Quantitative Proteomics Strategy Research Articles

Proteomics reveals reduced expression of transketolase in pyrimidine 5'-nucleotidase deficient patients.

To date, it remains a challenge to correctly and timely diagnose red blood cell (RBC) enzymopathies that result in hereditary nonspherocytic hemolytic anemia (HNSHA), the third most common of which is pyrimidine 5'-nucleotidase (P5N) deficiency with just over 100 cases recognized and confirmed worldwide. We have investigated the RBC proteome of a patient with P5N deficiency due to a homozygous frameshift mutation in the NT5C3A gene. Protein expression levels were analyzed against healthy controls and against patients with hemolytic anemia of different origin, to account for the patient's elevated reticulocyte versus RBC ratio. Stringent relative quantification of the patient's protein levels revealed reduced levels of P5N, and unexpectedly, also decreased levels of transketolase, an enzyme involved in the nonoxidative phase of the pentose phosphate pathway, one of the few key pathways active in RBCs. Immunoblotting of whole blood samples from this and other P5N-deficient patients with dissimilar mutations indicated that P5N deficiency was correlated with reduced transketolase levels. Consequently, insight into patient RBC proteomes illustrates potential benefit of coupling quantitative proteomics strategies with routine HNSHA diagnostic procedures. Proteomics facilitates finding novel biomarkers for HNSHA patients, for example, suffering from P5N deficiency, providing new prospects for future diagnosis and therapy.

Abstract 14: A switch in metabolic phenotype in vemurafenib-resistant melanoma cells to increased amino acid dependence serves as an alternate mechanism for survival

Abstract Despite considerable progress in the understanding of melanoma, and an increase in overall survival, resistance ultimately develops in most patients treated with BRAF inhibitors. To study the molecular mechanism(s) involved in acquired resistance, we generated a Vemurafenib-resistant A2-1b cell line in the BRAFV600E mutated SK-Mel28 melanoma cell background. Viability assays established IC50 of parental SK-Mel28 cells as 0.2 μM, while IC50>12 μM was noted for Vemurafenib-resistant A2-1b cells. Utilizing a quantitative proteomic strategy, profiling of A2-1b versus SK-Mel28 cells resulted in positive identification of 1720 proteins (≤1% FDR). Analysis of this data set identified a subset of 13 proteins, including Enolase 2, Triose-phosphate isomerase and Glyceraldehyde-3-phosphate dehydrogenase with a role in glycolysis, gluconeogenesis and NADH repair, to be differentially regulated in resistant cells. Additional studies validated significantly decreased Enolase 1 and 2 protein expression, suppressed glucose uptake and reduced lactic acid levels in resistant A2-1b cells. The pentose phosphate pathway, in contrast to citric acid cycle intermediates, was unaffected while the latter was significantly suppressed in resistant cells. The energy status of cells was similar as evidenced by equivalent total adenylate, guanylate, NADH, NADP+ and NADPH levels. Flux experiments measuring extracelluar acidification and oxygen consumption rates demonstrated that resistant A2-1b cells are unable to efficiently utilize intrinsic or exogenous fatty acids to meet energy demands. However, A2-1b cells maintained in media deprived of arginine and lysine exhibited increased basal and maximal respiration indicative of an energy reserve that relies on amino acid metabolism. Finally, metabolomics studies showing an overall increase in the absolute concentrations of gluconeogenic and ketogenic amino acids along with significantly elevated S6Kinase expression signify a shift from glucose to amino acids as the major energy source, in resistant A2-1b cells. Our data are consistent with a model of prolonged treatment response in which de-repression of ERK-MAPK pathway activity upon emergence of resistance to Vemurafenib is associated with adaptive changes in cellular metabolism. Sensitivity to BRAF inhibitors in the context of melanoma may not be defined by a reliance on glycolysis for survival, and that switch in metabolic phenotype can serve as an alternate mechanism for cell survival. Citation Format: Deeba N. Syed, Rahul K. Lall, Yong Wei Soon, Ahmed Aljohani, Changan Guo, Feng Liu, Frank L. Meyskens, James M. Ntambi, Hasan Mukhtar. A switch in metabolic phenotype in vemurafenib-resistant melanoma cells to increased amino acid dependence serves as an alternate mechanism for survival. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 14.

