Complement factor B is critical for sub-RPE deposit accumulation in a model of Doyne honeycomb retinal dystrophy with features of age-related macular degeneration.

EFEMP1 R345W is a dominant mutation causing Doyne honeycomb retinal dystrophy/malattia leventinese (DHRD/ML), a rare blinding disease with clinical pathology similar to age-related macular degeneration (AMD). Aged Efemp1  R345W/R345W knock-in mice (Efemp1ki/ki) develop microscopic deposits on the basal side of retinal pigment epithelial cells (RPE), an early feature in DHRD/ML and AMD. Here, we assessed the role of alternative complement pathway component factor B (FB) in the formation of these deposits. RNA-seq analysis of the posterior eyecups revealed increased unfolded protein response, decreased mitochondrial function in the neural retina (by 3 months of age) and increased inflammatory pathways in both neural retina and posterior eyecups (at 17 months of age) of Efemp1ki/ki mice compared with wild-type littermate controls. Proteomics analysis of eye lysates confirmed similar dysregulated pathways as detected by RNA-seq. Complement activation was increased in aged Efemp1ki/ki eyes with an approximately 2-fold elevation of complement breakdown products iC3b and Ba (P < 0.05). Deletion of the Cfb gene in female Efemp1ki/ki mice partially normalized the above dysregulated biological pathway changes and oral dosing of a small molecule FB inhibitor from 10 to 12 months of age reduced sub-RPE deposits by 65% (P = 0.029). In contrast, male Efemp1ki/ki mice had fewer sub-RPE deposits than age-matched females, no elevation of ocular complement activation and no effect of FB inhibition on sub-RPE deposits. The effects of FB deletion or inhibition on Efemp1ki/ki mice supports systemic inhibition of the alternative complement pathway as a potential treatment of dry AMD and DHRD/ML.

A highly multiplexed quantitative phosphosite assay for biology and preclinical studies.

Reliable methods to quantify dynamic signaling changes across diverse pathways are needed to better understand the effects of disease and drug treatment in cells and tissues but are presently lacking. Here, we present SigPath, a targeted mass spectrometry (MS) assay that measures 284 phosphosites in 200 phosphoproteins of biological interest. SigPath probes a broad swath of signaling biology with high throughput and quantitative precision. We applied the assay to investigate changes in phospho-signaling in drug-treated cancer cell lines, breast cancer preclinical models, and human medulloblastoma tumors. In addition to validating previous findings, SigPath detected and quantified a large number of differentially regulated phosphosites newly associated with disease models and human tumors at baseline or with drug perturbation. Our results highlight the potential of SigPath to monitor phosphoproteomic signaling events and to nominate mechanistic hypotheses regarding oncogenesis, response, and resistance to therapy.

A CRISPR Screen Reveals Resistance Mechanisms to CD3-Bispecific Antibody Therapy.

CD3-bispecific antibodies represent an important therapeutic strategy in oncology. These molecules work by redirecting cytotoxic T cells to antigen-bearing tumor cells. Although CD3-bispecific antibodies have been developed for several clinical indications, cases of cancer-derived resistance are an emerging limitation to the more generalized application of these molecules. Here, we devised whole-genome CRISPR screens to identify cancer resistance mechanisms to CD3-bispecific antibodies across multiple targets and cancer types. By validating the screen hits, we found that deficiency in IFNγ signaling has a prominent role in cancer resistance. IFNγ functioned by stimulating the expression of T-cell killing-related molecules in a cell type-specific manner. By assessing resistance to the clinical CD3-bispecific antibody flotetuzumab, we identified core fucosylation as a critical pathway to regulate flotetuzumab binding to the CD123 antigen. Disruption of this pathway resulted in significant resistance to flotetuzumab treatment. Proper fucosylation of CD123 was required for its normal biological functions. In order to treat the resistance associated with fucosylation loss, flotetuzumab in combination with an alternative targeting CD3-bispecific antibody demonstrated superior efficacy. Together, our study reveals multiple mechanisms that can be targeted to enhance the clinical potential of current and future T-cell-engaging CD3-bispecific antibody therapies.

Quantitative Proteomics of the Cancer Cell Line Encyclopedia.

