Immunodepletion plasma proteomics by tripleTOF 5600 and Orbitrap elite/LTQ-Orbitrap Velos/Q exactive mass spectrometers.

Plasma proteomic experiments performed rapidly and economically using several of the latest high-resolution mass spectrometers were compared. Four quantitative hyperfractionated plasma proteomics experiments were analyzed in replicates by two AB SCIEX TripleTOF 5600 and three Thermo Scientific Orbitrap (Elite/LTQ-Orbitrap Velos/Q Exactive) instruments. Each experiment compared two iTRAQ isobaric-labeled immunodepleted plasma proteomes, provided as 30 labeled peptide fractions, and 480 LC-MS/MS runs delivered >250 GB of data in 2 months. Several analysis algorithms were compared. At 1% false discovery rate, the relative comparative findings concluded that the Thermo Scientific Q Exactive Mass Spectrometer resulted in the highest number of identified proteins and unique sequences with iTRAQ quantitation. The confidence of iTRAQ fold-change for each protein is dependent on the overall ion statistics (Mascot Protein Score) attainable by each instrument. The benchmarking also suggested how to further improve the mass spectrometry parameters and HPLC conditions. Our findings highlight the special challenges presented by the low abundance peptide ions of iTRAQ plasma proteome because the dynamic range of plasma protein abundance is uniquely high compared with cell lysates, necessitating high instrument sensitivity.

Assessment of two immunodepletion methods: off-target effects and variations in immunodepletion efficiency may confound plasma proteomics.

Immunodepletion of abundant plasma proteins increases the depth of proteome penetration by mass spectrometry. However, the nature and extent of immunodepletion and the effect of off-target depletion on the quantitative comparison of the residual proteins have not been critically addressed. We performed mass spectrometry label-free quantitation to determine which proteins were immunodepleted and by how much. Two immunodepletion resins were compared: Qproteome (Qiagen) which removes albumin+immunoglobulins and Seppro IgY14+SuperMix (Sigma-Aldrich) which removes 14 target proteins plus a number of unidentified proteins. Plasma collected by P100 proteomic plasma collection tubes (BD) from 20 human subjects was individually immunodepleted to minimize potential variability, prior to pooling. The abundant proteins were quantified better when using only albumin+immunoglobulins removal (Qproteome), while lower abundance proteins were evaluated better using exhaustive immunodepletion (Seppro IgY14+SuperMix). The latter resin removed at least 155 proteins, 38% of the plasma proteome in protein number and 94% of plasma protein in mass. The depth of immunodepletion likely accounts for the effectiveness of this resin in revealing low abundance proteins. However, the more profound immunodepletion achieved with the IgY14+SuperMix may lead to false-positive fold-changes between comparison groups if the reproducibility and efficiency of the depletion of a given protein are not considered.

Challenges in Spinal Endoscopy.

The advent of any new technology or technique is fraught with challenges in the early stages of development and adoption. This situation is no different for spinal endoscopy, which has been continuously developing for decades and has only recently gained significant traction in North America. Patient selection can be challenging for even expert endoscopic surgeons, given the limited abilities of current technology for patients with multilevel disease. Anatomic limitations, such as iliac crest location and small foraminal dimensions, can restrict application of the transforaminal approach, considered the "workhorse" of endoscopic techniques. A paucity of dedicated training opportunities has led many to become late adopters or preclude exposure entirely, limiting the next generation of surgeons and expansion of the field. Finally, economic constraints, including capital expenses and issues with insurance reimbursement, have generated difficulties to widespread acquisition. Nonetheless, the future is bright for spinal endoscopy, with potential solutions to these issues already generating progress. In the present report, we have summarized these challenges and discussed some of the current steps underway to help alleviate their impact.

In-vivo Endoscopic Visualization of Patho-anatomy in Symptomatic Degenerative Conditions of the Lumbar Spine II: Intradiscal, Foraminal, and Central Canal Decompression.