Simultaneous quantification of α-lactalbumin and β-casein in human milk using ultra-performance liquid chromatography with tandem mass spectrometry based on their signature peptides and winged isotope internal standards

In recent years, there is an increasing need to measure the concentration of individual proteins in human milk, instead of total human milk proteins. Due to lack of human milk protein standards, there are only few quantification methods established. The objective of the present work was to develop a simple and rapid quantification method for simultaneous determination of α-lactalbumin and β-casein in human milk using signature peptides according to a modified quantitative proteomics strategy. The internal standards containing the signature peptide sequences were synthesized with isotope-labeled amino acids. The purity of synthesized peptides as standards was determined by amino acid analysis method and area normalization method. The contents of α-lactalbumin and β-casein in human milk were measured according to the equimolar relationship between the two proteins and their corresponding signature peptides. The method validation results showed a satisfied linearity (R2>0.99) and recoveries (97.2–102.5% for α-lactalbumin and 99.5–100.3% for β-casein). The limit of quantification for α-lactalbumin and β-casein was 8.0mg/100g and 1.2mg/100g, respectively. CVs for α-lactalbumin and β-casein in human milk were 5.2% and 3.0%. The contents of α-lactalbumin and β-casein in 147 human milk samples were successfully determined by the established method and their contents were 205.5–578.2mg/100g and 116.4–467.4mg/100g at different lactation stages. The developed method allows simultaneously determination of α-lactalbumin and β-casein in human milk. The quantitative strategy based on signature peptide should be applicable to other endogenous proteins in breast milk and other body fluids.

Pseudo isobaric peptide termini labelling for relative proteome quantification by SWATH MS acquisition.

The pseudo isobaric peptide termini labeling (IPTL) method is a remarkable strategy in quantitative proteomics, and has been efficiently applied in biological studies due to its high quantitative accuracy. However, irreproducible precursor ion selection caused by data dependent acquisition and the chromatographic shift caused by isotope effects limit the wide application of this method. Herein, we expand the use of pseudo IPTL to SWATH MS application and develop a novel quantitative strategy, termed SWATH-pseudo-IPTL, by which the relative quantification could be achieved by comparing the "complete" extracted ion chromatogram (XIC) intensity of MS/MS scan instead of a single intensity measurement in DDA-pseudo-IPTL which only reflected the peptide abundances at that given time. The quantitative analysis of various proportions of mixed HeLa samples revealed the strong accuracy and precision of our SWATH-pseudo-IPTL method, both of which were better than that of the DDA-pseudo-IPTL strategy. SWATH-pseudo-IPTL was also applied to the quantitative profiling of the proteome from human hepatocellular carcinoma cell lines with high and low metastatic potential, and most of the differentially expressed proteins were related to tumorigenesis and tumor metastasis, demonstrating the feasibility of this methodology for biological applications.

Label-free quantitative proteomic analysis of benzo(a)pyrene-transformed 16HBE cells serum-free culture supernatant and xenografted nude mice sera

