Urinary Proteomic Biomarkers of Trabecular Bone Volume Change during Army Basic Combat Training.

PURPOSE: Optimize a dMS-based urinary proteomic technique and evaluate the relationship between urinary proteome content and adaptive changes in bone microarchitecture during BCT. METHODS: Urinary proteomes were analyzed with an optimized dMS technique in two groups of 13 recruits (n = 26) at the beginning (Pre) and end (Post) of BCT. Matched by age (21 ± 4 yr), sex (16 W), and baseline tibial trabecular bone volume fractions (Tb.BV/TV), these groups were distinguished by the most substantial (High) and minimal (Low) improvements in Tb.BV/TV. Differential protein expression was analyzed with mixed permutation ANOVA and false discovery proportion-based adjustment for multiple comparisons. RESULTS: Tibial Tb.BV/TV increased from pre- to post-BCT in High (3.30 ± 1.64%, p < 0.0001) but not Low (-0.35 ± 1.25%, p = 0.4707). The optimized dMS technique identified 10,431 peptides from 1,368 protein groups that represented 165 integrative biological processes. 74 urinary proteins changed from pre- to post-BCT (p = 0.0019) and neutrophil mediated immunity was the most prominent ontology. Two proteins (Immunoglobulin heavy constant gamma 4 and C-type lectin domain family 4 member G) differed from pre- to post-BCT in High and Low (p = 0.0006). CONCLUSIONS: The dMS technique can identify more than 1000 urinary proteins. At least 74 proteins are responsive to BCT, and other principally immune system-related proteins show differential expression patterns that coincide with adaptive bone formation.

ATR promotes mTORC1 activation via de novo cholesterol synthesis in p16-low cancer cells.

DNA damage and cellular metabolism are intricately linked with bidirectional feedback. Two of the main effectors of the DNA damage response and control of cellular metabolism are ATR and mTORC1, respectively. Prior work has placed ATR upstream of mTORC1 during replication stress, yet the direct mechanism for how mTORC1 is activated in this context remain unclear. We previously published that p16-low cells have mTORC1 hyperactivation, which in part promotes their proliferation. Using this model, we found that ATR, but not ATM, is upstream of mTORC1 activation via de novo cholesterol synthesis and is associated with increased lanosterol synthase (LSS). Indeed, p16-low cells showed increased cholesterol abundance. Additionally, knockdown of either ATR or LSS decreased mTORC1 activity. Decreased mTORC1 activity due to ATR knockdown was rescued by cholesterol supplementation. Finally, using both LSS inhibitors and multiple FDA-approved de novo cholesterol synthesis inhibitors, we found that the de novo cholesterol biosynthesis pathway is a metabolic vulnerability of p16-low cells. Together, our data provide new evidence coupling the DNA damage response and cholesterol metabolism and demonstrate the feasibility of using FDA-approved cholesterol-lowering drugs in tumors with loss of p16.

Non-destructive monitoring method for leaf area of Brassica napus based on image processing and deep learning.

Introduction: Leaves are important organs for photosynthesis in plants, and the restriction of leaf growth is among the earliest visible effects under abiotic stress such as nutrient deficiency. Rapidly and accurately monitoring plant leaf area is of great importance in understanding plant growth status in modern agricultural production. Method: In this paper, an image processing-based non-destructive monitoring device that includes an image acquisition device and image process deep learning net for acquiring Brassica napus (rapeseed) leaf area is proposed. A total of 1,080 rapeseed leaf image areas from five nutrient amendment treatments were continuously collected using the automatic leaf acquisition device and the commonly used area measurement methods (manual and stretching methods). Results: The average error rate of the manual method is 12.12%, the average error rate of the stretching method is 5.63%, and the average error rate of the splint method is 0.65%. The accuracy of the automatic leaf acquisition device was improved by 11.47% and 4.98% compared with the manual and stretching methods, respectively, and had the advantages of speed and automation. Experiments on the effects of the manual method, stretching method, and splinting method on the growth of rapeseed are conducted, and the growth rate of rapeseed leaves under the stretching method treatment is considerably greater than that of the normal treatment rapeseed. Discussion: The growth rate of leaves under the splinting method treatment was less than that of the normal rapeseed treatment. The mean intersection over union (mIoU) of the UNet-Attention model reached 90%, and the splint method had higher prediction accuracy with little influence on rapeseed.

Prolonged heterogeneous liver enhancement accompanied by abdominal symptoms after sonographic contrast agent injection: a cross-sectional study.

