The use of mass spectrometry in a proteome-centered multiomics study of human pituitary adenomas.

A pituitary adenoma (PA) is a common intracranial neoplasm, and is a complex, chronic, and whole-body disease with multicausing factors, multiprocesses, and multiconsequences. It is very difficult to clarify molecular mechanism and treat PAs from the single-factor strategy model. The rapid development of multiomics and systems biology changed the paradigms from a traditional single-factor strategy to a multiparameter systematic strategy for effective management of PAs. A series of molecular alterations at the genome, transcriptome, proteome, peptidome, metabolome, and radiome levels are involved in pituitary tumorigenesis, and mutually associate into a complex molecular network system. Also, the center of multiomics is moving from structural genomics to phenomics, including proteomics and metabolomics in the medical sciences. Mass spectrometry (MS) has been extensively used in phenomics studies of human PAs to clarify molecular mechanisms, and to discover biomarkers and therapeutic targets/drugs. MS-based proteomics and proteoform studies play central roles in the multiomics strategy of PAs. This article reviews the status of multiomics, multiomics-based molecular pathway networks, molecular pathway network-based pattern biomarkers and therapeutic targets/drugs, and future perspectives for personalized, predeictive, and preventive (3P) medicine in PAs.

EVpedia: a community web portal for extracellular vesicles research.

MOTIVATION: Extracellular vesicles (EVs) are spherical bilayered proteolipids, harboring various bioactive molecules. Due to the complexity of the vesicular nomenclatures and components, online searches for EV-related publications and vesicular components are currently challenging. RESULTS: We present an improved version of EVpedia, a public database for EVs research. This community web portal contains a database of publications and vesicular components, identification of orthologous vesicular components, bioinformatic tools and a personalized function. EVpedia includes 6879 publications, 172 080 vesicular components from 263 high-throughput datasets, and has been accessed more than 65 000 times from more than 750 cities. In addition, about 350 members from 73 international research groups have participated in developing EVpedia. This free web-based database might serve as a useful resource to stimulate the emerging field of EV research. AVAILABILITY AND IMPLEMENTATION: The web site was implemented in PHP, Java, MySQL and Apache, and is freely available at http://evpedia.info.

Mass spectrometry analysis of nitrotyrosine-containing proteins.

Oxidative stress plays important roles in a wide range of diseases such as cancer, inflammatory disease, neurodegenerative disorders, etc. Tyrosine nitration in a protein is a chemically stable oxidative modification, and a marker of oxidative injuries. Mass spectrometry (MS) is a key technique to identify nitrotyrosine-containing proteins and nitrotyrosine sites in endogenous and synthetic nitroproteins and nitropeptides. However, in vivo nitrotyrosine-containing proteins occur with extreme low-abundance to severely challenge the use of MS to identify in vivo nitroproteins and nitrotyrosine sites. A preferential enrichment of nitroproteins and/or nitropeptides is necessary before MS analysis. Current enrichment methods include immuno-affinity techniques, chemical derivation of the nitro group plus target isolations, followed with tandem mass spectrometry analysis. This article reviews the MS techniques and pertinent before-MS enrichment techniques for the identification of nitrotyrosine-containing proteins. This article reviews future trends in the field of nitroproteomics, including quantitative nitroproteomics, systems biological networks of nitroproteins, and structural biology study of tyrosine nitration to completely clarify the biological functions of tyrosine nitration.

Deimination of linker histones links neutrophil extracellular trap release with autoantibodies in systemic autoimmunity.

Autoantibodies to nuclear antigens arise in human autoimmune diseases, but a unifying pathogenetic mechanism remains elusive. Recently we reported that exposure of neutrophils to inflammatory conditions induces the citrullination of core histones by peptidylarginine deiminase 4 (PAD4) and that patients with autoimmune disorders produce autoantibodies that recognize such citrullinated histones. Here we identify histone H1 as an additional substrate of PAD4, localize H1 within neutrophil extracellular traps, and detect autoantibodies to citrullinated H1 in 6% of sera from patients with systemic lupus erythematosus and Sjögren's syndrome. No preference for deiminated H1 was observed in healthy control sera and sera from patients with scleroderma or rheumatoid arthritis. We map binding to the winged helix of H1 and determine that citrulline 53 represents a key determinant of the autoantibody epitope. In addition, we quantitate RNA for H1 histone subtypes in mature human neutrophils and identify citrulline residues by liquid chromatography and tandem mass spectrometry. Our results indicate that deimination of linker histones generates new autoantibody epitopes with enhanced potential for stimulating autoreactive human B cells.-Dwivedi, N., Neeli, I., Schall, N., Wan, H., Desiderio, D. M., Csernok, E., Thompson, P. R., Dali, H., Briand, J.-P., Muller, S., Radic, M. Deimination of linker histones links neutrophil extracellular trap release with autoantibodies in systemic autoimmunity.