Proteins are essential agents of biological processes. To date, large-scale profiling of cell line collections including the Cancer Cell Line Encyclopedia (CCLE) has focused primarily on genetic information whereas deep interrogation of the proteome has remained out of reach. Here, we expand the CCLE through quantitative profiling of thousands of proteins by mass spectrometry across 375 cell lines from diverse lineages to reveal information undiscovered by DNA and RNA methods. We observe unexpected correlations within and between pathways that are largely absent from RNA. An analysis of microsatellite instable (MSI) cell lines reveals the dysregulation of specific protein complexes associated with surveillance of mutation and translation. These and other protein complexes were associated with sensitivity to knockdown of several different genes. These data in conjunction with the wider CCLE are a broad resource to explore cellular behavior and facilitate cancer research.

TRPS1 Is a Lineage-Specific Transcriptional Dependency in Breast Cancer.

Perturbed epigenomic programs play key roles in tumorigenesis, and chromatin modulators are candidate therapeutic targets in various human cancer types. To define singular and shared dependencies on DNA and histone modifiers and transcription factors in poorly differentiated adult and pediatric cancers, we conducted a targeted shRNA screen across 59 cell lines of 6 cancer types. Here, we describe the TRPS1 transcription factor as a strong breast cancer-specific hit, owing largely to lineage-restricted expression. Knockdown of TRPS1 resulted in perturbed mitosis, apoptosis, and reduced tumor growth. Integrated analysis of TRPS1 transcriptional targets, chromatin binding, and protein interactions revealed that TRPS1 is associated with the NuRD repressor complex. These findings uncover a transcriptional network that is essential for breast cancer cell survival and propagation.

Integrated microscale analysis system for targeted liquid chromatography mass spectrometry proteomics on limited amounts of enriched cell populations.

Limited samples, such as those that are in vivo sourced via biopsy, are closely representative of biological systems and contain valuable information for drug discovery. However, these precious samples are often heterogeneous and require cellular prefractionation prior to proteomic analysis to isolate specific subpopulations of interest. Enriched cells from in vivo samples are often very limited (<10(4) cells) and pose a significant challenge to proteomic nanoliquid chromatography mass spectrometry (nanoLCMS) sample preparation. To enable the streamlined analysis of these limited samples, we have developed an online cell enrichment, microscale sample preparation, nanoLCMS proteomics workflow by integrating fluorescence activated cell sorting (FACS), focused ultrasonication, microfluidics, immobilized trypsin digestion, and nanoLCMS. To assess the performance of the online FACS-Chip-LCMS workflow, 5000 fluorescent labeled cells were enriched from a 5% heterogeneous cell population and processed for LCMS proteomics in less than 2 h. Within these 5000 enriched cells, 30 peptides corresponding to 17 proteins spanning more than 4 orders of magnitude of cellular abundance were quantified using a QExactive MS. The results from the online FACS-Chip-LCMS workflow starting from 5000 enriched cells were directly compared to results from a traditional macroscale sample preparation workflow starting from 2.0 × 10(6) cells. The microscale FACS-Chip-LCMS workflow demonstrated high cellular enrichment efficiency and high peptide recovery across the wide dynamic range of targeted peptides. Overall the microscale FACS-Chip-LCMS workflow has shown effectiveness in efficiently preparing limited amounts of FACS enriched cells in an online manner for proteomic LCMS.

Human folliculin delays cell cycle progression through late S and G2/M-phases: effect of phosphorylation and tumor associated mutations.