To screen potential biomarkers of benzo(a)pyrene (BaP)-induced lung cancer, the proteomic profiles of BaP-transformed 16HBE cell line T-16HBE-C1 cells serum-free culture supernatant and xenografted nude mice sera were compared with those of 16HBE group by utilizing label-free quantitative proteomic strategy. By employing nano-LC−MS/MS technology followed by MaxQuant and Perseus processing, 489 differentially expressed proteins were identified between T-16HBE-C1 and 16HBE cells serum-free culture supernatant, and 49 significantly up-regulated proteins were identified in T-16HBE-C1 xenografted nude mice sera. Three proteins neuropilin-2 (NRP2), clusterin (CLU) and A-kinase anchor protein 12 (AKAP12) were up-regulated in the serum-free culture supernatant of T-16HBE-C1 cells. These 3 human proteins were present in the sera of nude mice xenografted with T-16HBE-C1 cells, but were undetectable in mice xenografted with 16HBE cells. The proteomic results of NRP2 and AKAP12 were confirmed by Western blotting and enzyme-linked immunosorbent assays, respectively. Moreover, the serum NRP2 levels were significantly elevated at the 4th day after tumor cell implantation and showed good positive correlation with tumor growth characterized by tumor volume. In conclusion, serum NRP2, CLU and AKAP12 could be potential biomarkers of BaP-induced lung cancer. The proteomic results will gain deeper insights into the mechanisms of BaP-induced carcinogenesis.

Triple SILAC quantitative proteomic analysis reveals differential abundance of cell signaling proteins between normal and lung cancer-derived exosomes

Exosomes are 30–100nm sized membrane vesicles released by cells into the extracellular space that mediate intercellular communication via transfer of proteins and other biological molecules. To better understand the role of these microvesicles in lung carcinogenesis, we employed a Triple SILAC quantitative proteomic strategy to examine the differential protein abundance between exosomes derived from an immortalized normal bronchial epithelial cell line and two non-small cell lung cancer (NSCLC) cell lines harboring distinct activating mutations in the cell signaling molecules: Kirsten rat sarcoma viral oncogene homolog (KRAS) or epidermal growth factor receptor (EGFR). In total, we were able to quantify 721 exosomal proteins derived from the three cell lines. Proteins associated with signal transduction, including EGFR, GRB2 and SRC, were enriched in NSCLC exosomes, and could actively regulate cell proliferation in recipient cells. This study's investigation of the NSCLC exosomal proteome has identified enriched protein cargo that can contribute to lung cancer progression, which may have potential clinical implications in biomarker development for patients with NSCLC. Biological significanceThe high mortality associated with lung cancer is a result of late-stage diagnosis of the disease. Current screening techniques used for early detection of lung cancer lack the specificity for accurate diagnosis. Exosomes are nano-sized extracellular vesicles, and the increased abundance of select protein cargo in exosomes derived from cancer cells may be used for diagnostic purposes. In this paper, we applied quantitative proteomic analysis to elucidate abundance differences in exosomal protein cargo between two NSCLC cell lines with distinctive oncogene mutations and an immortalized normal bronchial epithelial cell line. This study revealed proteins associated with cell adhesion, the extracellular matrix, and a variety of signaling molecules were enriched in NSCLC exosomes. The present data reveals a protein profile associated with NSCLC exosomes that suggests a role these vesicles have in the progression of lung carcinogenesis, as well as identifies several promising candidates that could be utilized as a multi-marker protein panel in a diagnostic platform for NSCLC.

Effect of Prolonged Simulated Microgravity on Metabolic Proteins in Rat Hippocampus: Steps toward Safe Space Travel.

Mitochondria are not only the main source of energy in cells but also produce reactive oxygen species (ROS), which result in oxidative stress when in space. This oxidative stress is responsible for energy imbalances and cellular damage. In this study, a rat tail suspension model was used in individual experiments for 7 and 21 days to explore the effect of simulated microgravity (SM) on metabolic proteins in the hippocampus, a vital brain region involved in learning, memory, and navigation. A comparative (18)O-labeled quantitative proteomic strategy was used to observe the differential expression of metabolic proteins. Forty-two and sixty-seven mitochondrial metabolic proteins were differentially expressed after 21 and 7 days of SM, respectively. Mitochondrial Complex I, III, and IV, isocitrate dehydrogenase and malate dehydrogenase were down-regulated. Moreover, DJ-1 and peroxiredoxin 6, which defend against oxidative damage, were up-regulated in the hippocampus. Western blot analysis of proteins DJ-1 and COX 5A confirmed the mass spectrometry results. Despite these changes in mitochondrial protein expression, no obvious cell apoptosis was observed after 21 days of SM. The results of this study indicate that the oxidative stress induced by SM has profound effects on metabolic proteins.

