Integrated proteotranscriptomics of breast cancer reveals globally increased protein-mRNA concordance associated with subtypes and survival.

Background: Transcriptome analysis of breast cancer discovered distinct disease subtypes of clinical significance. However, it remains a challenge to define disease biology solely based on gene expression because tumor biology is often the result of protein function. Here, we measured global proteome and transcriptome expression in human breast tumors and adjacent non-cancerous tissue and performed an integrated proteotranscriptomic analysis.

Protocol for the Analysis of Laser Capture Microdissected Fresh-Frozen Tissue Homogenates by Silver-Stained 1D SDS-PAGE.

The heterogeneity present in solid tumors adds significant difficulty to scientific analysis and improved understanding. Fundamentally, solid tumor formation consists of cancer cells proper along with stromal elements. The burgeoning malignant process is dependent upon modified stromal elements.

Preparation and Immunoaffinity Depletion of Fresh Frozen Tissue Homogenates for Mass Spectrometry-Based Proteomics in the Context of Drug Target/Biomarker Discovery.

The discovery of novel drug targets and biomarkers via mass spectrometry (MS)-based proteomic analysis of clinical specimens has proven to be challenging. The wide dynamic range of protein concentration in clinical specimens and the high background/noise originating from highly abundant proteins in tissue homogenates and serum/plasma encompass two major analytical obstacles. Immunoaffinity depletion of highly abundant blood-derived proteins from serum/plasma is a well-established approach adopted by numerous researchers; however, the utilization of this technique for immunodepletion of tissue homogenates obtained from fresh frozen clinical specimens is lacking.

Proinflammatory and lipid biomarkers mediate metabolically healthy obesity: A proteomics study.

Objective: The metabolically healthy obesity (MHO) phenotype is an important obesity subtype in which obesity is not accompanied by any metabolic comorbidity. However, the underlying molecular mechanisms remain elusive. In this study, a shotgun proteomics approach to identify circulating biomolecules and pathways associated with MHO was used.

Mammalian cell transient expression, non-affinity purification, and characterization of human recombinant IGFBP7, an IGF-1 targeting therapeutic protein.

Targeted inhibiting insulin-like growth factor 1 is an effective approach for cancer therapy. Insulin-like growth factor binding protein 7 (IGFBP7) is considered as a potential therapeutic protein. However, producing high quality of such non-IgG proteins in mammalian cells is still a challenge in biopharmaceutical development.

Mass spectrometry in cancer biomarker research: a case for immunodepletion of abundant blood-derived proteins from clinical tissue specimens.

The discovery of clinically relevant cancer biomarkers using mass spectrometry (MS)-based proteomics has proven difficult, primarily because of the enormous dynamic range of blood-derived protein concentrations and the fact that the 22 most abundant blood-derived proteins constitute approximately 99% of the total plasma protein mass. Immunodepletion of clinical body fluid specimens (e.g.

MYC-driven accumulation of 2-hydroxyglutarate is associated with breast cancer prognosis.

Metabolic profiling of cancer cells has recently been established as a promising tool for the development of therapies and identification of cancer biomarkers. Here we characterized the metabolomic profile of human breast tumors and uncovered intrinsic metabolite signatures in these tumors using an untargeted discovery approach and validation of key metabolites. The oncometabolite 2-hydroxyglutarate (2HG) accumulated at high levels in a subset of tumors and human breast cancer cell lines.

Trypsin-mediated ¹⁸O/¹⁶O labeling for biomarker discovery.

Differential (18)O/(16)O stable isotopic labeling that relies on post-digestion (18)O exchange is a simple and efficient method for the relative quantitation of proteins in complex mixtures. This method incorporates two (18)O atoms onto the C-termini of proteolytic peptides resulting in a 4 Da mass-tag difference between (18)O- and (16)O-labeled peptides. This allows for wide-range relative quantitation of proteins in complex mixtures using shotgun proteomics.

Proteomic analysis of frozen tissue samples using laser capture microdissection.

The discovery of effective cancer biomarkers is essential for the development of both advanced molecular diagnostics and new therapies/medications. Finding and exploiting useful clinical biomarkers for cancer patients is fundamentally linked to improving outcomes. Towards these aims, the heterogeneous nature of tumors represents a significant problem.

Post-digestion ¹⁸O exchange/labeling for quantitative shotgun proteomics of membrane proteins.