Identification of the proteomic variations of invasive relative to non-invasive non-functional pituitary adenomas.

The incomplete surgery section of invasive non-functional pituitary adenomas (NFPAs) carries the increased risks of complications and requires adjuvant radiotherapy and medications. It is necessary to clarify the molecular mechanisms and markers of invasiveness to guide the management of NFPA patients. The study aimed to proteomic variations of invasive and non-invasive NFPAs and sought the protein markers for invasive NFPAs. Invasive (n = 4) and non-invasive (n = 4) NFPA tissues were analyzed (n = 3-5/each tissue) with 2DE and PDQuest software. Twenty-four high-resolution 2DE gels were quantitatively compared to determine differentially expressed proteins (DEPs) between invasive and non-invasive NFPAs. Approximately 1200 protein spots were detected in each 2DE map, and 103 differential spots (64 upregulated and 39 downregulated) were identified. Among those 103 differential spots, 57 DEPs (30 upregulated and 27 downregulated) were characterized with peptide mass fingerprint and MS/MS. Gene-ontology (GO) and ingenuity pathway analyses of those DEPs revealed pathway networks including mitochondrial dysfunction, oxidative stress, mitogen-activated protein kinase signaling abnormality, TR/RXR activation, proteolysis abnormality, ketogenesis and ketolysis, cyclin-dependent kinase C signaling abnormality, and amyloid processing that were significantly associated with invasive characteristics of invasive NFPA. Those data demonstrate that proteomic variations exist between invasive and non-invasive NFPAs. 2DE-based comparative proteomics is an effective approach to identify proteomic variations and pathway network variations. Those findings will serve as a basis to understand the molecular mechanisms of invasive NFPAs and to discover protein markers to effectively manage patients with invasive NFPAs.

LC-MS/MS identification of the one-carbon cycle metabolites in human plasma.

The one-carbon cycle is composed of four major biologically important molecules: methionine (L-Met), S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), and homocysteine (Hcy). In addition to these key metabolites, there are multiple enzymes, vitamins, and cofactors that play essential roles in the cascade of the biochemical reactions that convert one metabolite into another in the cycle. Simultaneous quantitative measurement of four major metabolites can be used to detect possible aberrations in this vital cycle. Abnormalities in the one-carbon cycle might lead to hyper- or hypomethylation, homocystinemia, liver dysfunction, and accumulation of white-matter hyperintensities in the human brain. Previously published methods describe evaluation of several components of the one-carbon cycle, but none to our knowledge demonstrated simultaneous measurement of all four key molecules (L-Met, SAM, SAH, and Hcy). We describe a novel analytical method suitable for simultaneous identification and quantification of L-Met, SAM, SAH, and Hcy with LC-MS/MS. Moreover, we tested this method to identify these metabolites in human plasma collected from patients with multiple sclerosis and healthy individuals. In a pilot feasibility study, our results indicate that patients with multiple sclerosis showed abnormalities in the one-carbon cycle.

Proteomic analysis of microvesicles derived from human colorectal cancer ascites.

The presence of malignant ascites in the peritoneal cavity is a poor prognostic indicator of low survival rate. Various cancer cells, including those of colorectal cancer (CRC), release microvesicles (exosomes) into surrounding tissues and peripheral circulation including malignant ascites. Although recent progress has revealed that microvesicles play multiple roles in tumor progression, the protein composition and the pathological function of malignant ascites-derived microvesicles are still unknown. Here, we report the first global proteomic analyses of highly purified microvesicles derived from human CRC ascites. With 1-D SDS-PAGE and nano-LC-MS/MS analyses, we identified a total of 846 microvesicular proteins from ascites of three CRC patients with high confidence; 384 proteins were identified in at least two patients. We identified proteins that might function in tumor progression via disruption of epithelial polarity, migration, invasion, tumor growth, immune modulation, and angiogenesis. Furthermore, we identified several potential diagnostic markers of CRC including colon-specific surface antigens. Our proteomic analyses will help to elucidate diverse functions of microvesicles in cancer progression and will aid in the development of novel diagnostic tools for CRC.