Global Reprogramming of Host SUMOylation during Influenza Virus Infection.

SummaryDynamic nuclear SUMO modifications play essential roles in orchestrating cellular responses to proteotoxic stress, DNA damage, and DNA virus infection. Here, we describe a non-canonical host SUMOylation response to the nuclear-replicating RNA pathogen, influenza virus, and identify viral RNA polymerase activity as a major contributor to SUMO proteome remodeling. Using quantitative proteomics to compare stress-induced SUMOylation responses, we reveal that influenza virus infection triggers unique re-targeting of SUMO to 63 host proteins involved in transcription, mRNA processing, RNA quality control, and DNA damage repair. This is paralleled by widespread host deSUMOylation. Depletion screening identified ten virus-induced SUMO targets as potential antiviral factors, including C18orf25 and the SMC5/6 and PAF1 complexes. Mechanistic studies further uncovered a role for SUMOylation of the PAF1 complex component, parafibromin (CDC73), in potentiating antiviral gene expression. Our global characterization of influenza virus-triggered SUMO redistribution provides a proteomic resource to understand host nuclear SUMOylation responses to infection.

Heat-Treatment-Responsive Proteins in Different Developmental Stages of Tomato Pollen Detected by Targeted Mass Accuracy Precursor Alignment (tMAPA).

Recently, we have developed a quantitative shotgun proteomics strategy called mass accuracy precursor alignment (MAPA). The MAPA algorithm uses high mass accuracy to bin mass-to-charge (m/z) ratios of precursor ions from LC-MS analyses, determines their intensities, and extracts a quantitative sample versus m/z ratio data alignment matrix from a multitude of samples. Here, we introduce a novel feature of this algorithm that allows the extraction and alignment of proteotypic peptide precursor ions or any other target peptide from complex shotgun proteomics data for accurate quantification of unique proteins. This strategy circumvents the problem of confusing the quantification of proteins due to indistinguishable protein isoforms by a typical shotgun proteomics approach. We applied this strategy to a comparison of control and heat-treated tomato pollen grains at two developmental stages, post-meiotic and mature. Pollen is a temperature-sensitive tissue involved in the reproductive cycle of plants and plays a major role in fruit setting and yield. By LC-MS-based shotgun proteomics, we identified more than 2000 proteins in total for all different tissues. By applying the targeted MAPA data-processing strategy, 51 unique proteins were identified as heat-treatment-responsive protein candidates. The potential function of the identified candidates in a specific developmental stage is discussed.

ITMSQ: A software tool for N- and C-terminal fragment ion pairs based isobaric tandem mass spectrometry quantification.

Tandem MS (MS2) quantification using the series of N- and C-terminal fragment ion pairs generated from isobaric-labelled peptides was recently considered an accurate strategy in quantitative proteomics. However, the presence of multiplexed terminal fragment ion in MS2 spectra may reduce the efficiency of peptide identification, resulting in lower identification scores or even incorrect assignments. To address this issue, we developed a quantitative software tool, denoted isobaric tandem MS quantification (ITMSQ), to improve N- and C-terminal fragment ion pairs based isobaric MS2 quantification. A spectrum splitting module was designed to separate the MS2 spectra from different samples, increasing the accuracy of both identification and quantification. ITMSQ offers a convenient interface through which parameters can be changed along with the labelling method, and the result files and all of the intermediate files can be exported. We performed an analysis of in vivo terminal amino acid labelling labelled HeLa samples and found that the numbers of quantified proteins and peptides increased by 13.64 and 27.52% after spectrum splitting, respectively. In conclusion, ITMSQ provides an accurate and reliable quantitative solution for N- and C-terminal fragment ion pairs based isobaric MS2 quantitative methods.

Effects of simulated microgravity on the expression of presynaptic proteins distorting the GABA/glutamate equilibrium--A proteomics approach.

