The Differential Translation Capabilities of the Human DHFR2 Gene Indicates a Developmental and Tissue-Specific Endogenous Protein of Low Abundance.

A functional role has been ascribed to the human dihydrofolate reductase 2 (DHFR2) gene based on the enzymatic activity of recombinant versions of the predicted translated protein. However, the in vivo function is still unclear. The high amino acid sequence identity (92%) between DHFR2 and its parental homolog, DHFR, makes analysis of the endogenous protein challenging. This paper describes a targeted mass spectrometry proteomics approach in several human cell lines and tissue types to identify DHFR2-specific peptides as evidence of its translation. We show definitive evidence that the DHFR2 activity in the mitochondria is in fact mediated by DHFR, and not DHFR2. Analysis of Ribo-seq data and an experimental assessment of ribosome association using a sucrose cushion showed that the two main Ensembl annotated mRNA isoforms of DHFR2, 201 and 202, are differentially associated with the ribosome. This indicates a functional role at both the RNA and protein level. However, we were unable to detect DHFR2 protein at a detectable level in most cell types examined despite various RNA isoforms of DHFR2 being relatively abundant. We did detect a DHFR2-specific peptide in embryonic heart, indicating that the protein may have a specific role during embryogenesis. We propose that the main functionality of the DHFR2 gene in adult cells is likely to arise at the RNA level.

Development of a fast and simple method for the isolation of superparamagnetic iron oxide nanoparticles protein corona from protein-rich matrices.

The adsorption of proteins onto the surface of nanoparticle (NP) leads to the formation of the so-called "protein corona" as consisting both loosely and tightly bound proteins. It is well established that the biological identity of NPs that may be acquired after exposure to a biological matrix is mostly provided by the components of the hard corona as the pristine surface is generally less accessible for binding. For that reason, the isolation and the characterisation of the NP-corona complexes and identification of the associated biomolecules can help in understanding its biological behaviour. Established methods for the isolation of the NP-HC complexes are time-demanding and can lead to different results based on the isolation method applied. Herein, we have developed a fast and simple method using ferromagnetic beads isolated from commercial MACS column and used for the isolation of superparamagnetic NP following exposure to different types of biological milieu. We first demonstrated the ability to easily isolate superparamagnetic iron oxide NPs (IONPs) from different concentrations of human blood plasma, and also tested the method on the corona isolation using more complex biological matrices, such as culture medium containing pulmonary mucus where the ordinary corona methods cannot be applied. Our developed method showed less than 20% difference in plasma corona composition when compared with centrifugation. It also showed effective isolation of NP-HC complexes from mucus-containing culture media upon comparing with centrifugation and MACS columns, which failed to wash out the unbound proteins. Our study was supported with a full characterisation profile including dynamic light scattering, nanoparticle tracking analysis, analytical disk centrifuge, and zeta potentials. The biomolecules/ proteins composing the HC were separated by vertical gel electrophoresis and subsequently analysed by liquid chromatography-tandem mass spectrometry. In addition to our achievements in comparing different isolation methods to separate IONPs with corona from human plasma, this is the first study that provides a complete characterisation profile of particle protein corona after exposure in vitro to pulmonary mucus-containing culture media.

Proteomic and Metabolomic Analysis of Bone Marrow and Plasma from Patients with Extramedullary Multiple Myeloma Identifies Distinct Protein and Metabolite Signatures.

Multiple myeloma (MM) is an incurable haematological malignancy of plasma cells in the bone marrow. In rare cases, an aggressive form of MM called extramedullary multiple myeloma (EMM) develops, where myeloma cells enter the bloodstream and colonise distal organs or soft tissues. This variant is associated with refractoriness to conventional therapies and a short overall survival. The molecular mechanisms associated with EMM are not yet fully understood. Here, we analysed the proteome of bone marrow mononuclear cells and blood plasma from eight patients (one serial sample) with EMM and eight patients without extramedullary spread. The patients with EMM had a significantly reduced overall survival with a median survival of 19 months. Label-free mass spectrometry revealed 225 proteins with a significant differential abundance between bone marrow mononuclear cells (BMNCs) isolated from patients with MM and EMM. This plasma proteomics analysis identified 22 proteins with a significant differential abundance. Three proteins, namely vascular cell adhesion molecule 1 (VCAM1), pigment epithelium derived factor (PEDF), and hepatocyte growth factor activator (HGFA), were verified as the promising markers of EMM, with the combined protein panel showing excellent accuracy in distinguishing EMM patients from MM patients. Metabolomic analysis revealed a distinct metabolite signature in EMM patient plasma compared to MM patient plasma. The results provide much needed insight into the phenotypic profile of EMM and in identifying promising plasma-derived markers of EMM that may inform novel drug development strategies.

