Abstract: A National Institutes of Health (NIH) workshop was convened in Bethesda, MD on September 26–27, 2011, with representative scientific leaders in the field of proteomics and its applications to clinical settings. The main purpose of this workshop was to articulate ways in which the biomedical research community can capitalize on recent technology advances and synergize with ongoing efforts to advance the field of human proteomics. This executive summary and the following full report describe the main discussions and outcomes of the workshop.

Abstract: Background: Multiple sclerosis (MS) is a chronic inflammatory disorder of the central nervous system (CNS). It involves damage to the myelin sheath surrounding axons and to the axons themselves. MS most often presents with a series of relapses and remissions but then evolves over a variable period of time into a slowly progressive form of neurological dysfunction termed secondary progressive MS (SPMS). The reasons for this change in clinical presentation are unclear. The absence of a diagnostic marker means that there is a lag time of several years before the diagnosis of SPMS can be established. At the same time, understanding the mechanisms that underlie SPMS is critical to the development of rational therapies for this untreatable stage of the disease. Results: Using high performance liquid chromatography-coupled mass spectrometry (HPLC); we have established a highly specific and sensitive selected reaction monitoring (SRM) assay. Our multiplexed SRM assay has facilitated the simultaneous detection of surrogate peptides originating from 26 proteins present in cerebrospinal fluid (CSF). Protein levels in CSF were generally ~200-fold lower than that in human sera. A limit of detection (LOD) was determined to be as low as one femtomol. We processed and analysed CSF samples from a total of 22 patients with SPMS, 7 patients with SPMS treated with lamotrigine, 12 patients with non-inflammatory neurological disorders (NIND) and 10 healthy controls (HC) for the levels of these 26 selected potential protein biomarkers. Our SRM data found one protein showing significant difference between SPMS and HC, three proteins differing between SPMS and NIND, two proteins between NIND and HC, and 11 protein biomarkers showing significant difference between a lamotrigine-treated and untreated SPMS group. Principal component analysis (PCA) revealed that these 26 proteins were correlated, and could be represented by four principal components. Overall, we established an efficient platform to develop and verify protein biomarkers in CSF, which can be easily adapted to other proteins of interest related to neurodegenerative diseases. Conclusions: A highly specific and sensitive multiplex SRM-MS assay was established for development and verification of CSF protein biomarkers in SPMS. Five proteins were found to be expressed significantly differently between the three cohorts, SPMS, NIND and HC and 11 proteins associated with lamotrigine treatment, which we expect will further our current understanding of SPMS disease pathology and/or therapeutic intervention.

Abstract: The molecular events that lead to malignant transformation and subsequent metastasis of breast carcinoma include alterations in the cells at genome, transcriptome and proteome levels. In this study, we used publicly available gene expression databases to identify those candidate genes which are upregulated at the mRNA level in breast cancers but have not been systematically validated at the protein level. Based on an extensive literature search, we identified ribosome binding protein 1 (RRBP1) as a candidate that is upregulated at the mRNA level in five different studies but its protein expression had not been investigated. Immunohistochemical labeling of breast cancer tissue microarrays was carried out to determine the expression of RRBP1 in a large panel of breast cancers. We found that RRBP1 was overexpressed in 84% (177/219) of breast carcinoma cases tested. The subcellular localization of RRBP1 was mainly observed to be in the cytoplasm with intense staining in the perinuclear region. Our findings suggest that RRBP1 is an interesting molecule that can be further studied for its potential to serve as a breast cancer biomarker. This study also demonstrates how the integration of biological data from available resources in conjunction with systematic evaluation approaches can be successfully applied to clinical proteomics.

