A Fast-Tracking Sample Preparation Protocol for Proteomics of Formalin-Fixed Paraffin-Embedded Tumor Tissues.

Archived tumor specimens are routinely preserved by formalin fixation and paraffin embedding. Despite the conventional wisdom that proteomics might be ineffective due to the cross-linking and pre-analytical variables, these samples have utility for both discovery and targeted proteomics. Building on this capability, proteomics approaches can be used to maximize our understanding of cancer biology and clinical relevance by studying preserved tumor tissues annotated with the patients' medical histories.

Effects of multiple stressors on pancreatic human islets proteome reveal new insights into the pathways involved.

Pancreatic β-cell dysfunction is an early hallmark of type 1 diabetes mellitus. Among the potentially critical factors that cause β-cell dysfunction are cytokine attack, glucotoxicity, induction of endoplasmic reticulum (ER) or mitochondria stress. However, the exact molecular mechanism underlying β-cell's inability to maintain glucose homeostasis under severe stresses is unknown.

Deciphering Phenotypes from Protein Biomarkers for Translational Research with PIPER.

Liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM) has widespread clinical use for detection of inborn errors of metabolism, therapeutic drug monitoring, and numerous other applications. This technique detects proteolytic peptides as surrogates for protein biomarker expression, mutation, and post-translational modification in individual clinical assays and in cancer research with highly multiplexed quantitation across biological pathways. LC-MRM for protein biomarkers must be translated from multiplexed research-grade panels to clinical use.

HLA Allele-Specific Quantitative Profiling of Type 1 Diabetic B Lymphocyte Immunopeptidome.

Peptide ligands presented by human leukocyte antigen (HLA) molecules on the cell surface represent the immunopeptidome that could be utilized for identification of antigenic peptides for immunotherapy and prevention of autoimmune diseases. Although T-cells are well-known key players in the destruction of pancreatic beta-cells in type 1 diabetes (T1D), increasing evidence points toward a role for B-cells in disease pathogenesis. However, as antigen presenting cells, little is known about the comprehensive immunopeptidome of B cells and their changes in the context of T1D.

Pancreatic Tissue Proteomics Unveils Key Proteins, Pathways, and Networks Associated with Type 1 Diabetes.

Purpose: Type 1 diabetes (T1D) is characterized by autoimmune mediated self-destruction of the pancreatic islet beta cells and the resultant insulin deficiency. However, little is known about the underlying molecular pathogenesis at the pancreatic tissue level given the limited availability of clinical specimens.

Experimental Design: Quantitative proteomic studies is performed on age-matched T1D and healthy cadaveric pancreatic tissues (n = 18 each) using TMT 10plex-based isobaric labeling and BoxCar-based label-free LC-MS/MS approaches.

Mitochondrial lipid droplet formation as a detoxification mechanism to sequester and degrade excessive urothelial membranes.

The apical surface of the terminally differentiated mammalian urothelial umbrella cell is mechanically stable and highly impermeable, in part due to its coverage by urothelial plaques consisting of 2D crystals of uroplakin particles. The mechanism for regulating the uroplakin/plaque level is unclear. We found that genetic ablation of the highly tissue-specific sorting nexin Snx31, which localizes to plaques lining the multivesicular bodies (MVBs) in urothelial umbrella cells, abolishes MVBs suggesting that Snx31 plays a role in stabilizing the MVB-associated plaques by allowing them to achieve a greater curvature.

Engineered nanoparticles for imaging and drug delivery in colorectal cancer.

Colorectal cancer (CRC) is one of the deadliest diseases worldwide due to a lack of early detection methods and appropriate drug delivery strategies. Conventional imaging techniques cannot accurately distinguish benign from malignant tissue, leading to frequent misdiagnosis or diagnosis at late stages of the disease. Novel screening tools with improved accuracy and diagnostic precision are thus required to reduce the mortality burden of this malignancy.

Integrative omics connects N-glycoproteome-wide alterations with pathways and regulatory events in induced pluripotent stem cells.

Molecular-level differences ranging from genomes to proteomes, but not N-glycoproteomes, between human induced pluripotent stem cells (hiPSCs) and embryonic stem cells (hESCs) have been assessed to gain insights into cell reprogramming and induced pluripotency. Our multiplexed quantitative N-glycoproteomics study identified altered N-glycoproteins that significantly regulate cell adhesion processes in hiPSCs compared to hESCs. The integrative proteomics and functional network analyses of the altered N-glycoproteins revealed their significant interactions with known PluriNet (pluripotency-associated network) proteins.