The Birt-Hogg-Dube disease occurs as a result of germline mutations in the human Folliculin gene (FLCN), and is characterized by clinical features including fibrofolliculomas, lung cysts and multifocal renal neoplasia. Clinical and genetic evidence suggest that FLCN acts as a tumor suppressor gene. The human cell line UOK257, derived from the renal cell carcinoma of a patient with a germline mutation in the FLCN gene, harbors a truncated version of the FLCN protein. Reconstitution of the wild type FLCN protein into UOK257 cells delays cell cycle progression, due to a slower progression through the late S and G2/M-phases. Similarly, Flcn (-/-) mouse embryonic fibroblasts progress more rapidly through the cell cycle than wild type controls (Flcn (flox/flox)). The reintroduction of tumor-associated FLCN mutants (FLCN ΔF157, FLCN 1-469 or FLCN K508R) fails to delay cell cycle progression in UOK257 cells. Additionally, FLCN phosphorylation (on Serines 62 and 73) fluctuates throughout the cell cycle and peaks during the G2/M phase in cells treated with nocodazole. In keeping with this observation, the reintroduction of a FLCN phosphomimetic mutant into the UOK257 cell line results in faster progression through the cell cycle compared to those expressing the wild type FLCN protein. These findings suggest that the tumor suppression function of FLCN may be linked to its impact on the cell cycle and that FLCN phosphorylation is important for this activity. Additionally, these observations describe a novel in vitro assay for testing the functional significance of FLCN mutations and/or genetic polymorphisms.

Integrated proteomic, transcriptomic, and biological network analysis of breast carcinoma reveals molecular features of tumorigenesis and clinical relapse.

Gene and protein expression changes observed with tumorigenesis are often interpreted independently of each other and out of context of biological networks. To address these limitations, this study examined several approaches to integrate transcriptomic and proteomic data with known protein-protein and signaling interactions in estrogen receptor positive (ER+) breast cancer tumors. An approach that built networks from differentially expressed proteins and identified among them networks enriched in differentially expressed genes yielded the greatest success. This method identified a set of genes and proteins linking pathways of cellular stress response, cancer metabolism, and tumor microenvironment. The proposed network underscores several biologically intriguing events not previously studied in the context of ER+ breast cancer, including the overexpression of p38 mitogen-activated protein kinase and the overexpression of poly(ADP-ribose) polymerase 1. A gene-based expression signature biomarker built from this network was significantly predictive of clinical relapse in multiple independent cohorts of ER+ breast cancer patients, even after correcting for standard clinicopathological variables. The results of this study demonstrate the utility and power of an integrated quantitative proteomic, transcriptomic, and network analysis approach to discover robust and clinically meaningful molecular changes in tumors.

Microproteomic analysis of 10,000 laser captured microdissected breast tumor cells using short-range sodium dodecyl sulfate-polyacrylamide gel electrophoresis and porous layer open tubular liquid chromatography tandem mass spectrometry.

Precise proteomic profiling of limited levels of disease tissue represents an extremely challenging task. Here, we present an effective and reproducible microproteomic workflow for sample sizes of only 10,000 cells that integrates selective sample procurement via laser capture microdissection (LCM), sample clean-up and protein level fractionation using short-range SDS-PAGE, followed by ultrasensitive LC-MS/MS analysis using a 10 µm i.d. porous layer open tubular (PLOT) column. With 10,000 LCM captured mouse hepatocytes for method development and performance assessment, only 10% of the in-gel digest, equivalent to ∼1000 cells, was needed per LC-MS/MS analysis. The optimized workflow was applied to the differential proteomic analysis of 10,000 LCM collected primary and metastatic breast cancer cells from the same patient. More than 1100 proteins were identified from each injection with >1700 proteins identified from three LCM samples of 10,000 cells from the same patient (1123 with at least two unique peptides). Label free quantitation (spectral counting) was performed to identify differential protein expression between the primary and metastatic cell populations. Informatics analysis of the resulting data indicated that vesicular transport and extracellular remodeling processes were significantly altered between the two cell types. The ability to extract meaningful biological information from limited, but highly informative cell populations demonstrates the significant benefits of the described microproteomic workflow.

Histone deacetylase activity is necessary for left-right patterning during vertebrate development.