Background: To report the occurrence of abdominal symptoms in patients who presented with prolonged heterogeneous liver enhancement (PHLE) after injecting contrast agent SonoVue®. Methods: A total of 105 patients who indicated to have contrast-enhanced ultrasound (CEUS) examinations were consecutively observed. The liver scanning under ultrasound was performed before and after the contrast agent injection. Patients' basic information, clinical manifestations, and ultrasound images under B-mode and CEUS mode were respectively recorded. For patients exhibiting abdominal symptoms, the occurrence and last time of symptoms were recorded in detail. We subsequently compared the difference in clinical characteristics between patients with and without the PHLE phenomenon. Results: In 20 patients with the PHLE phenomenon, 13 showed abdominal symptoms. Eight patients (61.5%) appeared to have mild defecation sensation, and 5 (38.5%) showed apparent abdominal pain. The PHLE phenomenon began to appear within 15 minutes to 1.5 hours after the intravenous injection of SonoVue®. This phenomenon lasted for 30 minutes to 5 hours in ultrasound. Patients with severe abdominal symptoms showed large-area and diffuse PHLE patterns. Only sparse hyperechoic spots in the liver were detected in patients with mild discomfort. Abdominal discomfort resolved spontaneously in all patients. Meanwhile, the PHLE gradually disappeared without any medical treatment. In the PHLE-positive group, the proportion of patients with a history of gastrointestinal disease was significantly higher (P=0.02). Conclusions: Patients with the PHLE phenomenon can exhibit abdominal symptoms. We suggest gastrointestinal disorders may contribute to PHLE, which can be considered a harmless phenomenon that does not affect the safety profile of SonoVue®.

Contrast-enhanced ultrasound of ovarian steroid cell tumor, not otherwise specified: A case report.

Ovarian steroid cell tumor is a rare type of ovarian tumor, accounting for ~0.1% of all neoplasms of the ovary. Patients suffering from this type of tumor exhibit virilization due to high testosterone levels. The present study reported a case of an elderly female patient with high testosterone serum levels, resulting in hirsutism and deepening of the voice. Magnetic resonance imaging did not reveal any solid ovarian tumor. However, gray-scale ultrasound indicated suspicious solid nodules on the right ovary. A clear outline of the tumor, characterized by ring-shaped uniform enhancement, was revealed by contrast-enhanced ultrasound (CEUS) scanning. In addition, laparoscopic resection of both fallopian tubes and ovaries confirmed the right ovarian steroid cell tumor. After the operation, the patient's symptoms were completely relieved and testosterone levels returned to normal. In the present study, a case of ovarian steroid tumor diagnosed by CEUS was reported, supporting the significant role of CEUS in the detection of adnexal tumors.

NAD+ bioavailability mediates PARG inhibition-induced replication arrest, intra S-phase checkpoint and apoptosis in glioma stem cells.

Elevated expression of the DNA damage response proteins PARP1 and poly(ADP-ribose) glycohydrolase (PARG) in glioma stem cells (GSCs) suggests that glioma may be a unique target for PARG inhibitors (PARGi). While PARGi-induced cell death is achieved when combined with ionizing radiation, as a single agent PARG inhibitors appear to be mostly cytostatic. Supplementation with the NAD+ precursor dihydronicotinamide riboside (NRH) rapidly increased NAD+ levels in GSCs and glioma cells, inducing PARP1 activation and mild suppression of replication fork progression. Administration of NRH+PARGi triggers hyperaccumulation of poly(ADP-ribose) (PAR), intra S-phase arrest and apoptosis in GSCs but minimal PAR induction or cytotoxicity in normal astrocytes. PAR accumulation is regulated by select PARP1- and PAR-interacting proteins. The involvement of XRCC1 highlights the base excision repair pathway in responding to replication stress while enhanced interaction of PARP1 with PCNA, RPA and ORC2 upon PAR accumulation implicates replication associated PARP1 activation and assembly with pre-replication complex proteins upon initiation of replication arrest, the intra S-phase checkpoint and the onset of apoptosis.

The total mRNA concentration buffering system in yeast is global rather than gene-specific.