SILAC quantitative proteomics analysis of ivermectin-related proteomic profiling and molecular network alterations in human ovarian cancer cells.

The antiparasitic agent ivermectin offers more promises to treat a diverse range of diseases. However, a comprehensive proteomic analysis of ivermectin-treated ovarian cancer (OC) cells has not been performed. This study sought to identify ivermectin-related proteomic profiling and molecular network alterations in human OC cells. Stable isotope labeling with amino acids in cell culture (SILAC)-based quantitative proteomics was used to study the human OC TOV-21G cells. After TOV-21G cells underwent 10 passages in SILAC-labeled growth media, TOV-21G cells were treated with 10 ml of 20 µmol/L ivermectin in cell growing medium for 24 h. The SILAC-labeled proteins were digested with trypsin; tryptic peptides were identified with mass spectrometry (MS). Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis was used to mine signaling pathway alterations with ivermectin-related proteins in TOV-21G cells. Gene ontology (GO) analysis was used to explore biological functions of ivermectin-related proteins, including biological processes (BPs), cellular components (CCs), and molecular functions (MFs). The protein-protein interaction network was analyzed with molecular complex detection (MCODE) to identify hub modules. In total, 4,447 proteins were identified in ivermectin-treated TOV-21G cells. KEGG analysis revealed 89 statistically significant signaling pathways. Interestingly, the clustering analysis of these pathways showed that ivermectin was involved in various cancer pathogenesis processes, including modulation of replication, RNA metabolism, and translational machinery. GO analysis revealed 69 statistically significant CCs, 87 MFs, and 62 BPs. Furthermore, MCODE analysis identified five hub modules, including 147 hub molecules. Those hub modules involved ribosomal proteins, RNA-binding proteins, cell-cycle progression-related proteins, proteasome subunits, and minichromosome maintenance proteins. These findings demonstrate that SILAC quantitative proteomics is an effective method to analyze ivermectin-treated cells, provide the first ivermectin-related proteomic profiling and molecular network alterations in human OC cells, and provide deeper insights into molecular mechanisms and functions of ivermectin to inhibit OC cells.

Human growth hormone proteoform pattern changes in pituitary adenomas: Potential biomarkers for 3P medical approaches.

RELEVANCE: Human growth hormone (hGH) is synthesized, stored, and secreted by somatotroph cells in the pituitary gland, and promotes human growth and metabolism. Compared to a normal pituitary, a GH-secreting pituitary adenoma can secrete excessive GH to cause pathological changes in body tissues. GH proteoform changes would be associated with GH-related disease pathogenesis. PURPOSE: This study aimed to elucidate changes in GH proteoforms between GH-secreting pituitary adenomas and control pituitaries for the predictive diagnostics, targeted prevention, and personalization of medical services. METHODS: The isoelectric point (pI) and relative molecular mass (Mr) are two basic features of a proteoform that can be used to effectively array and detect proteoforms with two-dimensional gel electrophoresis (2DGE) and 2DGE-based western blot. GH proteoforms were characterized with liquid chromatography (LC) and mass spectrometry (MS). Phosphoproteomics, ubiquitinomics, acetylomics, and bioinformatics were used to analyze post-translational modifications (PTMs) of GH proteoforms in GH-secreting pituitary adenoma tissues and control pituitaries. RESULTS: Sixty-six 2D gel spots were found to contain hGH, including 46 spots (46 GH proteoforms) in GH-secreting pituitary adenomas and 35 spots (35 GH proteoforms) in control pituitaries. Further, 35 GH proteoforms in control pituitary tissues were matched with 35 of 46 GH proteoforms in GH-secreting pituitary adenoma tissues; and 11 GH proteoforms were presented in only GH-secreting pituitary adenoma tissues but not in control pituitary tissues. The matched 35 GH proteoforms showed quantitative changes in GH-secreting pituitary adenomas compared to the controls. The quantitative levels of those 46 GH proteoforms in GH-secreting pituitary adenomas were significantly different from those 35 GH proteoforms in control pituitaries. Meanwhile, different types of PTMs were identified among those GH proteoforms. Phosphoproteomics identified phosphorylation at residues Ser77, Ser132, Ser134, Thr174, and Ser176 in hGH. Ubiquitinomics identified ubiquitination at residue Lys96 in hGH. Acetylomics identified acetylation at reside Lys171 in hGH. Deamination was identified at residue Asn178 in hGH. CONCLUSION: These findings provide the first hGH proteoform pattern changes in GH-secreting pituitary adenoma tissues compared to control pituitary tissues, and the status of partial PTMs in hGH proteoforms. Those data provide in-depth insights into biological roles of hGH in GH-related diseases, and identify hGH proteoform pattern biomarkers for treatment of a GH-secreting pituitary adenoma in the context of 3P medicine -predictive diagnostics, targeted prevention, and personalization of medical services. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13167-021-00232-7.