The role of membrane proteins is critical for regulation of physiologic and pathologic cellular processes. Hence it is not surpassing that membrane proteins make ∼70% of contemporary drug targets. Quantitative profiling of membrane proteins using mass spectrometry (MS)-based proteomics is critical in a quest for disease biomarkers and novel cancer drugs.

Profiling the erythrocyte membrane proteome isolated from patients diagnosed with chronic obstructive pulmonary disease.

Structural and metabolic alterations in erythrocytes play an important role in the pathophysiology of Chronic Obstructive Pulmonary Disease (COPD). Whether these dysfunctions are related to the modulation of erythrocyte membrane proteins in patients diagnosed with COPD remains to be determined. Herein, a comparative proteomic profiling of the erythrocyte membrane fraction isolated from peripheral blood of smokers diagnosed with COPD and smokers with no COPD was performed using differential (16)O/(18)O stable isotope labeling.

Proteomic profiling of H-Ras-G12V induced hypertrophic cardiomyopathy in transgenic mice using comparative LC-MS analysis of thin fresh-frozen tissue sections.

Determination of disease-relevant proteomic profiles from limited tissue specimens, such as pathological biopsies and tissues from small model organisms, remains an analytical challenge and a much needed clinical goal. In this study, a transgenic mouse disease model of cardiac-specific H-Ras-G12V induced hypertrophic cardiomyopathy provided a system to explore the potential of using mass spectrometry (MS)-based proteomics to obtain a disease-relevant molecular profile from amount-limited specimens that are routinely used in pathological diagnosis. Our method employs a two-stage methanol-assisted solubilization to digest lysates prepared from 8-μm-thick fresh-frozen histological tissue sections of diseased/experimental and normal/control hearts.

Profiling solid tumor heterogeneity by LCM and biological MS of fresh-frozen tissue sections.

The heterogeneous nature of solid tumors represents a common problem in mass spectrometry (MS)-based analysis of fresh-frozen tissue specimens. Here, we describe a method that relies on synergy between laser capture microdissection (LCM) and MS for enhanced molecular profiling of solid tumors. This method involves dissection of homogeneous histologic cell types from thin fresh-frozen tissue sections via LCM, coupled with liquid chromatography (LC)-MS analysis.

An Efficient Stochastic Search for Bayesian Variable Selection with High-Dimensional Correlated Predictors.

We present a Bayesian variable selection method for the setting in which the number of independent variables or predictors in a particular dataset is much larger than the available sample size. While most existing methods allow some degree of correlations among predictors but do not consider these correlations for variable selection, our method accounts for correlations among the predictors in variable selection. Our correlation-based stochastic search (CBS) method, the hybrid-CBS algorithm, extends a popular search algorithm for high-dimensional data, the stochastic search variable selection (SSVS) method.

Optimized method for computing (18)O/(16)O ratios of differentially stable-isotope labeled peptides in the context of postdigestion (18)O exchange/labeling.

Differential (18)O/(16)O stable isotope labeling of peptides that relies on enzyme-catalyzed oxygen exchange at their carboxyl termini in the presence of H(2)(18)O has been widely used for relative quantitation of peptides/proteins. The role of tryptic proteolysis in bottom-up shotgun proteomics and low reagent costs have made trypsin-catalyzed (18)O postdigestion exchange a convenient and affordable stable isotope labeling approach. However, it is known that trypsin-catalyzed (18)O exchange at the carboxyl terminus is in many instances inhomogeneous/incomplete.

Combined blood/tissue analysis for cancer biomarker discovery: application to renal cell carcinoma.

A method that relies on subtractive tissue-directed shot-gun proteomics to identify tumor proteins in the blood of a patient newly diagnosed with cancer is described. To avoid analytical and statistical biases caused by physiologic variability of protein expression in the human population, this method was applied on clinical specimens obtained from a single patient diagnosed with nonmetastatic renal cell carcinoma (RCC). The proteomes extracted from tumor, normal adjacent tissue and preoperative plasma were analyzed using 2D-liquid chromatography-mass spectrometry (LC-MS).

Engineered human antibody constant domains with increased stability.

The immunoglobulin (Ig) constant CH2 domain is critical for antibody effector functions. Isolated CH2 domains are promising as scaffolds for construction of libraries containing diverse binders that could also confer some effector functions. However, previous work has shown that an isolated murine CH2 domain is relatively unstable to thermally induced unfolding.