Alteration in Levels of Specific miRNAs and Their Potential Protein Targets between Human Pancreatic Cancer Samples, Adjacent Normal Tissue, and Xenografts Derived from These Tumors.

Herein, we describe the global comparison of miRNAs in human pancreatic cancer tumors, adjacent normal tissue, and matched patient-derived xenograft models using microarray screening. RNA was extracted from seven tumor, five adjacent normal, and eight FI PDX tumor samples and analyzed by Affymetrix GeneChip miRNA 4.0 array. A transcriptome analysis console (TAC) was used to generate comparative lists of up- and downregulated miRNAs for the comparisons, tumor vs. normal and F1 PDX vs. tumor. Particular attention was paid to miRNAs that were changed in the same direction in both comparisons. We identified the involvement in pancreatic tumor tissue of several miRNAs, including miR4534, miR3154, and miR4742, not previously highlighted as being involved in this type of cancer. Investigation in the parallel mRNA and protein lists from the same samples allowed the elimination of proteins where altered expression correlated with corresponding mRNA levels and was thus less likely to be miRNA regulated. Using the remaining differential expression protein lists for proteins predicted to be targeted for differentially expressed miRNA on our list, we were able to tentatively ascribe specific protein changes to individual miRNA. Particularly interesting target proteins for miRs 615-3p, 2467-3p, 4742-5p, 509-5p, and 605-3p were identified. Prominent among the protein targets are enzymes involved in aldehyde metabolism and membrane transport and trafficking. These results may help to uncover vulnerabilities that could enable novel approaches to treating pancreatic cancer.

Label-Free Quantitative Proteomics Analysis of Adriamycin Selected Multidrug Resistant Human Lung Cancer Cells.

The development of drug resistance in lung cancer is a major clinical challenge, leading to a 5-year survival rate of only 18%. Therefore, unravelling the mechanisms of drug resistance and developing novel therapeutic strategies is of crucial importance. This study systematically explores the novel biomarkers of drug resistance using a lung cancer model (DLKP) with a series of drug-resistant variants. In-depth label-free quantitative mass spectrometry-based proteomics and gene ontology analysis shows that parental DLKP cells significantly differ from drug-resistant variants, and the cellular proteome changes even among the drug-resistant subpopulations. Overall, ABC transporter proteins and lipid metabolism were determined to play a significant role in the formation of drug resistance in DKLP cells. A series of membrane-related proteins such as HMOX1, TMB1, EPHX2 and NEU1 were identified to be correlated with levels of drug resistance in the DLKP subpopulations. The study also showed enrichment in biological processes and molecular functions such as drug metabolism, cellular response to the drug and drug binding. In gene ontology analysis, 18 proteins were determined to be positively or negatively correlated with resistance levels. Overall, 34 proteins which potentially have a therapeutic and diagnostic value were identified.

Nanoparticle Biomolecular Corona-Based Enrichment of Plasma Glycoproteins for N-Glycan Profiling and Application in Biomarker Discovery.

Biomolecular corona formation has emerged as a recurring and important phenomenon in nanomedicine that has been investigated for potential applications in disease diagnosis. In this study, we have combined the "personalized protein corona" with the N-glycosylation profiling that has recently gained considerable interest in human plasma biomarker discovery as a powerful early warning diagnostic and patient stratification tool. We envisioned that the protein corona formation could be exploited as an enrichment step that is critically important in both proteomic and proteoglycomic workflows. By using silica nanoparticles, plasma fibrinogen was enriched to a level in which its proteomic and glycomic "fingerprints" could be traced with confidence. Despite being a more simplified glycan profile compared to full plasma, the corona glycan profile revealed a fibrinogen-derived glycan peak that was found to potentially distinguish lung cancer patients from controls in a pilot study.

