Label-Free Quantification Mass Spectrometry Identifies Protein Markers of Chemotherapy Response in High-Grade Serous Ovarian Cancer.

Eighty percent of ovarian cancer patients initially respond to chemotherapy, but the majority eventually experience a relapse and die from the disease with acquired chemoresistance. In addition, 20% of patients do not respond to treatment at all, as their disease is intrinsically chemotherapy resistant. Data-independent acquisition nano-flow liquid chromatography-mass spectrometry (DIA LC-MS) identified the three protein markers: gelsolin (GSN), calmodulin (CALM1), and thioredoxin (TXN), to be elevated in high-grade serous ovarian cancer (HGSOC) tissues from patients that responded to chemotherapy compared to those who did not; the differential expression of the three protein markers was confirmed by immunohistochemistry.

A Protocol for the Acquisition of Comprehensive Proteomics Data from Single Cases Using Formalin-Fixed Paraffin Embedded Sections.

The molecular analysis of small or rare patient tissue samples is challenging and often limited by available technologies and resources, such as reliable antibodies against a protein of interest. Although targeted approaches provide some insight, here, we describe the workflow of two complementary mass spectrometry approaches, which provide a more comprehensive and non-biased analysis of the molecular features of the tissue of interest. Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) generates spatial intensity maps of molecular features, which can be easily correlated with histology.

Proteomic Analysis of Pre-Invasive Serous Lesions of the Endometrium and Fallopian Tube Reveals Their Metastatic Potential.

Serous endometrial cancer (SEC) and high grade serous ovarian cancer (HGSOC) are aggressive gynecological malignancies with high rates of metastasis and poor prognosis. Endometrial intraepithelial carcinoma (EIC), the precursor for SEC, and serous tubal intraepithelial carcinoma (STIC), believed to be the precursor lesion for HGSOC, can also be associated with intraabdominal spread. To provide insight into the etiology of these precancerous lesions and to explore the potential molecular mechanisms underlying their metastatic behavior, we performed a proteomic mass spectrometry analysis in a patient with synchronous EIC and STIC.

Structure, Mechanism, and Inhibition of Thioredoxin Reductase.

infections are associated with high mortality rates and high treatment costs. Limited available antifungals and increasing antifungal resistance highlight an urgent need for new antifungals. Thioredoxin reductase (TrxR) is essential for maintaining redox homeostasis and presents as a promising target for novel antifungals.

Translating N-Glycan Analytical Applications into Clinical Strategies for Ovarian Cancer.

Protein glycosylation, particularly N-linked glycosylation, is a complex posttranslational modification (PTM), which plays an important role in protein folding and conformation, regulating protein stability and activity, cell-cell interaction, and cell signaling pathways. This review focuses on analytical techniques, primarily MS-based techniques, to qualitatively and quantitatively assess N-glycosylation while successfully characterizing compositional, structural, and linkage features with high specificity and sensitivity. The analytical techniques explored in this review include LC-ESI-MS/MS and MALDI time-of-flight MS (MALDI-TOF-MS), which have been used to analyze clinical samples, such as serum, plasma, ascites, and tissue.

Targeted proteomic analysis of cognitive dysfunction in remitted major depressive disorder: Opportunities of multi-omics approaches towards predictive, preventive, and personalized psychiatry.

Unlabelled: In order to accelerate the understanding of pathophysiological mechanisms and clinical biomarker discovery and in psychiatry, approaches that integrate multiple -omics platforms are needed. We introduce a workflow that investigates a narrowly defined psychiatric phenotype, makes use of the potent and cost-effective discovery technology of gene expression microarrays, applies Weighted Gene Co-Expression Network Analysis (WGCNA) to better capture complex and polygenic traits, and finally explores gene expression findings on the proteomic level using targeted mass-spectrometry (MS) technologies. To illustrate the effectiveness of the workflow, we present a proteomic analysis of peripheral blood plasma from patient's remitted major depressive disorder (MDD) who experience ongoing cognitive deficits.

Novel IEF Peptide Fractionation Method Reveals a Detailed Profile of N-Terminal Acetylation in Chemotherapy-Responsive and -Resistant Ovarian Cancer Cells.