Approaching solid tumor heterogeneity on a cellular basis by tissue proteomics using laser capture microdissection and biological mass spectrometry.

The purpose of this study was to examine solid tumor heterogeneity on a cellular basis using tissue proteomics that relies on a functional relationship between Laser Capture Microdissection (LCM) and biological mass spectrometry (MS). With the use of LCM, homogeneous regions of cells exhibiting uniform histology were isolated and captured from fresh frozen tissue specimens, which were obtained from a human lymph node containing breast carcinoma metastasis. Six specimens approximately 50,000 cell each (three from tumor proper and three from tumor stroma) were collected by LCM.

Del-1, an endogenous leukocyte-endothelial adhesion inhibitor, limits inflammatory cell recruitment.

Leukocyte recruitment to sites of infection or inflammation requires multiple adhesive events. Although numerous players promoting leukocyte-endothelial interactions have been characterized, functionally important endogenous inhibitors of leukocyte adhesion have not been identified. Here we describe the endothelially derived secreted molecule Del-1 (developmental endothelial locus-1) as an anti-adhesive factor that interferes with the integrin LFA-1-dependent leukocyte-endothelial adhesion.

Proteomic analysis of traumatic brain injury: the search for biomarkers.

Although there are a number of causes of traumatic brain injury (TBI), the armed conflict in Iraq and Afghanistan has brought this disorder to the attention of the global community. A biomarker that would enable army medics to rapidly diagnose the severity of TBI on the battle-field would be a huge asset. Unfortunately, the study of TBI has not historically attracted the proteomic research community's interest as other disorders have, such as cancer.

Increased serum levels of complement C3a anaphylatoxin indicate the presence of colorectal tumors.

Background & Aims: Late diagnosis of colorectal carcinoma results in a significant reduction of average survival times. Yet despite screening programs, about 70% of tumors are detected at advanced stages (International Union Against Cancer stages III/IV). We explored whether detection of malignant disease would be possible through identification of tumor-specific protein biomarkers in serum samples.

Identification of the SELDI ProteinChip human serum retentate by microcapillary liquid chromatography-tandem mass spectrometry.

Surface-enhanced laser desorption/ionization (SELDI) time-of-flight (TOF) mass spectrometry (MS) has been widely applied for conducting biomarker research with the goal of discovering patterns of proteins and/or peptides from biological samples that reflect disease status. Many diseases, ranging from cancers of the colon, breast, and prostate to Alzheimer's disease, have been studied through serum protein profiling using SELDI-based methods. Although the results from SELDI-based diagnostic studies have generated a great deal of excitement and skepticism alike, the basis of the molecular identities of the features that underpin the diagnostic potential of the mass spectra is still largely unexplored.

Liquid Tissue: proteomic profiling of formalin-fixed tissues.

Identification and quantitation of candidate biomarker proteins in large numbers of individual tissues is required to validate specific proteins, or panels of proteins, for clinical use as diagnostic, prognostic, toxicological, or therapeutic markers. Mass spectrometry (MS) provides an exciting analytical methodology for this purpose. Liquid Tissue MS protein preparation allows researchers to utilize the vast, already existing, collections offormalin-fixed paraffin-embedded (FFPE) tissues for the procurement of peptides and the analysis across a variety of MS platforms.

Proteomic patterns: their potential for disease diagnosis.

Alterations in proteins abundance, structure, or function, act as useful indicators of pathological abnormalities prior to development of clinical symptoms and as such are often useful diagnostic and prognostic biomarkers. The underlying mechanism of diseases such as cancer are, however, quite complicated in that often multiple dysregulated proteins are involved. It is for this reason that recent hypotheses suggest that detection of panels of biomarkers may provide higher sensitivities and specificities for disease diagnosis than is afforded with single markers.

Proteomic patterns for early cancer detection.

The advent of proteomics has brought with it the hope of discovering novel biomarkers that can be used to diagnose diseases, predict susceptibility, and monitor progression. Much of this effort has focused on the mass spectral identification of the thousands of proteins that populate complex biosystems such as serum and tissues. A revolutionary approach in proteomic pattern analysis has emerged as an effective method for the early diagnosis of diseases such as ovarian, breast, and prostate cancer.