Gram-positive bacteria produce membrane vesicles: proteomics-based characterization of Staphylococcus aureus-derived membrane vesicles.

Although archaea, Gram-negative bacteria, and mammalian cells constitutively secrete membrane vesicles (MVs) as a mechanism for cell-free intercellular communication, this cellular process has been overlooked in Gram-positive bacteria. Here, we found for the first time that Gram-positive bacteria naturally produce MVs into the extracellular milieu. Further characterizations showed that the density and size of Staphylococcus aureus-derived MVs are both similar to those of Gram-negative bacteria. With a proteomics approach, we identified with high confidence a total of 90 protein components of S. aureus-derived MVs. In the group of identified proteins, the highly enriched extracellular proteins suggested that a specific sorting mechanism for vesicular proteins exists. We also identified proteins that facilitate the transfer of proteins to other bacteria, as well to eliminate competing organisms, antibiotic resistance, pathological functions in systemic infections, and MV biogenesis. Taken together, these observations suggest that the secretion of MVs is an evolutionally conserved, universal process that occurs from simple organisms to complex multicellular organisms. This information will help us not only to elucidate the biogenesis and functions of MVs, but also to develop therapeutic tools for vaccines, diagnosis, and antibiotics effective against pathogenic strains of Gram-positive bacteria.

Mass spectrometric identification of in vivo nitrotyrosine sites in the human pituitary tumor proteome.

The chemically stable tyrosine nitration of a protein involves the addition of a nitro group (-NO(2)) to the phenolic ring of a tyrosine residue, which may be associated with nervous system physiological and pathological processes. Identification of nitrotyrosine sites on a protein could clarify the functional significance of the modification. Due to the rarity of nitrotyrosine sites in a proteome, tandem mass spectrometry, coupled with different techniques that isolate and enrich nitrotyrosine-containing proteins from a pituitary proteome, is currently the most effective method for site identification. Commercially available nitrotyrosine polyclonal/monoclonal antibodies enable one to detect nitrotyrosine-containing proteins in a two-dimensional gel electrophoresis (2DGE) map, and to preferentially enrich nitrotyrosine-containing proteins with immunoprecipitation. Our present protocols have integrated different isolation/enrichment techniques (2DGE; Western blots; nitrotyrosine immunoaffinity precipitation) and two different tandem mass spectrometry methods (MALDI-MS/MS; ESI-MS/MS) to determine the amino acid sequence of nitrotyrosine-containing peptides that derive from nitrated proteins. Bioinformatics tools are then used to correlate nitrotyrosine sites with a functional domain/motif in order to understand the relationship between tyrosine nitration and the structural/functions of proteins.

Phosphoproteome analysis by in-gel isoelectric focusing and tandem mass spectrometry.

Protein phosphorylation is central to most signaling events in eukaryotic cells. Large-scale analysis of protein phosphorylation in vivo is a highly challenging undertaking that requires powerful analytical and bioinformatics tools; numerous phosphoproteomic methodologies that use various combinations of these tools have been developed recently. This chapter describes an in-gel isoelectric focusing-liquid chromatography-tandem mass spectrometry (IEF-LC-MS/MS) analytical strategy for phosphoproteome mapping. The strategy encompasses seven steps: (1) extraction of proteins from the biological system under study (e.g., a tissue); (2) separation of the protein mixture by isoelectric focusing in an immobilized pH gradient (IPG) strip; (3) protein fixation followed by sectioning of the IPG strip; (4) digestion of the proteins in each gel section; (5) enrichment of phosphopeptides in the digests by immobilized metal ion affinity chromatography; (6) analysis of the enriched digests by LC-MS/MS; and (7) identification of the phosphopeptides/proteins through database searches, and assignment of the sites of phosphorylation in these proteins.

MALDI-induced Fragmentation of Leucine enkephalin, Nitro-Tyr Leucine Enkaphalin, and d(5)-Phe-Nitro-Tyr Leucine Enkephalin.