Although acetylation is regarded as a common protein modification, a detailed proteome-wide profile of this post-translational modification may reveal important biological insight regarding differential acetylation of individual proteins. Here we optimized a novel peptide IEF fractionation method for use prior to LC-MS/MS analysis to obtain a more in depth coverage of N-terminally acetylated proteins from complex samples. Application of the method to the analysis of the serous ovarian cancer cell line OVCAR-5 identified 344 N-terminally acetylated proteins, 12 of which are previously unreported.

Annexin A2 and alpha actinin 4 expression correlates with metastatic potential of primary endometrial cancer.

The prediction of lymph node metastasis using clinic-pathological data and molecular information from endometrial cancers lacks accuracy and is therefore currently not routinely used in patient management. Consequently, although only a small percentage of patients with endometrial cancers suffer from metastasis, the majority undergo radical surgery including removal of pelvic lymph nodes. Upon analysis of publically available data and published research, we compiled a list of 60 proteins having the potential to display differential abundance between primary endometrial cancers with versus those without lymph node metastasis.

MALDI Mass Spectrometry Imaging Reveals Decreased CK5 Levels in Vulvar Squamous Cell Carcinomas Compared to the Precursor Lesion Differentiated Vulvar Intraepithelial Neoplasia.

Vulvar cancer is the fourth most common gynecological cancer worldwide. However, limited studies have been completed on the molecular characterization of vulvar squamous cell carcinoma resulting in a poor understanding of the disease initiation and progression. Analysis and early detection of the precursor lesion of HPV-independent vulvar squamous cell carcinoma (VSCC), differentiated vulvar intraepithelial neoplasia (dVIN), is of great importance given dVIN lesions have a high level of malignant potential.

Lymph node metastasis of primary endometrial cancers: Associated proteins revealed by MALDI imaging.

Metastasis is a crucial step of malignant progression and is the primary cause of death from endometrial cancer. However, clinicians presently face the challenge that conventional surgical-pathological variables, such as tumour size, depth of myometrial invasion, histological grade, lymphovascular space invasion or radiological imaging are unable to predict with accuracy if the primary tumour has metastasized. In the current retrospective study, we have used primary tumour samples of endometrial cancer patients diagnosed with (n = 16) and without (n = 27) lymph node metastasis to identify potential discriminators.

Proteomics of endometrial cancer diagnosis, treatment, and prognosis.

This review discusses the current status of proteomics technology in endometrial cancer diagnosis, treatment and prognosis. The first part of this review focuses on recently identified biomarkers for endometrial cancer, their importance in clinical use as well as the proteomic methods used in their discovery. The second part highlights some of the emerging mass spectrometry based proteomic technologies that promise to contribute to a better understanding of endometrial cancer by comparing the abundance of hundreds or thousands of proteins simultaneously.

State of the art of 2D DIGE.

Difference gel electrophoresis enables the accurate quantification of changes in the proteome including combinations of PTMs and protein isoform expression. Here, we review recent advances in study design, image acquisition, and statistical analysis. We also compare DIGE to established and emerging mass spectrometric analysis technologies.

Proteomic developments in the analysis of formalin-fixed tissue.

Retrospective proteomic studies, including those which aim to elucidate the molecular mechanisms driving cancer, require the assembly and characterization of substantial patient tissue cohorts. The difficulty of maintaining and accessing native tissue archives has prompted the development of methods to access archives of formalin-fixed tissue. Formalin-fixed tissue archives, complete with patient meta data, have accumulated for decades, presenting an invaluable resource for these retrospective studies.

Proangiogenic tumor proteins as potential predictive or prognostic biomarkers for bevacizumab therapy in metastatic colorectal cancer.

Tumor biomarkers to more accurately predict a patient's response to a given therapy are much needed in oncology practice. For metastatic colorectal cancer the anti-vascular endothelial growth factor (VEGF) monoclonal antibody bevacizumab is now commonly included in first-line therapy regimens and has led to modest but significant improvements in patient outcomes compared with chemotherapy. Given the modest gains there is a pressing need for predictive biomarkers to better identify patients who would benefit from this targeted therapy.