The patho-anatomy in an aging spine is partly defined by Rauschning's anatomic cryosections. Theories of pain generation and principles of minimally invasive spine surgery are suggested by close examination of these specimens. If the visualized patho-anatomy can be studied in vivo in a partially sedated patient by spinal probing, spinal pain can be better understood, and rational endoscopic treatment options may then evolve.1 A 1997 IRB-approved study provided evidence that endoscopic transforaminal surgery was feasible for the treatment of a wide spectrum of degenerative conditions in the lumbar spine. The technique incorporated evocative chromo-discography to correlate reproduction of pain with in-vivo probing of patho-anatomy. Laser and radiofrequency ablation augmented mechanical decompression to obtain pain relief.1-3 Endoscopic visualization of patho-anatomy ranging from annular tears to spondylolisthesis and stenosis provided clinical evidence that foraminal decompression, ablation, and irrigation could effectively treat these visualized painful conditions with minimal morbidity. This resulted in a better understanding of the pain generators in the lumbar spine, opening up options for surgical pain management.1-5 The procedure does not burn any bridges for more traditional surgical techniques. The learning curve may be steep for some and long for others, but results are very good, concomitant with each individual surgeon overcoming his personal learning curve.

Molecular mechanisms of action of imatinib mesylate in human ovarian cancer: a proteomic analysis.

BACKGROUND: Imatinib mesylate (Gleevec, Novartis, Basel, Switzerland) is a small-molecule tyrosine kinase inhibitor with activity against ABL, BCR-ABL, c-KIT, and PDGFR alpha. Several clinical trials have evaluated the efficacy and safety of imatinib in patients with ovarian carcinoma who have persistent or recurrent disease following front-line platinum/taxane based chemotherapy. However, there is limited pre-clinical and clinical data on the molecular targets and action of imatinib in ovarian cancer. MATERIALS AND METHODS: Human ovarian cancer cells (A2780) were treated with imatinib mesylate for either 6 or 24 h. We employed a 2D (two-dimensional) gel electrophoresis and mass spectrometry-based proteomics approach to identify protein expression patterns and signaling pathways that were altered in response to imatinib. Cells were analyzed for PDGFR alpha and AKT expression, which were then correlated with imatinib sensitivity. RESULTS: Using 2D gel electrophoresis of overlapping pH ranges from pH 4 to 11, about 4,000 protein spots could be analyzed reproducibly. Proteins whose levels changed between twofold to 30 fold were grouped according to whether changes were in the same direction at both time points of treatment with respect to the control, or changed their levels only at one of the time points. CONCLUSION: Differentially regulated proteins following imatinib treatment of A2780 cells involved the regulation of actin cytoskeleton, metabolic pathways, cell cycle, cell proliferation, apoptosis, cell junctions, and signal transduction. Thus, exposure of cells to imatinib produces complex changes in the cell that require further investigation.

Recombinant nucleases CEL I from celery and SP I from spinach for mutation detection.

BACKGROUND: The detection of unknown mutations is important in research and medicine. For this purpose, a mismatch-specific endonuclease CEL I from celery has been established as a useful tool in high throughput projects. Previously, CEL I-like activities were described only in a variety of plants and could not be expressed in an active form in bacteria. RESULTS: We describe expression of active recombinant plant mismatch endonucleases and modification of their activities. We also report the cloning of a CEL I ortholog from Spinacia oleracea (spinach) which we termed SP I nuclease. Active CEL I and SP I nucleases were expressed as C-terminal hexahistidine fusions and affinity purified from the cell culture media. Both recombinant enzymes were active in mutation detection in BRCA1 gene of patient-derived DNA. Native SP nuclease purified from spinach is unable to incise at single-nucleotide substitutions and loops containing a guanine nucleotide, but the recombinant SP I nuclease can cut at these sites. CONCLUSION: The insect cell-expressed CEL I orthologs may not be identical to their native counterparts purified from plant tissues. The present expression system should facilitate further development of CEL I-based mutation detection technologies.

Minimally invasive techniques for the management of lumbar disc herniation.

Traditionally, minimally invasive techniques for surgical discectomy have been defined as smaller incisions, tubular retractors, microscopically assisted tissue dissection, and conservative removal of only extruded or sequestered nucleus pulposus with preservation of the annulus. The first truly minimally invasive technique was chymopapain dissolution of the nucleus pulposus. Other percutaneous techniques followed; however, none were as efficacious as the gold standard of microlumbar discectomy until endoscopically visualized methods evolved to allow visualized mechanical discectomy through the foramen. In experienced hands, such a technique is as effective as microlumbar discectomy and results in less surgical morbidity for herniations that are appropriate for this minimally invasive endoscopic surgical portal that completely avoids traumatizing the normal anatomy of the dorsal musculature and ligamentous structures.

Haploinsufficiency in tumor predisposition syndromes: altered genomic transcription in morphologically normal cells heterozygous for VHL or TSC mutation.