Microgravity may cause cognition-related changes in the animal nervous system due to the resulting uneven flow of fluids in the body. These changes may restrict the long-term stay of humans in space for various purposes. In this study, a rat tail suspension model (30°) was used to explore the effects of 21 days of prolonged simulated microgravity (SM) on the expression of proteins involved in cognitive functions in the rat hippocampus. SM decreased the content of γ-aminobutyric acid (GABA) and increased the content of glutamate (Glu) in the rat hippocampus. A comparative (18)O-labeled quantitative proteomics strategy was applied to detect the differential expression of synaptic proteins under SM. Fifty-three proteins were found to be differentially expressed under SM. Microgravity induces difficulty in the formation of the SNARE complex due to the down-regulation of vesicle-associated membrane protein 3(VAMP3) and syntaxin-1A. Synaptic vesicle recycling may also be affected due to the dysregulation of syntaxin-binding protein 5 (tomosyn), rab3A and its effector rim2. Both processes are disturbed, indicating that presynaptic proteins mediate a GABA/Glu imbalance under SM. These findings provide clues for understanding the mechanism of the GABA/Glu equilibrium in the hippocampus induced by microgravity in space and represent steps toward safe space travel.

SILAC-based proteomic analysis reveals that salidroside antagonizes cobalt chloride-induced hypoxic effects by restoring the tricarboxylic acid cycle in cardiomyocytes

Hypoxic status alters the energy metabolism and induces cell injury in cardiomyocytes, and it further triggers the occurrence and development of cardiovascular diseases. Our previous studies have shown that salidroside (SAL) exhibits anti-hypoxic activity. However, the mechanisms remain obscure. In the present study, we successfully screened 92 different expression proteins in CoCl2-induced hypoxic conditions, 106 different expression proteins in the SAL-mediated anti-hypoxic group were compared with the hypoxic group using quantitative proteomics strategy, respectively. We confirmed that SAL showed a positive protective function involving the acetyl-CoA metabolic, tricarboxylic acid (TCA) cycle using bioinformatics analysis. We also demonstrated that SAL plays a critical role in restoring the TCA cycle and in protecting cardiomyocytes from oxidative injury via up-regulation expressions of PDHE1-B, ACO2, SUCLG1, SUCLG2 and down-regulation of MDH2. SAL also inhibited H9c2 cell apoptosis by inhibiting the activation of pro-apoptotic molecules caspase 3 and caspase 9 as well as activation of the anti-apoptotic molecular Bcl-2. Additionally, SAL also improved mitochondrial membrane potential (ΔΨm), reduced reactive oxygen species (ROS) and intercellular Ca2+ concentration ([Ca2+]i) accumulation and inhibited the excessive consumption of ATP in H9c2 cells.

Quantitative proteomic analysis for high-throughput screening of differential glycoproteins in hepatocellular carcinoma serum.

ObjectiveHepatocellular carcinoma (HCC) is a leading cause of cancer-related deaths. Novel serum biomarkers are required to increase the sensitivity and specificity of serum screening for early HCC diagnosis. This study employed a quantitative proteomic strategy to analyze the differential expression of serum glycoproteins between HCC and normal control serum samples.MethodsLectin affinity chromatography (LAC) was used to enrich glycoproteins from the serum samples. Quantitative mass spectrometric analysis combined with stable isotope dimethyl labeling and 2D liquid chromatography (LC) separations were performed to examine the differential levels of the detected proteins between HCC and control serum samples. Western blot was used to analyze the differential expression levels of the three serum proteins.ResultsA total of 2,280 protein groups were identified in the serum samples from HCC patients by using the 2D LC-MS/MS method. Up to 36 proteins were up-regulated in the HCC serum, whereas 19 proteins were down-regulated. Three differential glycoproteins, namely, fibrinogen gamma chain (FGG), FOS-like antigen 2 (FOSL2), and α-1,6-mannosylglycoprotein 6-β-N-acetylglucosaminyltransferase B (MGAT5B) were validated by Western blot. All these three proteins were up-regulated in the HCC serum samples.ConclusionA quantitative glycoproteomic method was established and proven useful to determine potential novel biomarkers for HCC.