The long-term objective of this study is to use MALDI MS and MS/MS to study the fragmentation pattern of in vitro nitrotyrosine-containing peptides in order to assist the interpretation of MS-identification of endogenous nitroproteins in human tissues and fluids. The short-term objective is to study synthetic leucine enkephalin, nitro-Tyr-leucine enkephalin, and d(5)-Phe-nitro-Tyr-leucine enkephalin with a vacuum matrix-assisted laser desorption/ionization linear ion-trap mass spectrometer (vMALDI-LTQ). The results demonstrated the UV laser-induced photochemical decomposition of the nitro group. Although photochemical decomposition decreased the ion intensity and complicated the MS spectrum, the recognition of that unique decomposition pattern unambiguously identified a nitrotyrosine. The a(4)- and b(4)-ions were the most-intense fragment ions found in the MS/MS spectra for those three synthetic peptides. Compared to the unmodified peptides, more collision energy optimized the fragmentation of the nitropeptide, increased the intensity of the a(4)-ion, and decreased the intensity of the b(4)-ion. Optimized laser fluence maximized the fragmentation of the nitropeptide. MS(3) analysis confirmed the MS(2)-derived amino acid sequence, but required much more sample. To detect a nitropeptide, the sensitivity of vMALDI-LTQ is 1 fmol for MS detection and 10 fmol for MS(2) detection; the S/N ratio was ca. 50:1 in those studies. Those data are important for an analysis of low-abundance endogenous nitroproteins, where preferential enrichment of nitroproteins and optimized mass spectrometry parameters are used.

Quantitative Analysis of Ubiquitinated Proteins in Human Pituitary and Pituitary Adenoma Tissues.

Protein ubiquitination is an important post-translational modification that is associated with multiple diseases, including pituitary adenomas (PAs). Protein ubiquitination profiling in human pituitary and PAs remains unknown. Here, we performed the first ubiquitination analysis with an anti-ubiquitin antibody (specific to K-ε-GG)-based label-free quantitative proteomics method and bioinformatics to investigate protein ubiquitination profiling between PA and control tissues. A total of 158 ubiquitinated sites and 142 ubiquitinated peptides in 108 proteins were identified, and five ubiquitination motifs were found. KEGG pathway network analysis of 108 ubiquitinated proteins identified four statistically significant signaling pathways, including PI3K-AKT signaling pathway, hippo signaling pathway, ribosome, and nucleotide excision repair. R software Gene Ontology (GO) analysis of 108 ubiquitinated proteins revealed that protein ubiquitination was involved in multiple biological processes, cellular components, and molecule functions. The randomly selected ubiquitinated 14-3-3 zeta/delta protein was further analyzed with Western blot, and it was found that upregulated 14-3-3 zeta/delta protein in nonfunctional PAs might be derived from the significantly decreased level of its ubiquitination compared to control pituitaries, which indicated a contribution of 14-3-3 zeta/delta protein to pituitary tumorigenesis. These findings provided the first ubiquitinated proteomic profiling and ubiquitination-involved signaling pathway networks in human PAs. This study offers new scientific evidence and basic data to elucidate the biological functions of ubiquitination in PAs, insights into its novel molecular mechanisms of pituitary tumorigenesis, and discovery of novel biomarkers and therapeutic targets for effective treatment of PAs.

Acetaldehyde dissociates the PTP1B-E-cadherin-beta-catenin complex in Caco-2 cell monolayers by a phosphorylation-dependent mechanism.

Interactions between E-cadherin, beta-catenin and PTP1B (protein tyrosine phosphatase 1B) are crucial for the organization of AJs (adherens junctions) and epithelial cell-cell adhesion. In the present study, the effect of acetaldehyde on the AJs and on the interactions between E-cadherin, beta-catenin and PTP1B was determined in Caco-2 cell monolayers. Treatment of cell monolayers with acetaldehyde induced redistribution of E-cadherin and beta-catenin from the intercellular junctions by a tyrosine phosphorylation-dependent mechanism. The PTPase activity associated with E-cadherin and beta-catenin was significantly reduced and the interaction of PTP1B with E-cadherin and beta-catenin was attenuated by acetaldehyde. Acetaldehyde treatment resulted in phosphorylation of beta-catenin on tyrosine residues, and abolished the interaction of beta-catenin with E-cadherin by a tyrosine kinase-dependent mechanism. Protein binding studies showed that the treatment of cells with acetaldehyde reduced the binding of beta-catenin to the C-terminal region of E-cadherin. Pairwise binding studies using purified proteins indicated that the direct interaction between E-cadherin and beta-catenin was reduced by tyrosine phosphorylation of beta-catenin, but was unaffected by tyrosine phosphorylation of E-cadherin-C. Treatment of cells with acetaldehyde also reduced the binding of E-cadherin to GST (glutathione S-transferase)-PTP1B. The pairwise binding study showed that GST-E-cadherin-C binds to recombinant PTP1B, but this binding was significantly reduced by tyrosine phosphorylation of E-cadherin. Acetaldehyde increased the phosphorylation of beta-catenin on Tyr-331, Tyr-333, Tyr-654 and Tyr-670. These results show that acetaldehyde induces disruption of interactions between E-cadherin, beta-catenin and PTP1B by a phosphorylation-dependent mechanism.