Long-term Ro60 humoral autoimmunity in primary Sjögren's syndrome is maintained by rapid clonal turnover.

Long-term humoral autoimmunity to RNA-protein autoantigens is considered a hallmark of systemic autoimmune diseases. We use high resolution Orbitrap mass spectrometric autoantibody sequencing to track the evolution of a Ro60-specific public clonotypic autoantibody in 4 patients with primary Sjögren's syndrome. This clonotype is specified by a VH3-23/VK3-20 heavy and light chain pairing.

Secreted human Ro52 autoantibody proteomes express a restricted set of public clonotypes.

Long-lived secreted autoantibody responses in systemic autoimmunity are generally regarded to be polyclonal and to express a diverse B-cell repertoire. Here, we have used a proteomic approach based on de novo sequencing to determine the clonality and V region structures of human autoantibodies directed against a prototypic systemic autoantigen, Ro52 (TRIM21). Remarkably, anti-Ro52 autoantibodies from patients with primary Sjögren's syndrome, systemic lupus erythematosus, systemic sclerosis or polymyositis were restricted to two IgG1 kappa clonotypes that migrated as a single species on isoelectric focusing; shared a common light chain paired with one of two closely-related heavy chains; and were public in unrelated patients.

Pathogenicity and proteomic signatures of autoantibodies to Ro and La.

Ro/SSA and La/SSB comprise a linked set of autoantigens that are clinically important members of the extractable nuclear antigen family and key translational biomarkers for lupus and primary Sjögren's syndrome. Autoantibodies directed against the Ro60 and La polypeptide components of the Ro/La ribonucleoprotein complex, and the structurally unrelated Ro52 protein, mediate tissue damage in the neonatal lupus syndrome, a model of passively acquired autoimmunity in humans in which the most serious manifestation is congenital heart block (CHB). Recent studies have concentrated on two distinct pathogenic mechanisms by which maternal anti-Ro/La autoantibodies can cause CHB: by forming immune complexes with apoptotic cells in developing fetal heart; and/or by acting as functional autoantibodies that cross-react with and inhibit calcium channels.

Increased Phospho-Keratin 8 Isoforms in Colorectal Tumors Associated with EGFR Pathway Activation and Reduced Apoptosis.

Hyperphosphorylated keratin (K) 8 acts as a phosphate "sponge" for stress-activated protein kinases thereby inhibiting pro-apoptotic molecules and thus apoptosis. MAP kinase/ERK1 has increased activity in colorectal cancer (CRC) and is known to phosphorylate K8. The aims were to identify the K8 isoforms abundantly present in colon tumors, using 2D difference gel electrophoresis (DIGE), to identify the modifications using mass spectrometry, and to validate the differential abundance of these isoforms in tumors relative to matched normal mucosae.

Desmin expression in colorectal cancer stroma correlates with advanced stage disease and marks angiogenic microvessels.

Introduction: Biomarkers that improve stratification of colorectal cancer patients for adjuvant therapy versus resection alone, or that are predictive of response to therapeutic agents, have the potential to greatly improve patient selection for such therapies. The aim was to determine proteins differentially expressed within the malignant epithelial glands and closely associated stromal elements compared to matched normal mucosa, and to characterise the over-expression of one such protein as a potential biomarker.

Methods: Protein from laser microdissected tumor and normal mucosa was analysed by two dimensional difference gel electrophoresis (2D DIGE) and mass spectrometry to determine differentially over expressed tumor proteins.

Molecular signature of a public clonotypic autoantibody in primary Sjögren's syndrome: a "forbidden" clone in systemic autoimmunity.

Objective: This study was undertaken to determine the molecular characteristics of clonotypic autoantibodies in the sera of patients with primary Sjögren's syndrome (SS). This characterization is hampered by the presence of mixed anti-Ro/La specificities that may conceal clonotypic species. In order to narrow clonotypic diversity, a positive selection step was performed on a peg-like determinant of Ro 60 (termed Ro 60-peg) prior to analysis of the autoantibody proteome.