In vitro and in vivo modulation of NADPH oxidase activity and reactive oxygen species production in human neutrophils by α1-antitrypsin.

Oxidative stress from innate immune cells is a driving mechanism that underlies COPD pathogenesis. Individuals with α-1 antitrypsin (AAT) deficiency (AATD) have a dramatically increased risk of developing COPD. To understand this further, the aim of this study was to investigate whether AATD presents with altered neutrophil NADPH oxidase activation, due to the specific lack of plasma AAT. Experiments were performed using circulating neutrophils isolated from healthy controls and individuals with AATD. Superoxide anion (O2 -) production was determined from the rate of reduction of cytochrome c. Quantification of membrane NADPH oxidase subunits was performed by mass spectrometry and Western blot analysis. The clinical significance of our in vitro findings was assessed in patients with AATD and severe COPD receiving intravenous AAT replacement therapy. In vitro, AAT significantly inhibited O2 - production by stimulated neutrophils and suppressed receptor stimulation of cyclic adenosine monophosphate and extracellular signal-regulated kinase (ERK)1/2 phosphorylation. In addition, AAT reduced plasma membrane translocation of cytosolic phox components of the NADPH oxidase. Ex vivo, AATD neutrophils demonstrated increased plasma membrane-associated p67phox and p47phox and significantly increased O2 - production. The described variance in phox protein membrane assembly was resolved post-AAT augmentation therapy in vivo, the effects of which significantly reduced AATD neutrophil O2 - production to that of healthy control cells. These results expand our knowledge on the mechanism of neutrophil-driven airways disease associated with AATD. Therapeutic AAT augmentation modified neutrophil NADPH oxidase assembly and reactive oxygen species production, with implications for clinical use in conditions in which oxidative stress plays a pathogenic role.

Copper toxicity of inflection point in human intestinal cell line Caco-2 dissected: influence of temporal expression patterns.

We previously described a non-monotonic dose response curve at low copper concentrations where 3.125 µM CuSO4 (the early inflection point) was more toxic than 25 µM CuSO4 in Caco-2 cells. We employed global proteomics to investigate this observation. The altered expression levels of a small number of proteins displaying a temporal response may provide the best indication of the underlying mechanism; more well-known copper response proteins including the metal binding metallothioneins (MT1X, MT1F, MT2A) and antioxidant response proteins including Heme oxygenase were upregulated to a similar level in both copper concentrations and so are less likely to underpin this phenomenon.The temporal response proteins include Granulins, AN1-type zinc finger protein 2A (ZFAND2A), and the heat shock proteins (HSPA6 and HSPA1B). Granulins were decreased after 4 h only in 25 µM CuSO4 but from 24 h, were decreased in both copper concentrations to a similar level. Induction of ZFAND2A and increases in HSPA6 and HSPA1B were observed at 24 h only in 25 µM CuSO4 but were present at 48 h in both copper conditions. The early expression of ZFAND2A, HSPs, and higher levels of α-crystallin B (CRYAB) correlated with lower levels of misfolded proteins in 25 µM CuSO4 compared to 3.125 µM CuSO4 at 48 h. These results suggest that 3.125 µM CuSO4 at early time points was unable to activate the plethora of stress responses invoked by the higher copper concentration, paradoxically exposing the Caco-2 cells to higher levels of misfolded proteins and greater proteotoxic stress.

Proteomic Analysis of Cell Lines and Primary Tumors in Pancreatic Cancer Identifies Proteins Expressed Only In Vitro and Only In Vivo.