Gene expression in eukaryotes does not follow a linear process from transcription to translation and mRNA degradation. Instead it follows a circular process in which cytoplasmic mRNA decay crosstalks with nuclear transcription. In many instances, this crosstalk contributes to buffer mRNA at a roughly constant concentration. Whether the mRNA buffering concept operates on the total mRNA concentration or at the gene-specific level, and if the mechanism to do so is a global or a specific one, remain unknown. Here we assessed changes in mRNA concentrations and their synthesis rates along the transcriptome of aneuploid strains of the yeast Saccharomyces cerevisiae We also assessed mRNA concentrations and their synthesis rates in nonsense-mediated decay (NMD) targets in euploid strains. We found that the altered synthesis rates in the genes from the aneuploid chromosome and the changes in their mRNA stabilities were not counterbalanced. In addition, the stability of NMD targets was not specifically compensated by the changes in synthesis rate. We conclude that there is no genetic compensation of NMD mRNA targets in yeast, and total mRNA buffering uses mostly a global system rather than a gene-specific one.

Validation and classification of RNA binding proteins identified by mRNA interactome capture.

RNA binding proteins (RBPs) take part in all steps of the RNA life cycle and are often essential for cell viability. Most RBPs have a modular organization and comprise a set of canonical RNA binding domains. However, in recent years a number of high-throughput mRNA interactome studies on yeast, mammalian cell lines, and whole organisms have uncovered a multitude of novel mRNA interacting proteins that lack classical RNA binding domains. Whereas a few have been confirmed to be direct and functionally relevant RNA binders, biochemical and functional validation of RNA binding of most others is lacking. In this study, we used a combination of NMR spectroscopy and biochemical studies to test the RNA binding properties of six putative RBPs. Half of the analyzed proteins showed no interaction, whereas the other half displayed weak chemical shift perturbations upon titration with RNA. One of the candidates we found to interact weakly with RNA in vitro is Drosophila melanogaster end binding protein 1 (EB1), a master regulator of microtubule plus-end dynamics. Further analysis showed that EB1's RNA binding occurs on the same surface as that with which EB1 interacts with microtubules. RNA immunoprecipitation and colocalization experiments suggest that EB1 is a rather nonspecific, opportunistic RNA binder. Our data suggest that care should be taken when embarking on an RNA binding study involving these unconventional, novel RBPs, and we recommend initial and simple in vitro RNA binding experiments.

Toxoplasma gondii association with host mitochondria requires key mitochondrial protein import machinery.

Host mitochondrial association (HMA) is a well-known phenomenon during Toxoplasma gondii infection of the host cell. The T. gondii locus mitochondrial association factor 1 (MAF1) is required for HMA and MAF1 encodes distinct paralogs of secreted dense granule effector proteins, some of which mediate the HMA phenotype (MAF1b paralogs drive HMA; MAF1a paralogs do not). To identify host proteins required for MAF1b-mediated HMA, we performed unbiased, label-free quantitative proteomics on host cells infected with type II parasites expressing MAF1b, MAF1a, and an HMA-incompetent MAF1b mutant. Across these samples, we identified ∼1,360 MAF1-interacting proteins, but only 13 that were significantly and uniquely enriched in MAF1b pull-downs. The gene products include multiple mitochondria-associated proteins, including those that traffic to the mitochondrial outer membrane. Based on follow-up endoribonuclease-prepared short interfering RNA (esiRNA) experiments targeting these candidate MAF1b-targeted host factors, we determined that the mitochondrial receptor protein TOM70 and mitochondria-specific chaperone HSPA9 were essential mediators of HMA. Additionally, the enrichment of TOM70 at the parasitophorous vacuole membrane interface suggests parasite-driven sequestration of TOM70 by the parasite. These results show that the interface between the T. gondii vacuole and the host mitochondria is characterized by interactions between a single parasite effector and multiple target host proteins, some of which are critical for the HMA phenotype itself. The elucidation of the functional members of this complex will permit us to explain the link between HMA and changes in the biology of the host cell.

Proteomic approach to discover human cancer viruses from formalin-fixed tissues.

The challenge of discovering a completely new human tumor virus of unknown phylogeny or sequence depends on detecting viral molecules and differentiating them from host molecules in the virus-associated neoplasm. We developed differential peptide subtraction (DPS) using differential mass spectrometry (dMS) followed by targeted analysis to facilitate this discovery. We validated this approach by analyzing Merkel cell carcinoma (MCC), an aggressive human neoplasm, in which ~80% of cases are caused by the human Merkel cell polyomavirus (MCV). Approximately 20% of MCC have a high mutational burden and are negative for MCV, but are microscopically indistinguishable from virus positive cases. Using 23 (12 MCV+, 11 MCV-) formalin-fixed MCC, DPS identified both viral and human biomarkers (MCV large T antigen, CDKN2AIP, SERPINB5, and TRIM29) that discriminate MCV+ and MCV- MCC. Statistical analysis of 498,131 dMS features not matching the human proteome by DPS revealed 562 (0.11%) to be upregulated in virus-infected samples. Remarkably, 4 (20%) of the top 20 candidate MS spectra originated from MCV T oncoprotein peptides and confirmed by reverse translation degenerate oligonucleotide sequencing. DPS is a robust proteomic approach to identify potentially novel viral sequences in infectious tumors when nucleic acid-based methods are not feasible.