BACKGROUND: Consistent asymmetry of the left-right (LR) axis is a crucial aspect of vertebrate embryogenesis. Asymmetric gene expression of the TGFß superfamily member Nodal related 1 (Nr1) in the left lateral mesoderm plate is a highly conserved step regulating the situs of the heart and viscera. In Xenopus, movement of maternal serotonin (5HT) through gap-junctional paths at cleavage stages dictates asymmetry upstream of Nr1. However, the mechanisms linking earlier biophysical asymmetries with this transcriptional control point are not known. RESULTS: To understand how an early physiological gradient is transduced into a late, stable pattern of Nr1 expression we investigated epigenetic regulation during LR patterning. Embryos injected with mRNA encoding a dominant-negative of Histone Deacetylase (HDAC) lacked Nr1 expression and exhibited randomized sidedness of the heart and viscera (heterotaxia) at stage 45. Timing analysis using pharmacological blockade of HDACs implicated cleavage stages as the active period. Inhibition during these early stages was correlated with an absence of Nr1 expression at stage 21, high levels of heterotaxia at stage 45, and the deposition of the epigenetic marker H3K4me2 on the Nr1 gene. To link the epigenetic machinery to the 5HT signaling pathway, we performed a high-throughput proteomic screen for novel cytoplasmic 5HT partners associated with the epigenetic machinery. The data identified the known HDAC partner protein Mad3 as a 5HT-binding regulator. While Mad3 overexpression led to an absence of Nr1 transcription and randomized the LR axis, a mutant form of Mad3 lacking 5HT binding sites was not able to induce heterotaxia, showing that Mad3's biological activity is dependent on 5HT binding. CONCLUSION: HDAC activity is a new LR determinant controlling the epigenetic state of Nr1 from early developmental stages. The HDAC binding partner Mad3 may be a new serotonin-dependent regulator of asymmetry linking early physiological asymmetries to stable changes in gene expression during organogenesis.

Ultrasensitive characterization of site-specific glycosylation of affinity-purified haptoglobin from lung cancer patient plasma using 10 µm i.d. porous layer open tubular liquid chromatography-linear ion trap collision-induced dissociation/electron transfer dissociation mass spectrometry.

Site-specific analysis of protein glycosylation is important for biochemical and clinical research efforts. Glycopeptide analysis using liquid chromatography-collision-induced dissociation/electron transfer dissociation mass spectrometry (LC-CID/ETD-MS) allows simultaneous characterization of the glycan structure and attached peptide site. However, due to the low ionization efficiency of glycopeptides during electrospray ionization, 200-500 fmol of sample per injection is needed for a single LC-MS run, which makes it challenging for the analysis of limited amounts of glycoprotein purified from biological matrixes. To improve the sensitivity of LC-MS analysis for glycopeptides, an ultranarrow porous layer open tubular (PLOT) LC column (2.5 m × 10 µm i.d.) was coupled to a linear ion trap (LTQ) collision-induced dissociation/electron transfer dissociation mass spectrometer to provide sensitive analysis of N-linked protein glycosylation heterogeneity. The potential of the developed method is demonstrated by the characterization of site-specific glycosylation using haptoglobin (Hpt) as a model protein. To limit the amount of haptoglobin to low picomole amounts of protein, we affinity purified it from 1 µL of pooled lung cancer patient plasma. A total of 26 glycoforms/glycan compositions on three Hpt tryptic glycopeptides were identified and quantified from 10 LC-MS runs with a consumption of 100 fmol of Hpt digest (13 ng of protein, 10 fmol per injection). Included in this analysis was the determination of the glycan occupancy level. At this sample consumption level, the high sensitivity of the PLOT LC-LTQ-CID/ETD-MS system allowed glycopeptide identification and structure determination, along with relative quantitation of glycans presented on the same peptide backbone, even for low abundant glycopeptides at the ∼100 amol level. The PLOT LC-MS system is shown to have sufficient sensitivity to allow characterization of site-specific protein glycosylation from trace levels of glycosylated proteins.

Substrates of the Arabidopsis thaliana protein isoaspartyl methyltransferase 1 identified using phage display and biopanning.