OBJECTIVES: A limited repertoire of good pancreatic ductal adenocarcinoma (PDAC) models is one of the main barriers in developing effective new PDAC treatments. We aimed to characterize 6 commonly used PDAC cell lines and compare them with PDAC patient tumor samples using proteomics. METHODS: Proteomic methods were used to generate an extensive catalog of proteins from 10 PDAC surgical specimens, 9 biopsies of adjacent normal tissue, and 6 PDAC cell lines. Protein lists were interrogated to determine what extent the proteome of the cell lines reflects the proteome of primary pancreatic tumors. RESULTS: We identified 7973 proteins from the cell lines, 5680 proteins from the tumor tissues, and 4943 proteins from the adjacent normal tissues. We identified 324 proteins unique to the cell lines, some of which may play a role in survival of cells in culture. Conversely, a list of 63 proteins expressed only in the patient samples, whose expression is lost in culture, may place limitations on the degree to which these model systems reflect tumor biology in vivo. CONCLUSIONS: Our work offers a catalog of proteins detected in each of the PDAC cell lines, providing a useful guide for researchers seeking model systems for PDAC functional studies.

Proteomic analysis of pancreatic ductal adenocarcinoma.

INTRODUCTION: Pancreatic ductal adenocarcinoma (PDAC), which represents approximately 80% of all pancreatic cancers, is a highly aggressive malignant disease and one of the most lethal among all cancers. Overall, the 5-year survival rate among all pancreatic cancer patients is less than 9%; these rates have shown little change over the past 30 years. A more comprehensive understanding of the molecular mechanisms underlying this complex disease is crucial to the development of new diagnostic tools for early detection and disease monitoring, as well as to identify new and more effective therapeutics to improve patient outcomes. AREA COVERED: We summarize recent advances in proteomic strategies and mass spectrometry to identify new biomarkers for early detection and monitoring of disease progression, predict response to therapy, and to identify novel proteins that have the potential to be 'druggable' therapeutic targets. An overview of proteomic studies that have been conducted to further our mechanistic understanding of metastasis and chemotherapy resistance in PDAC disease progression will also be discussed. EXPERT COMMENTARY: The results from these PDAC proteomic studies on a variety of PDAC sample types (e.g., blood, tissue, cell lines, exosomes, etc.) provide great promise of having a significant clinical impact and improving patient outcomes.

Characterisation and proteomic profiling of continuously exposed Cu-resistant variants of the Caco-2 cell line.

Studies in hepatic systems identify multiple factors involved in the generation of copper resistance. As the intestine is the route of exposure to dietary copper, we wanted to understand how intestinal cells overcome the toxic effects of high copper and what mechanisms of resistance develop. Using the intestinal cell line Caco-2, resistance was developed by serial subculture in 50 µM copper in inorganic (CuSO4) or organic (Cu proteinate) forms. Caco-2 variants exhibited resistance to copper and retained the non-monotonic dose response while displaying stable phenotypes following repeated subculture in the absence of copper. Phenotypic changes on exposure to copper in parental Caco-2 cells included significantly increased total protein yield, ROS, SOD, metallothionein expression, GSH and total glutathione. These phenotypic changes were not replicated in resistant variants on a per cell basis. Quantitative label-free LC-MS/MS proteomic analysis identified 1113 differentially expressed proteins (DEPs) between parental Caco-2 and resistant cells. With some exceptions, most of the DEPs were overexpressed to a low level around 2-fold suggesting resistance was supported by multiple small changes in protein expression. These variants may be a useful tool in studying the toxicity of stress responses in further Cu-related studies.

PRDX3 is associated with metastasis and poor survival in uveal melanoma.

