Spectral Counting Approach Research Articles

Mapping Protein-Protein Interactions Using Data-Dependent Acquisition without Dynamic Exclusion.

Systematic analysis of affinity-purified samples by liquid chromatography coupled to mass spectrometry (LC-MS) requires high coverage, reproducibility, and sensitivity. While data-independent acquisition (DIA) approaches improve the reproducibility of protein-protein interaction detection as compared to standard data-dependent acquisition approaches, the need for library generation reduces their throughput, and analysis pipelines are still being optimized. In this study, we report the development of a simple and robust approach, termed turboDDA, to improve interactome analysis using spectral counting and data-dependent acquisition (DDA) by eliminating the dynamic exclusion (DE) step and optimizing the acquisition parameters. Using representative interaction and proximity proteomics samples, we detected increases in identified interactors of 18-71% compared to all samples analyzed by standard DDA with dynamic exclusion and for most samples analyzed by DIA with the MSPLIT-DIA spectral counting approach. In summary, turboDDA provides better sensitivity and identifies more high-confident interactors than the optimized DDA with DE and comparable or better sensitivity than DIA spectral counting approaches.

In-Depth Proteomic Characterization of Classical and Non-Classical Monocyte Subsets.

Monocytes are bone marrow-derived leukocytes that are part of the innate immune system. Monocytes are divided into three subsets: classical, intermediate and non-classical, which can be differentiated by their expression of some surface antigens, mainly CD14 and CD16. These cells are key players in the inflammation process underlying the mechanism of many diseases. Thus, the molecular characterization of these cells may provide very useful information for understanding their biology in health and disease. We performed a multicentric proteomic study with pure classical and non-classical populations derived from 12 healthy donors. The robust workflow used provided reproducible results among the five participating laboratories. Over 5000 proteins were identified, and about half of them were quantified using a spectral counting approach. The results represent the protein abundance catalogue of pure classical and enriched non-classical blood peripheral monocytes, and could serve as a reference dataset of the healthy population. The functional analysis of the differences between cell subsets supports the consensus roles assigned to human monocytes.

Spectral Counting Approach to Measure Selectivity of High-Resolution LC-MS Methods for Environmental Analysis.

Advances in high-resolution mass spectrometers have allowed for the development of nontargeted screening methods, where data sets can be archived and retrospectively mined as new environmental contaminants are identified. We have developed a spectral counting approach to calculate the selectivities of LC-MS acquisition modes taking mass accuracy, sample matrix, and the analyte properties into account. The selectivities of high-resolution MS (HRMS) alone or in combination with all-ion-fragmentation (AIF), data-independent-acquisition (DIA), and data-dependent-acquisition (DDA) modes, performed on a Q-Exactive Orbitrap were compared by retrospectively screening surface water samples for 95 pharmaceuticals. Samples were reanalyzed using targeted LC-MS/MS to confirm the accuracy of each acquisition method and to quantitate the 29 putatively detected drugs. LC-HRMS provided the lowest calculated selectivities and accordingly produced the highest number of false positives (6). In contrast, DDA provided the highest selectivities, yielding only one false positive; however, it was bias toward the most intense signals resulting in the detection of only 10 compounds. AIF had lower selectivities than traditional LC-MS/MS, produced one false positive and did not detect 6 confirmed compounds. Because of the high-quality archived data, DIA selectivities were better than traditional LC-MS/MS, showed no bias toward the most intense signals, achieved low limits of detection, and confidently detected the greatest number of pharmaceuticals (22) with only one false positive. This spectral counting method can be used across different instrument platforms or samples and provides a robust and empirical estimation of selectivities to give more confident detection of trace analytes.

DDX3Y, a Male-Specific Region of Y Chromosome Gene, May Modulate Neuronal Differentiation.