The role of protein isoaspartyl methyltransferase (PIMT) in repairing a wide assortment of damaged proteins in a host of organisms has been inferred from the affinity of the enzyme for isoaspartyl residues in a plethora of amino acid contexts. The identification of PIMT target proteins in plant seeds, where the enzyme is highly active and proteome long-lived, has been hindered by large amounts of isoaspartate-containing storage proteins. Mature seed phage display libraries circumvented this problem. Inclusion of the PIMT co-substrate, S-adenosylmethionine (AdoMet), during panning permitted PIMT to retain aged phage in greater numbers than controls lacking co-substrate or when PIMT protein binding was poisoned with S-adenosyl homocysteine. After four rounds, phage titer plateaued in AdoMet-containing pans, whereas titer declined in both controls. This strategy identified 17 in-frame PIMT target proteins, including a cupin-family protein similar to those identified previously using on-blot methylation. All recovered phage had at least one susceptible Asp or Asn residue. Five targets were recovered independently. Two in-frame targets were produced in Escherichia coli as recombinant proteins and shown by on-blot methylation to acquire isoAsp, becoming a PIMT target. Both gained isoAsp rapidly in solution upon thermal insult. Mutant analysis of plants deficient in any of three in-frame PIMT targets resulted in demonstrable phenotypes. An over-representation of clones encoding proteins involved in protein production suggests that the translational apparatus comprises a subgroup for which PIMT-mediated repair is vital for orthodox seed longevity. Impaired PIMT activity would hinder protein function in these targets, possibly resulting in poor seed performance.

Shotgun proteomic analysis of yeast-elicited California poppy (Eschscholzia californica) suspension cultures producing enhanced levels of benzophenanthridine alkaloids.

The California poppy, Eschscholzia californica, produces benzophenanthridine alkaloids (BPAs), an important class of biologically active compounds. Cell cultures of E. californica were investigated as an alternative and scalable method for producing these valuable compounds; treatment with yeast extract increased production from low levels to 23 mg/g dry weight (DW) of BPAs. A shotgun proteomic analysis of E. californica cell cultures was undertaken to explore changes in metabolism associated with enhanced BPA production. We implemented differential centrifugation and then shotgun proteomics based on nanoliquid chromatography/mass spectrometry (nano-LC-MS/MS) for peptide separation and analysis. A unigene database available for E. californica was translated and utilized for protein identification. Approximately 646 proteins (3% false discovery rate at the protein level) were identified. Differentially abundant proteins observed with elicitation included enzymes involved in (S)-adenosyl methionine (SAM) biosynthesis and BPA biosynthesis. These results demonstrate (1) the identification of proteins from a medicinal plant using shotgun proteomics combined with a well-annotated, translated unigene database and (2) the potential utility of proteomics for exploring changes in metabolism associated with enhanced secondary metabolite production.

In situ proteomic analysis of human breast cancer epithelial cells using laser capture microdissection: annotation by protein set enrichment analysis and gene ontology.

Identification of molecular signatures that allow detection of the transition from normal breast epithelial cells to malignant invasive cells is a critical component in the development of diagnostic, therapeutic, and preventative strategies for human breast cancer. Substantial efforts have been devoted to deciphering breast cancer etiology at the genome level, but only a limited number of studies have appeared at the proteome level. In this work, we compared individual in situ proteome profiles of nonpatient matched nine noncancerous, normal breast epithelial (NBE) samples with nine estrogen receptor (ER)-positive (luminal subtype), invasive malignant breast epithelial (MBE) samples by combining laser capture microdissection (LCM) and quantitative shotgun proteomics. A total of 12,970 unique peptides were identified from the 18 samples, and 1623 proteins were selected for quantitative analysis using spectral index (SpI) as a measure of protein abundance. A total of 298 proteins were differentially expressed between NBE and MBE at 95% confidence level, and this differential expression correlated well with immunohistochemistry (IHC) results reported in the Human Protein Atlas (HPA) database. To assess pathway level patterns in the observed expression changes, we developed protein set enrichment analysis (PSEA), a modification of a well-known approach in gene expression analysis, Gene Set Enrichment Analysis (GSEA). Unlike single gene-based functional term enrichment analyses that only examines pathway overrepresentation of proteins above a given significance threshold, PSEA applies a weighted running sum statistic to the entire expression data to discover significantly enriched protein groups. Application of PSEA to the expression data in this study revealed not only well-known ER-dependent and cellular morphology-dependent protein abundance changes, but also significant alterations of downstream targets for multiple transcription factors (TFs), suggesting a role for specific gene regulatory pathways in breast tumorigenesis. A parallel GOMiner analysis revealed both confirmatory and complementary data to PSEA. The combination of the two annotation approaches yielded extensive biological feature mapping for in depth analysis of the quantitative proteomic data.