[Values of the Quantitative Parameters of Contrast-enhanced Ultrasound in the Diagnosis of Thyroid Benign and Malignant Nodules].

Objective To investigate the value of contrast-enhanced ultrasound(CEUS)quantitative parameters in the diagnosis of thyroid benign and malignant nodules. Methods The CEUS features of 85 histopathologically confirmed thyroid nodules were quantitatively analyzed using five parameters including rising time(RT),time to peak(TTP),area under the curve(AUC),maximum intensity(Imax),and mean transit time(mTT).The dynamic vascular pattern(DVP)curves were also drawn. Results The Imax(Z=-7.08,P=0.01)and AUC(Z=-2.03,P=0.04)of thyroid malignant nodules were significantly smaller than those of thyroid tissue,and the Imax(Z=-1.35,P=0.02)and AUC(Z=-0.21,P=0.02)of thyroid benign nodules were significantly larger than those of thyroid tissue.There were significant differences between thyroid benign and malignant nodules in Imax(Z=-4.16,P=0.00),AUC(Z=-3.01,P=0.01),and DVP curve types(P=0.00).RT(Z=-0.28,P=0.62),TTP(Z=-0.10,P=0.89),and mTT(Z=-0.79,P=0.05)were not significantly different between thyroid benign and malignant nodules. Conclusion The quantitative parameters of CEUS,especially Imax and AUC parameters,are valuable in the diagnosis of benign and malignant thyroid nodules.

The N-cadherin interactome in primary cardiomyocytes as defined using quantitative proximity proteomics.

The junctional complexes that couple cardiomyocytes must transmit the mechanical forces of contraction while maintaining adhesive homeostasis. The adherens junction (AJ) connects the actomyosin networks of neighboring cardiomyocytes and is required for proper heart function. Yet little is known about the molecular composition of the cardiomyocyte AJ or how it is organized to function under mechanical load. Here, we define the architecture, dynamics and proteome of the cardiomyocyte AJ. Mouse neonatal cardiomyocytes assemble stable AJs along intercellular contacts with organizational and structural hallmarks similar to mature contacts. We combine quantitative mass spectrometry with proximity labeling to identify the N-cadherin (CDH2) interactome. We define over 350 proteins in this interactome, nearly 200 of which are unique to CDH2 and not part of the E-cadherin (CDH1) interactome. CDH2-specific interactors comprise primarily adaptor and adhesion proteins that promote junction specialization. Our results provide novel insight into the cardiomyocyte AJ and offer a proteomic atlas for defining the molecular complexes that regulate cardiomyocyte intercellular adhesion. This article has an associated First Person interview with the first authors of the paper.

Altered Levels of Visinin-Like Protein 1 Correspond to Regional Neuronal Loss in Alzheimer Disease and Frontotemporal Lobar Degeneration.

Recent studies have implicated the neuronal calcium-sensing protein visinin-like 1 protein (Vilip-1) as a peripheral biomarker in Alzheimer disease (AD), but little is known about expression of Vilip-1 in the brains of patients with AD. We used targeted and quantitative mass spectrometry to measure Vilip-1 peptide levels in the entorhinal cortex (ERC) and the superior frontal gyrus (SF) from cases with early to moderate stage AD, frontotemporal lobar degeneration (FTLD), and cognitively and neuropathologically normal elderly controls. We found that Vilip-1 levels were significantly lower in the ERC, but not in SF, of AD subjects compared to normal controls. In FTLD cases, Vilip-1 levels in the SF were significantly lower than in normal controls. These findings suggest a unique role for cerebrospinal fluid Vilip-1 as a biomarker of ERC neuron loss in AD.

Structure of Ctk3, a subunit of the RNA polymerase II CTD kinase complex, reveals a noncanonical CTD-interacting domain fold.