Proteomics-Based Analysis of Protein Complexes in Pluripotent Stem Cells and Cancer Biology.

A protein complex consists of two or more proteins that are linked together through protein-protein interactions. The proteins show stable/transient and direct/indirect interactions within the protein complex or between the protein complexes. Protein complexes are involved in regulation of most of the cellular processes and molecular functions.

Interleukin-25 Mediates Transcriptional Control of PD-L1 via STAT3 in Multipotent Human Mesenchymal Stromal Cells (hMSCs) to Suppress Th17 Responses.

Multipotent human mesenchymal stromal cells (hMSCs) harbor immunomodulatory properties that are therapeutically relevant. One of the most clinically important populations of leukocytes is the interleukin-17A (IL-17A)-secreting T (Th17) lymphocytes. However, mechanisms of hMSC and Th17 cell interactions are incompletely resolved.

Loss of PTPRM associates with the pathogenic development of colorectal adenoma-carcinoma sequence.

Identification and functional analysis of genes from genetically altered chromosomal regions would suggest new molecular targets for cancer diagnosis and treatment. Here we performed a genome-wide analysis of chromosomal copy number alterations (CNAs) in matching sets of colon mucosa-adenoma-carcinoma samples using high-throughput oligonucleotide microarray analysis. In silico analysis of NCBI GEO and TCGA datasets allowed us to uncover the significantly altered genes (p ≤ 0.

Functional phosphoproteomics of oncogenic KRAS signaling.

Identification of oncogene-mediated phosphorylation events is essential to understanding the molecular determinants responsible for cancer development and progression. Here, we identify KRAS-regulated phosphorylation events using label-free quantitation-based comparative phosphoproteomics analyses of immortalized human bronchial epithelial cells that express oncogenic KRAS as well as cells that do not. Further, we demonstrate integration of the identified phosphorylation events with the Pathway Interaction Database to infer KRAS-regulated pathways, which may have implications in KRAS-associated lung adenocarcinoma development.

Quantitative proteomics of protein complexes and their implications for cell reprograming and pluripotency.

Pluripotency of embryonic stem cells (ESCs)/induced pluripotent stem cells (iPSCs) and reprograming of somatic cells (SCs) to pluripotency are governed by known and unknown factors. These factors, including protein complexes, are poorly described at the proteome level. Here, we established the quantitative proteomic profiles across three types of cells (iPSCs, ESCs, and SCs) using OFFGEL fractionation coupled with LTQ-Orbitrp analysis.

Label-free quantitative proteomics and N-glycoproteomics analysis of KRAS-activated human bronchial epithelial cells.

Mutational activation of KRAS promotes various malignancies, including lung adenocarcinoma. Knowledge of the molecular targets mediating the downstream effects of activated KRAS is limited. Here, we provide the KRAS target proteins and N-glycoproteins using human bronchial epithelial cells with and without the expression of activated KRAS (KRAS(V12)).

Phosphoproteomics identifies oncogenic Ras signaling targets and their involvement in lung adenocarcinomas.

Background: Ras is frequently mutated in a variety of human cancers, including lung cancer, leading to constitutive activation of MAPK signaling. Despite decades of research focused on the Ras oncogene, Ras-targeted phosphorylation events and signaling pathways have not been described on a proteome-wide scale.

Methodology/principal Findings: By functional phosphoproteomics, we studied the molecular mechanics of oncogenic Ras signaling using a pathway-based approach.

IDEAL-Q, an automated tool for label-free quantitation analysis using an efficient peptide alignment approach and spectral data validation.

In this study, we present a fully automated tool, called IDEAL-Q, for label-free quantitation analysis. It accepts raw data in the standard mzXML format as well as search results from major search engines, including Mascot, SEQUEST, and X!Tandem, as input data. To quantify as many identified peptides as possible, IDEAL-Q uses an efficient algorithm to predict the elution time of a peptide unidentified in a specific LC-MS/MS run but identified in other runs.