Tumor suppressor genes and their effector pathways have been identified for many dominantly heritable cancers, enabling efforts to intervene early in the course of disease. Our approach on the subject of early intervention was to investigate gene expression patterns of morphologically normal "one-hit" cells before they become hemizygous or homozygous for the inherited mutant gene which is usually required for tumor formation. Here, we studied histologically non-transformed renal epithelial cells from patients with inherited disorders that predispose to renal tumors, including von Hippel-Lindau (VHL) disease and Tuberous Sclerosis (TSC). As controls, we studied histologically normal cells from non-cancerous renal epithelium of patients with sporadic clear cell renal cell carcinoma (ccRCC). Gene expression analyses of VHLmut/wt or TSC1/2mut/wt versus wild-type (WT) cells revealed transcriptomic alterations previously implicated in the transition to precancerous renal lesions. For example, the gene expression changes in VHLmut/wt cells were consistent with activation of the hypoxia response, associated, in part, with the "Warburg effect". Knockdown of any remaining VHL mRNA using shRNA induced secondary expression changes, such as activation of NFκB and interferon pathways, that are fundamentally important in the development of RCC. We posit that this is a general pattern of hereditary cancer predisposition, wherein haploinsufficiency for VHL or TSC1/2, or potentially other tumor susceptibility genes, is sufficient to promote development of early lesions, while cancer results from inactivation of the remaining normal allele. The gene expression changes identified here are related to the metabolic basis of renal cancer and may constitute suitable targets for early intervention.

In-vivo endoscopic visualization of patho-anatomy in painful degenerative conditions of the lumbar spine.

The degenerative processes in an aging spine have been defined traditionally only by our knowledge of the biology of disc and facet degeneration, as well as interpretation of post-mortem cryosections by forensic anatomist Wolfgang Rauschning, M.D. In this chapter, visualization of in-vivo patho-anatomy in a degenerating disc and spinal segment is demonstrated at surgery using the Yeung Endoscopic Spine System (Y.E.S.S.), (Richard Wolf Surgical Instrument Company, Vernon Hills, IL, USA). An Institutional Review Board (IRB)-approved study of endoscopic treatment for degenerative conditions of the lumbar spine incorporated intraoperative probing under local anesthesia and endoscopic treatment of the visualized patho-anatomy. An intraoperative evocative chromo-discogram, using indigocarmine, was used to elicit discogenic pain and label the fissured and degenerative nucleus pulposus for surgical removal and thermal modulation. Painful patho-anatomy was probed in a conscious patient. The most common endoscopic finding was Inflammatory tissue in the disc and annulus. Inflammation was correlated with the presence of annular tears. Patho-physiologic changes that affect the exiting nerve, which contains the Dorsal Root Ganglion (DRG), was associated with stenotic and chemical irritation. Unavoidable postoperative dysesthesia was associated with the presence of an inflammatory membrane, and removal or thermal coagulation of "anomalous" furcal nerves in the foramen that branched off of the exiting spinal nerve. Neo-angiogenesis and neurogenesis in the inflammatory membrane present in the foraminal triangle was a new finding not reported in traditional clinical studies. Visualization and treatment of pathologic findings inside (annular tears) and outside the disc in Herniated Nucleus Pulposus (HNP), synovial cysts, foraminal stenosis, central stenosis, spondylolisthesis, is demonstrated. The endoscopic foraminal approach to the spine and disc is a technique that provides access to patho-anatomy in the lumbar spine not usually feasible with traditional surgical methods. Favorable surgical results allow for continued evolution of the endoscopic method, concomitant with the continued evolution of endoscopic spinal surgery.

Enhanced cisplatin cytotoxicity by disturbing the nucleotide excision repair pathway in ovarian cancer cell lines.