Prolactin Variants in Human Pituitaries and Pituitary Adenomas Identified With Two-Dimensional Gel Electrophoresis and Mass Spectrometry.

Human prolactin (hPRL) plays multiple roles in growth, metabolism, development, reproduction, and immunoregulation, which is an important protein synthesized in a pituitary. Two-dimensional gel electrophoresis (2DE) is an effective method in identity of protein variants for in-depth insight into functions of that protein. 2DE, 2DE-based PRL-immunoblot, mass spectrometry, and bioinformatics were used to analyze hPRL variants in human normal (control; n = 8) pituitaries and in five subtypes of pituitary adenomas [NF- (n = 3)-, FSH+ (n = 3)-, LH+ (n = 3)-, FSH+/LH+ (n = 3)-, and PRL+ (n = 3)-adenomas]. Six hPRL variants were identified with different isoelectric point (pI)-relative molecular mass (Mr ) distribution on a 2DE pattern, including variants V1 (pI 6.1; 26.0 kDa), V2 (pI 6.3; 26.4 kDa), V3 (pI 6.3; 27.9 kDa), V4 (pI 6.5; 26.1 kDa), V5 (pI 6.8; 25.9 kDa), and V6 (pI 6.7; 25.9 kDa). Compared to controls, except for variants V2-V6 in PRL-adenomas, V2 in FSH+-adenomas, and V3 in NF--adenomas, the other PRL variants were significantly downregulated in each subtype of pituitary adenomas. Moreover, the pattern of those six PRL variants was significantly different among five subtypes of pituitary adenomas relative to control pituitaries. Different hPRL variants might be involved in different types of PRL receptor-signaling pathways in a given condition. Those findings clearly revealed the existence of six hPRL variants in human pituitaries, and the pattern changes of six hPRL variants among different subtypes of pituitary adenomas, which provide novel clues to further study the functions, and mechanisms of action, of hPRL in human pituitary and in PRL-related diseases, and the potential clinical value in pituitary adenomas.

Novel molecular signaling and classification of human clinically nonfunctional pituitary adenomas identified by gene expression profiling and proteomic analyses.

Pituitary adenomas comprise 10% of intracranial tumors and occur in about 20% of the population. They cause significant morbidity by compression of regional structures or the inappropriate expression of pituitary hormones. Their molecular pathogenesis is unclear, and the current classification of clinically nonfunctional tumors does not reflect any molecular distinctions between the subtypes. To further elucidate the molecular changes that contribute to the development of these tumors and reclassify them according to the molecular basis, we investigated 11 nonfunctional pituitary adenomas and eight normal pituitary glands, using 33 oligonucleotide GeneChip microarrays. We validated microarray results with the reverse transcription real-time quantitative PCR, using a larger number of nonfunctional adenomas. We also used proteomic analysis to examine protein expression in these nonfunctional adenomas. Microarray analysis identified significant increases in the expression of 115 genes and decreases in 169 genes, whereas proteomic analysis identified 21 up-regulated and 29 down-regulated proteins. We observed changes in expression of SFRP1, TLE2, PITX2, NOTCH3, and DLK1, suggesting that the developmental Wnt and Notch pathways are activated and important for the progression of nonfunctional pituitary adenomas. We further analyzed gene expression profiles of all nonfunctional pituitary subtypes to each other and identified genes that were affected uniquely in each subtype. These results show distinct gene and protein expression patterns in adenomas, provide new insight into the pathogenesis and molecular classification of nonfunctional pituitary adenomas, and suggest that therapeutic targeting of the Notch pathway could be effective for these tumors.