Abstract: Tuberculous meningitis is a frequent extrapulmonary disease caused by Mycobacterium tuberculosis and is associated with high mortality rates and severe neurological sequelae. In an earlier study employing DNA microarrays, we had identified genes that were differentially expressed at the transcript level in human brain tissue from cases of tuberculous meningitis. In the current study, we used a quantitative proteomics approach to discover protein biomarkers for tuberculous meningitis. To compare brain tissues from confirmed cased of tuberculous meningitis with uninfected brain tissue, we carried out quantitative protein expression profiling using iTRAQ labeling and LC-MS/MS analysis of SCX fractionated peptides on Agilent’s accurate mass QTOF mass spectrometer. Through this approach, we identified both known and novel differentially regulated molecules. Those described previously included signal-regulatory protein alpha (SIRPA) and protein disulfide isomerase family A, member 6 (PDIA6), which have been shown to be overexpressed at the mRNA level in tuberculous meningitis. The novel overexpressed proteins identified in our study included amphiphysin (AMPH) and neurofascin (NFASC) while ferritin light chain (FTL) was found to be downregulated in TBM. We validated amphiphysin, neurofascin and ferritin light chain using immunohistochemistry which confirmed their differential expression in tuberculous meningitis. Overall, our data provides insights into the host response in tuberculous meningitis at the molecular level in addition to providing candidate diagnostic biomarkers for tuberculous meningitis.

Abstract: Background Central nervous system (CNS) infection is a nearly universal feature of untreated systemic HIV infection with a clinical spectrum that ranges from chronic asymptomatic infection to severe cognitive and motor dysfunction. Analysis of cerebrospinal fluid (CSF) has played an important part in defining the character of this evolving infection and response to treatment. To further characterize CNS HIV infection and its effects, we applied advanced high-throughput proteomic methods to CSF to identify novel proteins and their changes with disease progression and treatment.

Abstract: Pathogenesis of liver damage in patients with HIV and HCV co-infection is complex and multifactorial. Although global awareness regarding HIV-1/HCV co-infection is increasing little is known about the pathophysiology that mediates the rapid progression to hepatic disease in the co-infected individuals. In this study, we investigated the proteome profiles of peripheral blood mononuclear cells from HIV-1 mono-, HCV mono-, and HIV-1/HCV co-infected patients. The results of high-resolution 2D gel electrophoresis and PD quest software quantitative analysis revealed that several proteins were differentially expressed in HIV-1, HCV, and HIV-1/HCV co-infection. Liquid chromatography-mass spectrometry and Mascot database matching (LC-MS/MS analysis) successfully identified 29 unique and differentially expressed proteins. These included cytoskeletal proteins (tropomyosin, gelsolin, DYPLSL3, DYPLSL4 and profilin-1), chaperones and co-chaperones (HSP90-beta and stress-induced phosphoprotein), metabolic and pre-apoptotic proteins (guanosine triphosphate [GTP]-binding nuclear protein Ran, the detoxifying enzyme glutathione S-transferase (GST) and Rho GDP-dissociation inhibitor (Rho-GDI), proteins involved in cell prosurvival mechanism, and those involved in matrix synthesis (collagen binding protein 2 [CBP2]). The six most significant and relevant proteins were further validated in a group of mono- and co-infected patients (n = 20) at the transcriptional levels. The specific pro- and anti- apoptotic protein signatures revealed in this study could facilitate the understanding of apoptotic and protective immune-mediated mechanisms underlying HIV-1 and HCV co-infection and their implications on liver disease progression in co-infected patients.

Abstract: Background Pancreatic cancer is among the most lethal malignancies worldwide. This study aimed to identify a novel prognostic biomarker, facilitating treatment selection, using mass spectrometry (MS)-based proteomic analysis with formalin-fixed paraffin-embedded (FFPE) tissue.

Abstract: Aspergillus is a leading causative agent for fungal morbidity and mortality in immuno-compromised patients. To identify a putative target to design or identify new antifungal drug, against Aspergillus is required. In our previous work, we have analyzed the various biochemical pathways, and we found Ketol Acid Reducto-Isomerase (KARI) an enzyme involves in the amino acid biosynthesis, could be a better target. This enzyme was found to be unique by comparing to host proteome through BLASTp analysis. A homology based model of KARI was generated by Swiss model server. The generated model had been validated by PROCHECK and WHAT IF programs. The Zinc library was generated within the limitation of the Lipinski rule of five, for docking study. Based on the dock-score six molecules have been studied for ADME/TOX analysis and subjected for pharmacophore model generation. The Zinc ID of the potential inhibitors is ZINC00720614, ZINC01068126, ZINC0923, ZINC02090678, ZINC00663057 and ZINC02284065 and found to be pharmacologically active agonist and antagonist of KARI. This study is an attempt to Insilco evaluation of the KARI as a drug target and the screened inhibitors could help in the development of the better drug against Aspergillus.