Improved sample preparation method for glycan analysis of glycoproteins by CE-LIF and CE-MS.

CE is a high-resolution separation technique broadly used in the biotechnology industry for carbohydrate analysis. The standard sample preparation protocol for CE analysis of glycans released from glycoproteins generally requires derivatization times of overnight at 37 degrees C, using > or =100 fold excess of fluorophore reagent, 8-aminopyrene-1,3,6-trisulfonic-acid, if the sample is unknown, or it is a regulated biotherapeutic product, possibly containing terminal sialic acid(s). In this paper, we report on significant improvements for the standard CE sample preparation method of glycan analysis. By replacing the conventionally used acetic acid catalyst with citric acid, as low as 1:10 glycan to fluorophore molar ratio (versus the typical 1:> or =100 ratio) maintained the >95% derivatization yield at 55 degrees C with only 50 min reaction time. Terminal sialic acid loss was negligible at 55 degrees C during the derivatization process, and indicating that the kinetics of labeling at 55 degrees C was faster than the loss of sialic acid from the glycan. The reduced relative level of 8-aminopyrene-1,3,6-trisulfonic-acid simplified the removal of excess reagent, important in both CE-LIF (electrokinetic injection bias) and CE-MS (ion suppression). Coupling CE- ESI-MS confirmed that the individual peaks separated by CE corresponded to single glycans and increased the confidence of structural assignment based on glucose unit values.

Desulfurization of cysteine-containing peptides resulting from sample preparation for protein characterization by mass spectrometry.

In this study, we have examined two cysteine modifications resulting from sample preparation for protein characterization by mass spectrometry (MS): (1) a previously observed conversion of cysteine into dehydroalanine, now found in the case of disulfide mapping and (2) a novel modification corresponding to conversion of cysteine into alanine. Using model peptides, the conversion of cysteine into dehydroalanine via beta-elimination of a disulfide bond was seen to result from the conditions of typical tryptic digestion (37 degrees C, pH 7.0-9.0) without disulfide reduction and alkylation. Furthermore, the surprising conversion of cysteine into alanine was shown to occur by heating cysteine-containing peptides in the presence of a phosphine (tris(2-carboxyethyl)phosphine hydrochloride (TCEP)). The formation of alanine from cysteine, investigated by performing experiments in H(2)O or D(2)O, suggested a radical-based desulfurization mechanism unrelated to beta-elimination. Importantly, an understanding of the mechanism and conditions favorable for cysteine desulfurization provides insight for the establishment of improved sample preparation procedures of protein analysis.

Profiling the glycoforms of the intact alpha subunit of recombinant human chorionic gonadotropin by high-resolution capillary electrophoresis-mass spectrometry.

With the rapid growth of complex heterogeneous biological molecules, effective techniques that are capable of rapid characterization of biologics are essential to ensure the desired product characteristics. To address this need, we have developed a method for analysis of intact glycoproteins based on high-resolution capillary electrophoretic separation coupled to an LTQ-FT mass spectrometer. We evaluated the performance of this method on the alpha subunit of mouse cell line-derived recombinant human chorionic gonadotrophin (r-alpha hCG), a protein that is glycosylated at two sites and is part of the clinically relevant gonadotrophin family. Analysis of r-alpha hCG, using capillary electrophoresis (CE) with a separation time under 20 min, resulted in the identification of over 60 different glycoforms with up to nine sialic acids. High-resolution CE-Fourier transform mass spectrometry (FT-MS) allowed separation and analysis of not only intact glycoforms with different numbers of sialic acids but also intact glycoforms that differed by the number and extent of neutral monosaccharides. The high mass resolution of the FT-MS enabled a limited mass range to be targeted for the examination of the protein glycoforms, simplifying the analysis without sacrificing accuracy. In addition, the limited mass range resulted in a fast scan speed that enhanced the reproducibility of the relative quantitation of individual glycoforms. The intact glycoprotein analysis was complemented with the analysis of the tryptic glycopeptides and glycans of r-alpha hCG to enable the assignment of glycan structures to individual sites, resulting in a detailed characterization of the protein. Samples of r-alpha hCG obtained from a CHO cell line were also analyzed and briefly shown to be significantly different from the murine cell line product. Taken together, the results suggest that the CE coupled to high-resolution FT-MS can be one of the effective tools for in-process monitoring as well as for final product characterization.