Ovarian cancer is the leading cause of death among women from gynecological malignancies inthe United States. Resistance to the chemotherapeutic agent cisplatin isa major limitation for the successful treatment of ovarian cancer. In an effort to overcome the cisplatin resistance problem in ovarian cancer treatment, we have sought to enhance cisplatin cytotoxicity by perturbing the nucleotide excision repair (NER) pathway. The NER pathway is responsible for repairing cisplatin bound to DNA. Expression of one of the NER components, ERCC1, is correlated with cisplatin drug resistance. Hence, we targeted ERCC1 by antisense RNA methodologies, and we show that we could sensitize a relatively sensitive A2780 cell line and also the highly resistant OVCAR10 cell line to cisplatin by expressing antisense ERCC1 RNA in them as measured with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays. The A2780 cell lines expressing antisense ERCC1 had 1.9-8.1-fold enhancements in cisplatin sensitivity. The OVCAR10 antisense ERCC1 cell lines had IC(50) values ranging from 2.28 microM to 2.7 microM cisplatin as compared with 9.52 micro M for control OVCAR10 cells. The OVCAR10 antisense ERCC1 cells also show reduced DNA-damage repair capacity as assessed by host cell reactivation. Furthermore, immunocompromised mice transplanted with the antisense cell lines survived longer than the mice bearing control cells after response to cisplatin treatment. These data suggest that it is possible to substantially enhance the cisplatin cytotoxicity by disturbing the NER pathway in cisplatin-resistant cell lines and to enhance the survival capacity of mice in an ovarian cancer xenograft model.

Enzymatic mutation detection technologies.

Mutation is as necessary for life as fidelity is in DNA replication. The study of mutations reveals the normal functions of genes, messages, proteins, the causes of many diseases, and the variability of responses among individuals. Indeed, recent mutations that have not yet become polymorphisms are often deleterious and pertinent to the disease history of afflicted individuals. This review discusses the principles behind a variety of methods for the detection of mutations and factors that should be considered in future methods design. One enzymatic approach in particular using orthologs of the CEL I nuclease that show high specificity for all mismatches, appears to be easy and robust. Further developments of this and other methods will allow mutation detection to become an integral component of individualized medicine.

Altered gene expression in phenotypically normal renal cells from carriers of tumor suppressor gene mutations.

BACKGROUND: The inherently complex signaling networks of tumors result from genetic and epigenetic alterations that occur during cancer initiation and progression. METHODS: In an attempt to identify early molecular changes associated with dominantly inherited predisposition to "two-hit" renal tumors, the expression profiles of primary cultures of phenotypically normal renal epithelial cells from individuals bearing a germline mutation in either the von Hippel-Lindau (VHL) or the tuberous sclerosis complex (TSC) gene were compared to that of renal epithelial cells from control nonmutation carriers by microarray analysis. RESULTS: Reliability of the microarray data from pooled samples was confirmed by real-time RT-PCR. Principal Component Analysis revealed substantial differences in the gene expression profiles of the renal epithelial cells from VHL and TSC mutation carriers. In several instances, the microarray data confirm our present knowledge of the cellular pathways affected by biallelic VHL and TSC mutations. CONCLUSIONS: These findings demonstrate that heterozygosity for a mutant tumor suppressor gene may alter the expression profiles of phenotypically normal epithelial cells in a gene-specific manner. Detectable effects of "one-hit" represent early molecular changes in tumorigenesis that may serve as targets for chemopreventive intervention.

Evaluation of dual-labeled fluorescent DNA probe purity versus performance in real-time PCR.

Real-time PCR technology using dual-labeled fluorescent oligonucleotide probes allows for sensitive, specific, and quantitative determination of mRNA or DNA targets. Historically, dual-labeled probes have been the most expensive reagent in real-time PCR because of the postsynthesis high-performance liquid chromatography (HPLC) and/or gel purification steps required due to limitations in traditional synthesis chemistry. The recent availability of quencher reagents that allow the 3' quencher incorporation as part of the on-machine synthesis has presented the possibility that probes, when carefully synthesized, may be used without extensive postsynthesis purification. This would substantially reduce cost, making the synthesis of dual-labeled fluorescent probes affordable to any DNA synthesis laboratory. The Nucleic Acids Research Group (NARG) of the Association of Biomolecular Resource Facilities (ABRF) (Santa Fe, NM, USA) tested the hypothesis that now any DNA synthesis laboratory is capable of making quality dual-labeled fluorescent probes suitable for real-time PCRs without the need for postsynthesis purification. Members of the DNA synthesis community synthesized dual-labeled human beta-actin probes and submitted them for quality and functional analysis. We found that probes that were at least 20% pure had the same efficiency as those near 100% purity, but the sensitivity of the assay was reduced as the level of purity decreased.

Posterolateral transforaminal selective endoscopic discectomy and thermal annuloplasty for chronic lumbar discogenic pain: a minimal access visualized intradiscal surgical procedure.