Single drop microextraction using silver nanoparticles as electrostatic probes for peptide analysis in atmospheric pressure matrix-assisted laser desorption/ionization mass spectrometry and comparison with gold electrostatic probes and silver hydrophobic probes.

Single drop microextraction using tetraalkylammonium bromide coated silver nanoparticles (SDME-AgNPs) prepared in toluene has been successfully applied as electrostatic affinity probes to preconcentrate peptide mixtures in biological samples prior to atmospheric pressure matrix-assisted laser desorption/ionization ion trap mass spectrometry (AP-MALDI-MS) analysis. This approach is based on the isoelectric point (pI) of peptides and surface charge of AgNPs. Using the SDME-AgNPs technique, from a peptide mixture, Met- and Leu-enkephalins (Met-enk and Leu-enk) were extracted into a droplet of toluene containing AgNPs, but not the neutral peptides (gramicidins).

CD44 engagement promotes matrix-derived survival through the CD44-SRC-integrin axis in lipid rafts.

CD44 is present in detergent-resistant, cholesterol-rich microdomains, called lipid rafts, in many types of cells. However, the functional significance of CD44 in lipid rafts is still unknown. We have previously demonstrated that osteopontin-mediated engagement of CD44 spliced variant isoforms promotes an extracellular matrix-derived survival signal through integrin activation.

Osteopontin promotes integrin activation through outside-in and inside-out mechanisms: OPN-CD44V interaction enhances survival in gastrointestinal cancer cells.

Osteopontin (OPN) and splice variants of CD44 (CD44(V)) have independently been identified as markers for tumor progression. In this study, we show that both OPN and CD44(V) are frequently overexpressed in human gastric cancer and that OPN-engaged CD44(V) ligation confers cells an increased survival mediated through integrin activation. First, we show that OPN treatment confers cells an increased resistance to UV-induced apoptosis.

Identification of peptides using gold nanoparticle-assisted single-drop microextraction coupled with AP-MALDI mass spectrometry.

A novel technique, gold nanoparticle-assisted single-drop microextraction (SDME) combined with atmospheric pressure matrix-assisted laser desorption/ionization mass spectrometry (AP-MALDI-MS) for the identification of peptides has been described. The SDME of peptides from aqueous solution was achieved using gold nanoparticles prepared in toluene as the acceptor phase. A simple phenomenon of isoelectric point (pI) of the peptides has been utilized successfully to extract the peptides into a single drop of nanogold in toluene.

The effects of salt stress on photosynthetic electron transport and thylakoid membrane proteins in the cyanobacterium Spirulina platensis.

The response of Spirulina (Arthrospira) platensis to high salt stress was investigated by incubating the cells in light of moderate intensity in the presence of 0.8 M NaCl. NaCl caused a decrease in photosystem II (PSII) mediated oxygen evolution activity and increase in photosystem I (PSI) activity and the amount of P700.

An application of electrospray ionization tandem mass spectrometry to probe the interaction of Ca2+/Mg2+/Zn2+ and Cl- with gramicidin A.

The peptide, gramicidin A (GrA), has been demonstrated to interact with divalent salts (CaCl2, MgCl2, and ZnCl2) using electrospray ionization mass spectrometry (ESI-MS). The ESI-MS analysis revealed different complexes formed due to the interaction of Val-GrA and Ile-GrA with divalent salts: [Val or Ile-GrA-H+M]+, [Val or Ile-GrA+MCl]+ and [Val or Ile-GrA+M]2+, where M is Ca or Mg or Zn. All these complexes have been subjected to collisionally activated dissociation (CAD).

Probing the interaction of kojic acid antibiotics with iron(III) chloride by using electrospray tandem mass spectrometry.

Kojic acid, 5-hydroxy-2-(hydroxymethyl)-4H-pyran-4-one, has been used extensively as a clinical iron-chelating drug although the nature of the complexes of iron and kojic acid has not been established. In this article we demonstrate the complexation of kojic acid with iron(III) chloride by using electrospray ionization mass spectrometry (ESI-MS). The ESI-MS analysis revealed different reactions between iron(III) chloride and kojic acid (M), and the mass spectrum exhibited four complexes: [Fe+2(M-H)]+, [Fe+3(M-H)+H]+, [Fe2+4(M-H)+Cl]+, and [Fe2+5(M-H)]+.