AIMS: Uveal melanoma (UM) is the most common primary intraocular malignancy in adults, and 40% develop fatal metastatic disease. Overexpression of thioredoxin-dependent peroxidase reductase (PRDX3) has been implicated in several cancers, including prostate, breast, colorectal and lung cancer. The aim of this study was to compare the immunohistochemical expression of PRDX3 in formalin-fixed, paraffin-embedded (FFPE) primary UM tissues of patients who did and did not develop metastatic disease. METHODS: Immunohistochemical staining of PRDX3 was performed on FFPE tissue microarray samples of 92 primary UM tumours from patients who did and did not develop metastatic disease. The immunohistochemical staining was assessed by two observers who were blinded to all clinicopathological and cytogenetic details including metastatic/non-metastatic information. Based on a scoring system, expression of PRDX3 was graded as high or low. RESULTS: There were 55 tumours (59.8%) from patients who developed metastatic disease, while 37 (40.2%) were from patients who did not develop metastasis. A statistically significant difference in PRDX3 expression was observed in patients who did and did not develop metastasis (p=0.001). A significant positive correlation between high PRDX3 expression and metastasis was observed (p=0.001). A significant negative correlation between PRDX3 expression and survival was found (p=0.005). Kaplan-Meier survival analysis showed a statistically significant difference in overall survival between tumours that demonstrated low and high expression of PRDX3 (67.61 vs 130.64 months, respectively, p=0.013). CONCLUSIONS: High immunohistochemical expression of PRDX3 in primary UM tissue is associated with metastasis and poor survival.

Increased growth rate and productivity following stable depletion of miR-7 in a mAb producing CHO cell line causes an increase in proteins associated with the Akt pathway and ribosome biogenesis.

Cell line engineering using microRNAs represents a desirable route for improving the efficiency of recombinant protein production by CHO cells. In this study we generated stable CHO DP12 cells expressing a miR-7 sponge transcript which sequesters miR-7 from its endogenous targets. Depletion of miR-7 results in a 65% increase in cell growth and >3-fold increase in yield of secreted IgG protein. Quantitative labelfree LC-MS/MS proteomic profiling was carried out to identify the targets of miR-7 and understand the functional drivers of the improved CHO cell phenotypes. Subcellular enrichment and total proteome analysis identified more than 3000 proteins per fraction resulting in over 5000 unique proteins identified per timepoint analysed. Early stage culture analysis identified 117 proteins overexpressed in miR-7 depleted cells. A subset of these proteins are involved in the Akt pathway which could be the underlying route for cell density improvement and may be exploited more specifically in the future. Late stage culture identified 160 proteins overexpressed in miR-7 depleted cells with some of these involved in ribosome biogenesis which may be causing the increased productivity through improved translational efficiency. This is the first in-depth proteomic profiling of the IgG producing CHO DP12 cell line stably depleted of miR-7. SIGNIFICANCE: Chinese hamster ovary (CHO) cells are the mammalian cell expression system of choice for production of recombinant therapeutic proteins. There is much research ongoing to characterise CHO cell factories through the application of systems biology approaches that will enable a fundamental understanding of CHO cell physiology, and as a result, a better knowledge and understanding of recombinant protein production. This study profiles the proteomic effects of microRNA-7 depletion on the IgG producing CHO DP12 cell line. This is one of the very few studies that attempts to identify the functioning proteins driving improved CHO cell phenotypes resulting from microRNA manipulation. Using subcellular enrichment and total proteome analysis we identified over 5000 unique proteins in miR-7 depleted CHO cells. This work has identified a cohort of proteins involved in the Akt pathway and ribosome biogenesis. These proteins may drive improved CHO cell phenotypes and are of great interest for future work.

Depletion of endogenous miRNA-378-3p increases peak cell density of CHO DP12 cells and is correlated with elevated levels of ubiquitin carboxyl-terminal hydrolase 14.

miRNAs are potent molecular regulators of cellular behaviour. The manipulation of these small non-coding RNAs has been used to enhance industrially relevant phenotypes in Chinese Hamster Ovary (CHO) cells. We investigated the stable depletion of six miRNAs; miR-204-5p, 338-3p, 378-3p, 409-3p, 455-3p and 505-3p, robustly associated with cell growth rate from a previous profiling study. Inhibition of endogenous miR-378-3p function by miRNA-sponge-decoy improved peak cell density by 59%. Quantitative label free LC-MS/MS proteomic analysis of the fractionated cell cultures at day 4 and 8 of batch culture found 216 cytosolic and 114 membrane-associated proteins differentially expressed with stable miR-378-3p depletion. qRT-PCR of 8 genes; Clic4, Hnrnpa1, Prdx1, Actn4, Usp14, Srxn1, Canx and Gnb1, with unidirectional differential protein expression over the two time points of analysis was carried out. In-silico predictive algorithms; TargetScan and miRDB, were used to decipher possible direct targets of miR-378-3p. The Ubiquitin carboxyl-terminal hydrolase 14 (Usp14) protein was identified in the cytosolic fractions at both timepoints as differentially expressed with an increased abundance of 1.58-fold in the miR-378-3p depleted cells on day 8. Usp14 is a deubiquitinase (DUB) with previous reports of its up-regulation leading to increased proliferation of cancer cells. Overexpression of Usp14 in CHO cells had significant effects on cell growth supporting a role for Usp14 in the increased peak cell density seen with miR-378-3p depletion. This study highlights miR-378-3p as a novel engineering candidate for improving CHO cell growth. The use of subcellular fractionation also improved proteome coverage in the identification of novel miRNA targets.