Abstract 1179: Unique metabolic profile of Vemurafenib-resistant melanoma cells: a quantitative proteomics approach

Abstract Malignant melanoma is noted for its aggressive clinical behavior and propensity for lethal metastasis. Despite the remarkable success of BRAF inhibitor Vemurafenib in clinics, emergence of resistance remains a limiting factor in increasing overall survival. Thus, comprehensive studies are needed to increase the biological understanding of BRAF-resistant tumors in order to develop effective therapeutic regimens. To study the molecular mechanism(s) involved in acquired resistance, we generated a Vemurafenib-resistant cell line in the BRAFV600E mutated SK-Mel28 melanoma cell background. Incubation of SK-Mel28 cells with Vemurafenib (0.1 μM) resulted in an initial loss of cell viability after which majority of cells resumed proliferation. Thereafter, cells were exposed to gradually increasing Vemurafenib concentrations and allowed to proliferate in the presence of 5 μM Vemurafenib, to generate the A2-1b resistant cell line. Viability assays established the IC50 of parental SK-Mel28 cells as 0.2 μM, while IC50 > than 12 μM was noted for the Vemurafenib-resistant A2-1b population. We utilized a quantitative proteomics strategy, employing label-free nano-LC-MS/MS technology on a Q-exactive, to compare the proteome of A2-1b with SK-Mel28 cells. The data searched against the human proteome using the Sequest search engine and further analyzed with SIEVE software resulted in the positive identification of 1720 proteins (≤1% FDR). This data set containing proteins with only uniquely identified peptides with a high level of confidence (p<0.05) was uploaded into Ingenuity Pathway Analysis and Panther softwares, with number of peptides and corresponding ratio between the two groups, set as observational parameters. Notably, the largest fraction (49%) of identified proteins belonged to cellular metabolic processes. Glycolysis, gluconeogenesis and NADH repair were the major pathways modulated in Vemurafenib-resistant A2-1b cells. In addition, TCA cycle and unfolded protein response was significantly affected. A subset of 13 proteins, linked to these pathways and not previously associated with acquired resistance was identified. The upregulated proteins included CALD1, OR4A15, PTPRK, CALU, IDH3A, RNH1, CLIC4, SPAG17, SND1 and VCP while ENO2, TPI1 and GAPDH were significantly downregulated. In summary, the proteomic approach applied for the first time to study acquired resistance to BRAF inhibition identified novel targets which upon further validation in appropriate models has the potential to provide valuable therapeutic strategies against Vemurafenib-resistant melanomas. Citation Format: Deeba N. Syed, Rahul K. Lall, Iram Majeed, Feng Liu, Frank L. Meyskens, Hasan Mukhtar. Unique metabolic profile of Vemurafenib-resistant melanoma cells: a quantitative proteomics approach. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1179. doi:10.1158/1538-7445.AM2015-1179

ITRAQ-Based Quantitative Proteomic Analysis of the Initiation of Head Regeneration in Planarians.

The planarian Dugesia japonica has amazing ability to regenerate a head from the anterior ends of the amputated stump with maintenance of the original anterior-posterior polarity. Although planarians present an attractive system for molecular investigation of regeneration and research has focused on clarifying the molecular mechanism of regeneration initiation in planarians at transcriptional level, but the initiation mechanism of planarian head regeneration (PHR) remains unclear at the protein level. Here, a global analysis of proteome dynamics during the early stage of PHR was performed using isobaric tags for relative and absolute quantitation (iTRAQ)-based quantitative proteomics strategy, and our data are available via ProteomeXchange with identifier PXD002100. The results showed that 162 proteins were differentially expressed at 2 h and 6 h following amputation. Furthermore, the analysis of expression patterns and functional enrichment of the differentially expressed proteins showed that proteins involved in muscle contraction, oxidation reduction and protein synthesis were up-regulated in the initiation of PHR. Moreover, ingenuity pathway analysis showed that predominant signaling pathways such as ILK, calcium, EIF2 and mTOR signaling which were associated with cell migration, cell proliferation and protein synthesis were likely to be involved in the initiation of PHR. The results for the first time demonstrated that muscle contraction and ILK signaling might played important roles in the initiation of PHR at the global protein level. The findings of this research provide a molecular basis for further unraveling the mechanism of head regeneration initiation in planarians.

