCs). All tumors were already invasive at the time of presentation, and in most cases, the histopathological grade could not be determined with certainty. The more differentiated of the tumors included SCC 589-1, a lesion showing extensive keratinization, and 536-1, a pure SCC that resembled normal skin growing invasively into the muscle. Both tumors expressed keratins 5, 6, 10, 14, 16, 17, and 20, as well as the differentiation-associated proteins psoriasin, psoriasis-associated fatty acid-binding protein (PA-FABP), and galectin 7. SCC 589-1, however, exhibited higher levels of keratin 10, PA-FABP, and galectin 7 and, in addition, expressed keratins 13, 15, and 19, which were not detected in the pure SCC. Involucrin, glutathione S-transferase pi, stratifin (14-3-3 sigma), and the SCC antigen 1, on the other hand, were less abundant in SCC 589-1. In comparison, less-differentiated tumors did not express keratin 10 and were characterized by a decreased expression of keratin 14, psoriasin, PA-FABP, galectin 7, and stratifin (14-3-3 sigma). Indeed, two of these lesions (553-1 and 651-1) could be readily lined up in order of decreasing degree of differentiation based on the expression of these markers. The degree of differentiation of the other two SCCs could not be assessed with certainty because they may represent special cases (SCC 646-1, solid tumor; SCC 485-1, special differentiation pattern). All six SCCs externalized psoriasin to the urine, supporting the contention that this protein, alone or in combination with other polypeptides, may represent a useful marker for the early detection of these lesions.

Biomarkers, Tumor↗