Abstract: Background The diagnosis and management of BK virus (BKV) reactivation following renal transplantation continues to be a significant clinical problem. Following reactivation of latent virus, impaired cellular immunity enables sustained viral replication to occur in urothelial cells, which potentially leads to the development of BKV-associated nephropathy (BKVAN). Current guidelines recommend regular surveillance for BKV reactivation through the detection of infected urothelial cells in urine (decoy cells) or viral nucleic acid in urine or blood. However, these methods have variable sensitivity and cannot routinely distinguish between different viral subtypes. We therefore asked whether mass spectrometry might be able to overcome these limitations and provide an additional non-invasive technique for the surveillance of BKV and identification of recipients at increased risk of BKVAN.

Abstract: Autosomal dominant polycystic kidney disease (ADPKD) is responsible for 10% of cases of the end stage renal disease Early diagnosis, especially of potential fast progressors would be of benefit for efficient planning of therapy Urine excreted proteome has become a promising field of the search for marker patterns of renal diseases including ADPKD Up to now however, only the low molecular weight fraction of ADPKD proteomic fingerprint was studied The aim of our study was to characterize the higher molecular weight fraction of urinary proteome of ADPKD population in comparison to healthy controls as a part of a general effort aiming at exhaustive characterization of human urine proteome in health and disease, preceding establishment of clinically useful disease marker panel We have analyzed the protein composition of urine retentate (>10 kDa cutoff) from 30 ADPKD patients and an appropriate healthy control group by means of a gel-free relative quantitation of a set of more than 1400 proteins We have identified an ADPKD-characteristic footprint of 155 proteins significantly up- or downrepresented in the urine of ADPKD patients We have found changes in proteins of complement system, apolipoproteins, serpins, several growth factors in addition to known collagens and extracellular matrix components For a subset of these proteins we have confirmed the results using an alternative analytical technique Obtained results provide basis for further characterization of pathomechanism underlying the observed differences and establishing the proteomic prognostic marker panel

Abstract: We aimed to investigate the use of ovarian cyst fluid as a source for biomarker discovery and to find novel biomarkers for use in the diagnosis of epithelial ovarian tumors. Ovarian cyst fluids from 218 women were collected and 192 (benign n = 129, malignant n = 63) were analyzed using mass spectrometry. 1180 peaks were detected, 221 of which were differently expressed between benign and malignant ovarian tumors. Seventeen peaks had receiver operating curve and area under the curve values >0.70; the majority of these represented peaks for apolipoproteins C-III and C-I (ApoC-I), transthyretin (TTR), serum amyloid A4 (SAA4), and protein C inhibitor (PCI). ApoC-III, PCI, and serum CA125, with an ROC AUC 0.94 was the best combination for diagnosing epithelial ovarian cancer. ApoC-III and PCI was analyzed with ELISA in the original cohort (n = 40) and in 40 new cyst fluid samples for confirmation with an independent method and validation. Results from MS and ELISA for ApoC-III correlated well (p = 0.04). In the validation set, ApoC-III was significantly (p = 0.001) increased in the malignant epithelial ovarian cancers. Fluid from ovarian cysts connected directly to the primary tumor harbor many possible new tumor-specific biomarkers. Biomarkers found in ovarian cyst fluid may be used as molecular imaging targets for early diagnostics and prediction of therapy. Plasma abundant proteins are also influencing the cystic fluid proteome. Methods for isolating less frequent cyst fluid proteins are needed.

Abstract: Background In approximately 80% of patients, ovarian cancer is diagnosed when the patient is already in the advanced stages of the disease. CA125 is currently used as the marker for ovarian cancer; however, it lacks specificity and sensitivity for detecting early stage disease. There is a critical unmet need for sensitive and specific routine screening tests for early diagnosis that can reduce ovarian cancer lethality by reliably detecting the disease at its earliest and treatable stages.

Abstract: Background Prostatitis is an inflammation of the prostate gland which affects approximately 10% of men. Despite its frequency, diagnosing prostatitis and monitoring patient response to treatment remains frustrating. As the prostate contributes a substantial percentage of proteins to seminal plasma, we hypothesized that a protein biomarker of prostatitis might be found by comparing the seminal plasma proteome of patients with and without prostatitis.