A Comparative Quantitative LC-MS/MS Profiling Analysis of Human Pancreatic Adenocarcinoma, Adjacent-Normal Tissue, and Patient-Derived Tumour Xenografts.

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers worldwide; it develops in a relatively symptom-free manner, leading to rapid disease progression and metastasis, leading to a 5-year survival rate of less than 5%. A lack of dependable diagnostic markers and rapid development of resistance to conventional therapies are among the problems associated with management of the disease. A better understanding of pancreatic tumour biology and discovery of new potential therapeutic targets are important goals in pancreatic cancer research. This study describes the comparative quantitative LC-MS/MS proteomic analysis of the membrane-enriched proteome of 10 human pancreatic ductal adenocarcinomas, 9 matched adjacent-normal pancreas and patient-derived xenografts (PDXs) in mice (10 at F1 generation and 10 F2). Quantitative label-free LC-MS/MS data analysis identified 129 proteins upregulated, and 109 downregulated, in PDAC, compared to adjacent-normal tissue. In this study, analysing peptide MS/MS data from the xenografts, great care was taken to distinguish species-specific peptides definitively derived from human sequences, or from mice, which could not be distinguished. The human-only peptides from the PDXs are of particular value, since only human tumour cells survive, and stromal cells are replaced during engraftment in the mouse; this list is, therefore, enriched in tumour-associated proteins, some of which might be potential therapeutic or diagnostic targets. Using human-specific sequences, 32 proteins were found to be upregulated, and 113 downregulated in PDX F1 tumours, compared to primary PDAC. Differential expression of CD55 between PDAC and normal pancreas, and expression across PDX generations, was confirmed by Western blotting. These data indicate the value of using PDX models in PDAC research. This study is the first comparative proteomic analysis of PDAC which employs PDX models to identify patient tumour cell-associated proteins, in an effort to find robust targets for therapeutic treatment of PDAC.

Neutrophil Membrane Cholesterol Content is a Key Factor in Cystic Fibrosis Lung Disease.

BACKGROUND: Identification of mechanisms promoting neutrophil trafficking to the lungs of patients with cystic fibrosis (CF) is a challenge for next generation therapeutics. Cholesterol, a structural component of neutrophil plasma membranes influences cell adhesion, a key step in transmigration. The effect of chronic inflammation on neutrophil membrane cholesterol content in patients with CF (PWCF) remains unclear. To address this we examined neutrophils of PWCF to evaluate the cause and consequence of altered membrane cholesterol and identified the effects of lung transplantation and ion channel potentiator therapy on the cellular mechanisms responsible for perturbed membrane cholesterol and increased cell adhesion. METHODOLOGY: PWCF homozygous for the ΔF508 mutation or heterozygous for the G551D mutation were recruited (n=48). Membrane protein expression was investigated by mass spectrometry. The effect of lung transplantation or ivacaftor therapy was assessed by ELISAs, and calcium fluorometric and µ-calpain assays. FINDINGS: Membranes of CF neutrophils contain less cholesterol, yet increased integrin CD11b expression, and respond to inflammatory induced endoplasmic reticulum (ER) stress by activating µ-calpain. In vivo and in vitro, increased µ-calpain activity resulted in proteolysis of the membrane cholesterol trafficking protein caveolin-1. The critical role of caveolin-1 for adequate membrane cholesterol content was confirmed in caveolin-1 knock-out mice. Lung transplant therapy or treatment of PWCF with ivacaftor, reduced levels of circulating inflammatory mediators and actuated increased caveolin-1 and membrane cholesterol, with concurrent normalized neutrophil adhesion. INTERPRETATION: Results demonstrate an auxiliary benefit of lung transplant and potentiator therapy, evident by a reduction in circulating inflammation and controlled neutrophil adhesion.