MiR-200c dampens cancer cell migration via regulation of protein kinase A subunits.

Expression of miR-200c is a molecular switch to determine cellular fate towards a mesenchymal or epithelial phenotype. miR-200c suppresses the early steps of tumor progression by preventing epithelial-mesenchymal transition (EMT) and intravasation of tumor cells. Unraveling the underlying molecular mechanisms might pinpoint to novel therapeutic options. To better understand these mechanisms it is crucial to identify targets of miR-200c. Here, we employ a combination of quantitative proteomic and bioinformatic strategies to identify novel miR-200c targets. We identify and confirm two subunits of the central cellular kinase protein kinase A (PKA), namely PRKAR1A and PRKACB, to be directly regulated by miR-200c. Notably, siRNA-mediated downregulation of both proteins phenocopies the migratory behavior of breast cancer cells after miR-200c overexpression. Patient data from publicly accessible databases supports a miR-200c-PKA axis. Thus, our study identifies the PKA heteroprotein as an important mediator of miR-200c induced repression of migration in breast cancer cells. By bioinformatics, we define a miRNA target cluster consisting of PRKAR1A, PRKAR2B, PRKACB, and COF2, which is targeted by a group of 14 miRNAs.

Quantitative Proteomics Analysis Reveals Novel Insights into Mechanisms of Action of Long Noncoding RNA Hox Transcript Antisense Intergenic RNA (HOTAIR) in HeLa Cells*

Long noncoding RNAs (lncRNAs), which have emerged in recent years as a new and crucial layer of gene regulators, regulate various biological processes such as carcinogenesis and metastasis. HOTAIR (Hox transcript antisense intergenic RNA), a lncRNA overexpressed in most human cancers, has been shown to be an oncogenic lncRNA. Here, we explored the role of HOTAIR in HeLa cells and searched for proteins regulated by HOTAIR. To understand the mechanism of action of HOTAIR from a systems perspective, we employed a quantitative proteomic strategy to systematically identify potential targets of HOTAIR. The expression of 170 proteins was significantly dys-regulated after inhibition of HOTAIR, implying that they could be potential targets of HOTAIR. Analysis of this data at the systems level revealed major changes in proteins involved in diverse cellular components, including the cytoskeleton and the respiratory chain. Further functional studies on vimentin (VIM), a key protein involved in the cytoskeleton, revealed that HOTAIR exerts its effects on migration and invasion of HeLa cells, at least in part, through the regulation of VIM expression. Inhibition of HOTAIR leads to mitochondrial dysfunction and ultrastructural alterations, suggesting a novel role of HOTAIR in maintaining mitochondrial function in cancer cells. Our results provide novel insights into the mechanisms underlying the function of HOTAIR in cancer cells. We expect that the methods used in this study will become an integral part of functional studies of lncRNAs.