CTDK-I is a yeast kinase complex that phosphorylates the C-terminal repeat domain (CTD) of RNA polymerase II (Pol II) to promote transcription elongation. CTDK-I contains the cyclin-dependent kinase Ctk1 (homologous to human CDK9/CDK12), the cyclin Ctk2 (human cyclin K), and the yeast-specific subunit Ctk3, which is required for CTDK-I stability and activity. Here we predict that Ctk3 consists of a N-terminal CTD-interacting domain (CID) and a C-terminal three-helix bundle domain. We determine the X-ray crystal structure of the N-terminal domain of the Ctk3 homologue Lsg1 from the fission yeast Schizosaccharomyces pombe at 2.0 Å resolution. The structure reveals eight helices arranged into a right-handed superhelical fold that resembles the CID domain present in transcription termination factors Pcf11, Nrd1, and Rtt103. Ctk3 however shows different surface properties and no binding to CTD peptides. Together with the known structure of Ctk1 and Ctk2 homologues, our results lead to a molecular framework for analyzing the structure and function of the CTDK-I complex.

BRCA1 deficiency in ovarian cancer is associated with alteration in expression of several key regulators of cell motility - A proteomics study.

Functional loss of expression of breast cancer susceptibility gene 1(BRCA1) has been implicated in genomic instability and cancer progression. There is emerging evidence that BRCA1 gene product (BRCA1) also plays a role in cancer cell migration. We performed a quantitative proteomics study of EOC patient tumor tissues and identified changes in expression of several key regulators of actin cytoskeleton/cell adhesion and cell migration (CAPN1, 14-3-3, CAPG, PFN1, SPTBN1, CFN1) associated with loss of BRCA1 function. Gene expression analyses demonstrate that several of these proteomic hits are differentially expressed between early and advanced stage EOC thus suggesting clinical relevance of these proteins to disease progression. By immunohistochemistry of ovarian tumors with BRCA1(+/+) and BRCA1(null) status, we further verified our proteomic-based finding of elevated PFN1 expression associated with BRCA1 deficiency. Finally, we established a causal link between PFN1 and BRCA1-induced changes in cell migration thus uncovering a novel mechanistic basis for BRCA1-dependent regulation of ovarian cancer cell migration. Overall, findings of this study open up multiple avenues by which BRCA1 can potentially regulate migration and metastatic phenotype of EOC cells.

Evaluation of a 4-protein serum biomarker panel-biglycan, annexin-A6, myeloperoxidase, and protein S100-A9 (B-AMP)-for the detection of esophageal adenocarcinoma.

BACKGROUND: Esophageal adenocarcinoma (EAC) is associated with a dismal prognosis. The identification of cancer biomarkers can advance the possibility for early detection and better monitoring of tumor progression and/or response to therapy. The authors present results from the development of a serum-based, 4-protein (biglycan, myeloperoxidase, annexin-A6, and protein S100-A9) biomarker panel for EAC. METHODS: A vertically integrated, proteomics-based biomarker discovery approach was used to identify candidate serum biomarkers for the detection of EAC. Liquid chromatography-tandem mass spectrometry analysis was performed on formalin-fixed, paraffin-embedded tissue samples that were collected from across the Barrett esophagus (BE)-EAC disease spectrum. The mass spectrometry-based spectral count data were used to guide the selection of candidate serum biomarkers. Then, the serum enzyme-linked immunosorbent assay data were validated in an independent cohort and were used to develop a multiparametric risk-assessment model to predict the presence of disease. RESULTS: With a minimum threshold of 10 spectral counts, 351 proteins were identified as differentially abundant along the spectrum of Barrett esophagus, high-grade dysplasia, and EAC (P<.05). Eleven proteins from this data set were then tested using enzyme-linked immunosorbent assays in serum samples, of which 5 proteins were significantly elevated in abundance among patients who had EAC compared with normal controls, which mirrored trends across the disease spectrum present in the tissue data. By using serum data, a Bayesian rule-learning predictive model with 4 biomarkers was developed to accurately classify disease class; the cross-validation results for the merged data set yielded accuracy of 87% and an area under the receiver operating characteristic curve of 93%. CONCLUSIONS: Serum biomarkers hold significant promise for the early, noninvasive detection of EAC.

Erlotinib, erlotinib-sulindac versus placebo: a randomized, double-blind, placebo-controlled window trial in operable head and neck cancer.