CLUE-TIPS, clustering methods for pattern analysis of LC-MS data.

Liquid Chromatography Mass Spectrometry (LC-MS) based proteomics is an important tool in detecting changes in peptide/protein abundances in samples potentially leading to the discovery of disease biomarker candidates. We present CLUE-TIPS (Clustering Using Euclidean distance in Tanimoto Inter-Point Space), an approach that compares complex proteomic samples for similarity/dissimilarity analysis. In CLUE-TIPS, an intersample distance feature map is generated from filtered, aligned and binarized raw LC-MS data by applying the Tanimoto distance metric to obtain normalized similarity scores between all sample pairs for each m/z value. We developed clustering and visualization methods for the intersample distance map to analyze various samples for differences at the sample level as well as the individual m/z level. An approach to query for specific m/z values that are associated with similarity/dissimilarity patterns in a set of samples was also briefly described. CLUE-TIPS can also be used as a tool in assessing the quality of LC-MS runs. The presented approach does not rely on tandem mass-spectrometry (MS/MS), isotopic labels or gels and also does not rely on feature extraction methods. CLUE-TIPS suite was applied to LC-MS data obtained from plasma samples collected at various time points and treatment conditions from immunosuppressed mice implanted with MCF-7 human breast cancer cells. The generated raw LC-MS data was used for pattern analysis and similarity/dissimilarity detection. CLUE-TIPS successfully detected the differences/similarities in samples at various time points taken during the progression of tumor, and also recognized differences/similarities in samples representing various treatment conditions.

TAT peptide and its conjugates: proteolytic stability.

The proteolytic cleavage of TATp, TATp-PEG(1000)-PE conjugate (TATp-conjugate), and TATp as TATp-conjugate in mixed micelles made of TATp-conjugate and PEG(5000)-PE (2.5% mol of TATp-conjugate, TATp-Mic) were studied by HPLC with fluorescent detection using fluorenylmethyl chloroformate (FMOC) labeling and by MALDI-TOF MS analysis. The cleavage kinetics were analyzed in human blood plasma and in trypsin-containing phosphate buffered saline (PBS), pH 7.4, to simulate the proteolytic activity of human plasma. The trypsinolysis of free TATp, TATp-conjugate, and TATp-Mic revealed that the main initial fragmentation is an endocleavage at the carboxyl terminus resulting in an Arg-Arg (RR) dimer. The trypsinolysis followed pseudo-first-order kinetics. The cleavage of the free TATp was relatively fast with a half-life of a few minutes (t(1/2) ∼ 3.5 min). The TATp-conjugate showed more stability with about a 3-fold increase in half-life (t(1/2) ∼ 10 min). TATp in TATp-Mic was highly protected against proteolysis with an over 100-fold increase in half-life (t(1/2) ∼ 430 min). The shielding of TATp by PEG moieties in the proposed TATp-Mic is of great importance for its potential use as a cell-penetrating moiety for multifunctional "smart" drug delivery systems with detachable PEG.

Optimization of poly(GMA-co-EDMA) monolithic support for trypsin nanoreactor fabrication.

Fabrication of poly(glycidyl methacrylate-co-ethylene dimethacrylate) [also referred to as poly(GMA-co-EDMA)] monoliths was optimized as supporting material for trypsin digestion nanoreactors. Reaction parameters, such as polymerization time, porogen concentration, and monomer to crosslinker ratios, were evaluated in respect to the permeability of the resulting monolith and their effect on digestion efficiency, estimated by mass spectrometric analysis of a model protein cytochrome C. The structural homogeneity of the resulting monolithic support was checked by scanning electron microscopy. The best nanoreactor performance, measured by the reduction of nanoreactor backpressure and increased sequence coverage of cytochrome C, was achieved with 8% 2-octanol (porogen) 20%/20% glycidyl methacrylate to ethylene glycol dimethacrylate ratio and 5 h of polymerization time. Digestion of as low as 3 microg of cytochrome C with 77% sequence coverage was obtained using the optimized trypsin nanoreactor.