Global cPILOT analysis of the APP/PS‐1 mouse liver proteome

A quantitative proteomics strategy called combined precursor isotopic labeling and isobaric tagging (cPILOT) was designed to discover alterations in the amyloid precursor protein/presenilin-1 (APP/PS-1) mouse liver proteome. The multiplexing strategy allows simultaneous quantitation of 12 samples in a single experiment. For cPILOT samples, six APP/PS-1 and six heterozygous mouse livers were modified using precursor dimethylation (pH 2.5) followed by isobaric tagging (pH 8.0). Samples were pooled, fractioned with strong cation exchange, and analyzed using RPLC-MS(3) for protein identification and relative quantitation. In order to increase proteome coverage, a two-tiered data collection strategy was employed. Six duplex precursor dimethylation experiments were also performed to verify cPILOT protein quantitation. The combination of cPILOT with precursor dimethylation data resulted in 2437 total liver proteins identified and 77 differentially expressed proteins in APP/PS-1 liver. Differentially expressed proteins are involved in metabolic processes such as B-oxidation, pyruvate metabolism, and glucose regulation. cPILOT expands protein quantitation using isobaric tags and can be applied to any clinical laboratory interested in enhanced multiplexing strategies. Differentially expressed proteins in APP/PS-1 mouse liver suggest the potential use of ketone bodies to alleviate metabolic dysregulation in Alzheimer's disease brain. Our work also suggests alterations in the alanine cycle potentially leading to hyperammonia production, may contribute to Alzheimer's disease pathogenesis.

Improving label-free quantitative proteomics strategies by distributing shared peptides and stabilizing variance.

In a previous study, we demonstrated that spectral counts-based label-free proteomic quantitation could be improved by distributing peptides shared between multiple proteins. Here, we compare four quantitative proteomic approaches, namely, the normalized spectral abundance factor (NSAF), the normalized area abundance factor (NAAF), normalized parent ion intensity abundance factor (NIAF), and the normalized fragment ion intensity abundance factor (NFAF). We demonstrate that label-free proteomic quantitation methods based on chromatographic peak area (NAAF), parent ion intensity in MS1 (NIAF), and fragment ion intensity (NFAF) are also improved when shared peptides are distributed on the basis of peptides unique to each isoform. To stabilize the variance inherent to label-free proteomic quantitation data sets, we use cyclic-locally weighted scatter plot smoothing (LOWESS) and linear regression normalization (LRN). Again, all four methods are improved when cyclic-LOWESS and LRN are applied to reduce variation. Finally, we demonstrate that absolute quantitative values may be derived from label-free parameters such as spectral counts, chromatographic peak area, and ion intensity when using spiked-in proteins of known amounts to generate standard curves.

Experimental study on differences in clivus chordoma bone invasion: an iTRAQ-based quantitative proteomic analysis.

Although a bone tumor, significant differences in the extent of bone invasion exist in skull base chordoma, which directly affect the extent of surgical resection, and have an impact on its prognosis. However, the underlying mechanism of the phenomenon is not clearly understood. Therefore, we used an iTRAQ-based quantitative proteomics strategy to identify potential molecular signatures, and to find predictive markers of discrepancy in bone invasion of clivus chordoma. According to bone invasive classification criteria, 35 specimens of clivus chordoma were calssified to be either endophytic type (Type I) or exophytic type (Type II). An initial screening of six specimens of endophytic type and six of exophytic was performed, and 250 differentially expressed proteins were identified. Through the GO and IPA analysis, we found evidence that the expression of inflammatory activity-associated proteins up-regulated in endophytic type, whereas the expression of cell motility-associated proteins up-regulated in exophytic ones. Moreover, TGFβ1 and mTOR signal pathway seemed to be related with bone invasion. Thus, TGFβ1, PI3K, Akt, mTOR, and PTEN were validated in the following 23 samples by immune histochemistry and Western blot. The expression levels of TGFβ1 and PTEN were significantly lower in the endophytic type than in the exophytic ones. It was found that TGFβ1 may play an important role in its bone invasion. The mechanisms may be related with conducting an increased inflammatory cell response and a decline in cytoskeletal protein expression. PTEN is confirmed to be associated with the degree of bone invasion. The PI3K/AKT/mTOR signaling pathway might be associated with the bone invasion, but still needs a larger sample size to be verified These results, for the first time, not only demonstrate the biological changes that occur in different growth patterns from the perspective of proteomics, but also provide novel markers that may help to reveal the mechanisms behind clivus chordomas.