BACKGROUND CONTEXT: Chronic lumbar discogenic pain (CLDP) impairs the patient's physical abilities to function within the normal physiologic loading ranges of activities of daily living. The pathogenesis of CLDP is multifactorial and not well understood. Conservative therapeutic regimens often fail to achieve sufficient pain relief. Surgical options vary greatly in surgical invasiveness as well as outcome. Definitive surgical treatment is often 360-degree fusion. The morbidity associated with this approach is significant, considering that only 65% to 80% of patients obtain satisfactory clinical results. This has spawned interest in minimally invasive surgical options, such as intradiscal electrothermal therapy (IDET; ORATEC Interventions, Inc., Menlo Park, CA), but results are conflicting. PURPOSE: The authors describe their surgical technique of minimal access posterolateral transforaminal selective endoscopic discectomy (SED) and bipolar radiofrequency thermal annuloplasty to treat CLDP. The procedure's rationale is based on the hypothesis that annular defects are the focal points of chronic exposure between neural sensory receptors in the defect and the inflammatogenic nucleus pulposus. In contrast to other percutaneous procedures, this technique allows direct visualization and targeting of the disc nucleus and annular fissures. Our 2-year clinical result is reported. STUDY DESIGN/SETTING: This is a retrospective review of consecutive surgical cases performed by one surgeon (ATY). The procedures were carried out from January 1997 to December 1999. Each patient has a minimum postoperative follow-up of 2 years. PATIENT SAMPLE: A total of 113 patients met the generally accepted clinical criteria for chronic lumbar discogenic pain and were selected for the procedure. OUTCOME MEASURES: Two outcome measures were used for clinical assessment: a surgeon-based modified MacNab method and a patient-based questionnaire. A mandatory poor result was given to any patient who had repeat spine surgery at the same level or has indicated dissatisfaction with the surgical result on the questionnaire response. METHOD: After meeting CLDP selection criteria, provocation contrast/indigo carmine dye discography was performed. This test was used to confirm the suspected discs as pain generators. The subject surgery then followed. Only cases with one and two levels of confirmed painful discs were entered into the study. The nonoperating author (PMT) analyzed the data. RESULTS: Using the surgeon assessment method, 17 patients (15%) had excellent results, 32 patients (28.3%) had good results, 34 patients (30.1%) had fair results and 30 patients (26.5%) had poor results. Of the 30 patients in the poor result group, 12 reported either no improvement or worsening, and refused further surgical treatment. Of the remaining 18 patients in the poor group, 8 had spinal fusion, 3 had laminectomy and 7 had repeat spinal endoscopic surgery. The patient-based questionnaire yielded similar percentages in each category. However, only 73.5% of the 113 patients returned the survey questionnaire. There were no aborted procedures, unexpected hemorrhage, device-related complications, neurologic deficits, perioperative deaths or late instability. CONCLUSIONS: Posterolateral transforaminal SED and radiofrequency thermal annuloplasty were used to interrupt the purported annular defect pain sensitization process, thought to be necessary in the genesis of chronic lumbar discogenic pain. Lack of clinical benefit from the subject procedure did not degrade any subsequent surgical or nonsurgical treatment options. The experience gained from this study warrants further investigation into the cellular and molecular processes that provided back pain relief in these patients.

Advances in endoscopic disc and spine surgery: foraminal approach.

Endoscopic spine surgery is evolving rapidly due to improvements in surgical technique, endoscope design, and instrumentation. The current technique expands on the basic features and principles of Kambin's access to the spine through the triangular zone. A standardized method for foraminal surgery, the Yeung Endoscopic Spine System (YESS) (Richard Wolf Surgical Instrument Company, Vernon Hills, Illinois, USA) technique is proposed: (1) A protocol for optimal instrument placement by identifying the skin window, annular window, anatomic disc center, and disc inclination plane through topographical coordinates calculated by lines drawn on the skin from the C-Arm image. Adjustments in the trajectory are made to accommodate individual anatomic considerations and the pathologic disorders to be accessed. (2) Evocative Chromo-Discography (Richard Wolf Surgical Instrument Company, Vernon Hills, Illinois, USA). (3) Selective Endoscopic Discectomy (Richard Wolf Surgical Instrument Company, Vernon Hills, Illinois, USA). (4) Thermal discoplasty and annuloplasty. (5) Endoscopic foraminoplasty. (6) Accessing the epidural space in the axilla between the traversing and exiting nerve root. (7) Partially resecting the posterior annulus to get beneath the herniated fragment, if needed. This technique allows access to the epidural space from the lumbar disc as far cephalad as the middle of the vertebral body or approximately 2-3 mm caudally. The foraminal approach is routinely accessible from T-10 to L4-5. L5-S1 can be accessed with special techniques that include foraminoplasty of the lateral facet. Surgical results continue to improve, consistent with refinement of indications and techniques for specific conditions treatable by this endoscopic method.