PURPOSE: The EGF receptor (EGFR) and COX2 pathways are upregulated in head and neck squamous cell carcinoma (HNSCC). Preclinical models indicate synergistic antitumor activity from dual blockade. We conducted a randomized, double-blind, placebo-controlled window trial of erlotinib, an EGFR inhibitor; erlotinib plus sulindac, a nonselective COX inhibitor; versus placebo. EXPERIMENTAL DESIGN: Patients with untreated, operable stage II-IVb HNSCC were randomized 5:5:3 to erlotinib, erlotinib-sulindac, or placebo. Tumor specimens were collected before and after seven to 14 days of treatment. The primary endpoint was change in Ki67 proliferation index. We hypothesized an ordering effect in Ki67 reduction: erlotinib-sulindac > erlotinib > placebo. We evaluated tissue microarrays by immunohistochemistry for pharmacodynamic modulation of EGFR and COX2 signaling intermediates. RESULTS: From 2005-2009, 47 patients were randomized for the target 39 evaluable patients. Thirty-four tumor pairs were of sufficient quality to assess biomarker modulation. Ki67 was significantly decreased by erlotinib or erlotinib-sulindac (omnibus comparison, two-sided Kruskal-Wallis, P = 0.04). Wilcoxon pairwise contrasts confirmed greater Ki67 effect in both erlotinib groups (erlotinib-sulindac vs. placebo, P = 0.043; erlotinib vs. placebo, P = 0.027). There was a significant trend in ordering of Ki67 reduction: erlotinib-sulindac > erlotinib > placebo (two-sided exact Jonckheere-Terpstra, P = 0.0185). Low baseline pSrc correlated with greater Ki67 reduction (R(2) = 0.312, P = 0.024). CONCLUSIONS: Brief treatment with erlotinib significantly decreased proliferation in HNSCC, with additive effect from sulindac. Efficacy studies of dual EGFR-COX inhibition are justified. pSrc is a potential resistance biomarker for anti-EGFR therapy, and warrants investigation as a molecular target.

Global analysis of eukaryotic mRNA degradation reveals Xrn1-dependent buffering of transcript levels.

The rates of mRNA synthesis and degradation determine cellular mRNA levels and can be monitored by comparative dynamic transcriptome analysis (cDTA) that uses nonperturbing metabolic RNA labeling. Here we present cDTA data for 46 yeast strains lacking genes involved in mRNA degradation and metabolism. In these strains, changes in mRNA degradation rates are generally compensated by changes in mRNA synthesis rates, resulting in a buffering of mRNA levels. We show that buffering of mRNA levels requires the RNA exonuclease Xrn1. The buffering is rapidly established when mRNA synthesis is impaired, but is delayed when mRNA degradation is impaired, apparently due to Xrn1-dependent transcription repressor induction. Cluster analysis of the data defines the general mRNA degradation machinery, reveals different substrate preferences for the two mRNA deadenylase complexes Ccr4-Not and Pan2-Pan3, and unveils an interwoven cellular mRNA surveillance network.

Distinct profiles of oxidative stress-related and matrix proteins in adult bone and soft tissue osteosarcoma and desmoid tumors: a proteomics study.

Osteosarcomas rarely occur in older adults. Proteomics has not been reported to date in osteosarcoma occurring in the older adult population. This proteomic investigation was conducted to identify differentially expressed proteins in osteosarcoma occurring in various backgrounds from older adults. Desmoid tumors, known to recur locally but not metastasize, were also analyzed. Protein digests isolated from formalin-fixed, paraffin-embedded tumor tissue specimen representing 14 primary osteosarcomas of soft tissue and bone and 18 desmoid tumors were analyzed by high-resolution liquid chromatography-tandem mass spectrometry for protein identification and relative quantification by spectral counting. Elevated abundance levels of several proteins including heat shock protein 90 (HSP90), elastin microfibril interface-located protein 1, and clusterin were identified in osteosarcoma with slight differences in proteomic profiles. Desmoids had an abundance of collagen II and periostin only. The findings were confirmed by immunohistochemical staining for HSP90 and clusterin in the experimental samples and additionally in 16 posttherapy conventional osteosarcomas in tissue microarrays constructed from heterogeneous sarcomas and benign lesions. All osteosarcomas were positive for HSP90 and clusterin to a variable extent. One case of well-differentiated parosteal osteosarcoma was negative. Thirty of 75 other high-grade sarcomas including cases of chondrosarcoma were positive for HSP90. Low-grade and benign lesions and scars and 18 desmoid tumors had little or no expression of these proteins. HSP90 and clusterin represent candidate markers of aggressiveness in osteosarcoma occurring in older adults and may be indicative of drug resistance.