Novel panel of protein biomarkers to predict response to bortezomib-containing induction regimens in multiple myeloma patients.

BACKGROUND: Multiple myeloma (MM) is a complex heterogeneous disease. Various risk stratification models have been recommended including cytogenetic and FISH analysis to identify high-risk patients who may benefit from novel treatments, but such facilities are not widely available. The International Scoring System (ISS) using beta-2-microglobulin and albumin remains a widely used prognostic scoring system in many clinical practices; however it is not useful in predicting response to treatment in MM. The aim of this study is to identify clinically useful biomarkers to predict response to treatment containing bortezomib. METHODS: 17 MM patient serum samples (9 responders/8 non-responders) were used for the discovery phase (label-free mass spectrometry) and an additional 20 MM patient serum samples were used for the ELISA-based validation phase (14 responders/6 non-responders). RESULTS: CLU and ANG mean levels were higher in the responders group, while Complement C1q had lower concentrations. The combination of all standard biomarkers (albumin, beta-2-microglobulin (ß2M), paraprotein and kappa/lambda (K/L) ratio) had an AUC value of 0.71 with 65% correct classification, while an overall combination of new candidate protein biomarkers with standard biomarkers had an AUC value of 0.89 with 85.3% correct classification. CONCLUSIONS: A combination of new and standard biomarkers consisting of CLU, ANG, C1Q, albumin, ß2M, paraprotein and K/L ratio may have potential as a novel panel of biomarkers to predict MM response to treatment containing bortezomib. GENERAL SIGNIFICANCE: Use of this biomarker panel could facilitate a more personalized therapy approach and to minimize unnecessary side effects from ineffective drugs.

Identification of the metabolic alterations associated with the multidrug resistant phenotype in cancer and their intercellular transfer mediated by extracellular vesicles.

Multidrug resistance (MDR) is a serious obstacle to efficient cancer treatment. Overexpression of P-glycoprotein (P-gp) plays a significant role in MDR. Recent studies proved that targeting cellular metabolism could sensitize MDR cells. In addition, metabolic alterations could affect the extracellular vesicles (EVs) cargo and release. This study aimed to: i) identify metabolic alterations in P-gp overexpressing cells that could be involved in the development of MDR and, ii) identify a potential role for the EVs in the acquisition of the MDR. Two different pairs of MDR and their drug-sensitive counterpart cancer cell lines were used. Our results showed that MDR (P-gp overexpressing) cells have a different metabolic profile from their drug-sensitive counterparts, demonstrating decreases in the pentose phosphate pathway and oxidative phosphorylation rate; increases in glutathione metabolism and glycolysis; and alterations in the methionine/S-adenosylmethionine pathway. Remarkably, EVs from MDR cells were capable of stimulating a metabolic switch in the drug-sensitive cancer cells, towards a MDR phenotype. In conclusion, obtained results contribute to the growing knowledge about metabolic alterations in MDR cells and the role of EVs in the intercellular transfer of MDR. The specific metabolic alterations identified in this study may be further developed as targets for overcoming MDR.

Clinical Proteomics: Liquid Chromatography-Mass Spectrometry (LC-MS) Purification Systems.

Liquid chromatography-mass spectrometry (LC-MS) has become a routine powerful technology in clinical proteomic studies for protein identification, protein characterization and the discovery of biomarkers. In this chapter, we describe two protocol methods to analyze clinical patient samples using a resin based depletion column followed by either protein In-gel enzymatic digestion or protein in-solution enzymatic digestion and then analysis by one-dimensional reverse-phase chromatography or two-dimensional strong cation exchange (SCX)-reverse-phase chromatography (RPC).