Potential prognostic biomarkers of pancreatic cancer.

OBJECTIVES: We evaluated whether pancreatic main duct fluid can provide protein biomarkers with prognostic value. METHODS: Mass spectrometry proteomics was applied to as little as 20µL of fluid collected at the time of tumor surgical resection. Biomarker proteins identified for 27 patients were correlated with clinical outcomes. RESULTS: Thirteen patients had pancreatic ductal adenocarcinomas, 4 had intraductal papillary mucinous neoplasm with in situ adenocarcinoma, 5 had ampullary adenocarcinomas, 2 had intraductal papillary mucinous neoplasms, and 3 had benign diseases. In pathologic stage II or higher pancreatic ductal adenocarcinoma, moderate or high expression of S100A8 or S100A9 proteins was associated with a median disease recurrence-free survival of 5.8 months compared with 17.3 months in patients with low expression (P = 0.002). Median overall survival was 12.6 versus 27 months for patients with moderate to high versus low S100A8 and A9 expression (P = 0.02). CONCLUSIONS: This analysis suggests distinct proteomic signatures for pancreatic cancer. Patients in our study with elevated levels of S100A8 or A9 in the ductal fluid, a near absence of pancreatic enzymes, and high levels of mucins were found to have significantly worse prognosis. Although further validation is needed to corroborate these findings, analysis of pancreatic ductal fluid is a promising tool for identifying biomarkers of interest.

Isobaric labeling and data normalization without requiring protein quantitation.

Isobaric multiplexed quantitative proteomics can complement high-resolution sample isolation techniques. Here, we report a simple workflow exponentially modified protein abundance index (emPAI)-MW deconvolution (EMMOL) for normalizing isobaric reporter ratios within and between experiments, where small or unknown amounts of protein are used. EMMOL deconvolutes the isobaric tags for relative and absolute quantification (iTRAQ) data to yield the quantity of each protein of each sample in the pool, a new approach that enables the comparison of many samples without including a channel of reference standard. Moreover, EMMOL allows using a sufficient quantity of control sample to facilitate the peptide fractionation (isoelectric-focusing was used in this report), and mass spectrometry MS/MS sequencing yet relies on the broad dynamic range of iTRAQ quantitation to compare relative protein abundance. We demonstrated EMMOL by comparing four pooled samples with 20-fold range differences in protein abundance and performed data normalization without using prior knowledge of the amounts of proteins in each sample, simulating an iTRAQ experiment without protein quantitation prior to labeling. We used emPAI, the target protein MW, and the iTRAQ reporter ratios to calculate the amount of each protein in each of the four channels. Importantly, the EMMOL-delineated proteomes from separate iTRAQ experiments can be assorted for comparison without using a reference sample. We observed no compression of expression in iTRAQ ratios over a 20-fold range for all protein abundances. To complement this ability to analyze minute samples, we report an optimized iTRAQ labeling protocol for using 5 µg protein as the starting material.

APC +/- alters colonic fibroblast proteome in FAP.

Here we compared the proteomes of primary fibroblast cultures derived from morphologically normal colonic mucosa of familial adenomatous polyposis (FAP) patients with those obtained from unaffected controls. The expression signature of about 19% of total fibroblast proteins separates FAP mutation carriers from unaffected controls (P < 0.01). More than 4,000 protein spots were quantified by 2D PAGE analysis, identifying 368 non-redundant proteins and 400 of their isoforms. Specifically, all three classes of cytoskeletal filaments and their regulatory proteins were altered as were oxidative stress response proteins. Given that FAP fibroblasts showed heightened sensitivity to transformation by KiMSV and SV40 including elevated levels of the p53 protein, events controlled in large measure by the Ras suppressor protein-1 (RSU-1) and oncogenic DJ-1, here we show decreased RSU1 and augmented DJ-1 expression in both fibroblasts and crypt-derived epithelial cells from morphologically normal colonic mucosa of FAP gene-carriers. The results indicate that heterozygosity for a mutant APC tumor suppressor gene alters the proteomes of both colon-derived normal fibroblasts in a gene-specific manner, consistent with a "one-hit" effect.