Although it is apparent that chromosome complement mediates sexually dimorphic expression patterns of some proteins that lead to functional differences, there has been insufficient evidence following the manipulation of the male-specific region of the Y chromosome (MSY) gene expression during neural development. In this study, we profiled the expression of 23 MSY genes and 15 of their X-linked homologues during neural cell differentiation of NTERA-2 human embryonal carcinoma cell line (NT2) cells in three different developmental stages using qRT-PCR, Western blotting, and immunofluorescence. The expression level of 12 Y-linked genes significantly increased over neural differentiation, including RBMY1, EIF1AY, DDX3Y, HSFY1, BPY2, PCDH11Y, UTY, RPS4Y1, USP9Y, SRY, PRY, and ZFY. We showed that siRNA-mediated knockdown of DDX3Y, a DEAD box RNA helicase enzyme, in neural progenitor cells impaired cell cycle progression and increased apoptosis, consequently interrupting differentiation. Label-free quantitative shotgun proteomics based on a spectral counting approach was then used to characterize the proteomic profile of the cells after DDX3Y knockdown. Among 917 reproducibly identified proteins detected, 71 proteins were differentially expressed following DDX3Y siRNA treatment compared with mock treated cells. Functional grouping indicated that these proteins were involved in cell cycle, RNA splicing, and apoptosis, among other biological functions. Our results suggest that MSY genes may play an important role in neural differentiation and demonstrate that DDX3Y could play a multifunctional role in neural cell development, probably in a sexually dimorphic manner.

Abstract 5306: Shotgun proteomic analysis of human head and neck squamous cell carcinoma cell line SQ20B with diminished AHSG expression

Abstract The Alpha-Heremans-Schmid Glycoprotein (AHSG) has tumor promoting properties in animal models of breast cancer and lung cancer (1, 2). These cancer cells do not synthesize AHSG, instead utilizing the liver-generated glycoprotein that is abundant in serum. We have reported that head and neck squamous cell carcinoma (HNSCC) cell lines synthesize AHSG (in press) and have also detected abundant AHSG in primary HNSCC tumors (unpublished). Growth in serum-free medium and in vitro tumorigenic properties, including proliferation, adhesion and migration, are diminished in the HNSCC SQ20B cell line modified with AHSG-specific shRNA to express only twenty percent of the wild-type SQ20B cell line (SQ20B-AH20) compared to SQ20B modified with empty vector alone and expressing the wild-type amount of AHSG (SQ20B-EV) (in press). Here we have used shotgun proteomic analysis to identify additional proteins whose expression may also affect these in vitro properties of tumorigenesis associated with AHSG. The two cell lines were cultured in serum-free medium to avoid the contribution of exogenous AHSG in serum. Cell lysates were obtained, and the proteins were separated in polyacrylamide gels in duplicate. Each full protein lane was cut into 10 pieces, in-gel digested with trypsin and analyzed with tandem liquid chromatography mass spectrometry (LC-MS). MS data were analyzed against a recent human protein database. Proteins differentially expressed were quantified using a spectral counting approach. A total of 1386 distinct protein groups were identified in the two samples. Forty-eight proteins were differentially expressed in these samples (z score > 3, which corresponds to p ≤ 0.001, and Benjamini-Hochberg [statistical] FDR < 0.05). Proteins associated with the cytoskeleton, adhesion and apoptosis were over-represented in the group of differentially expressed proteins. Here we have shown that the human HNSCC cell line SQ20B exhibits changes in the proteome when AHSG expression is diminished. These proteins are critical for the tumorigenic properties of adhesion and migration. These data will help to identify mechanisms of tumorigenesis associated with AHSG in HNSCC.

Statistically enhanced spectral counting approach to TCDD cardiac toxicity in the adult zebrafish heart.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a persistent environmental pollutant and teratogen that produces cardiac toxicity in the developing zebrafish. Here we adopted a label free quantitative proteomic approach based on normalized spectral abundance factor (NSAF) to investigate the disturbance of the cardiac proteome induced by TCDD in the adult zebrafish heart. The protein expression level changes between heart samples from TCDD-treated and control zebrafish were systematically evaluated by a large scale MudPIT analysis, which incorporated triplicate analyses for both control and TCDD-exposed heart proteomic samples to overcome the data-dependent variation in shotgun proteomic experiments and obtain a statistically significant protein data set with improved quantification confidence. A total of 519 and 443 proteins were identified in hearts collected from control and TCDD-treated zebrafish, respectively, among which 106 proteins showed statistically significant expression changes. After correcting for the experimental variation between replicate analyses by statistical evaluation, 55 proteins exhibited NSAF ratios above 2 and 43 proteins displayed NSAF ratios smaller than 0.5, with statistical significance by t test (p < 0.05). The proteins identified as altered by TCDD encompass a wide range of biological functions including calcium handling, myocardium cell architecture, energy production and metabolism, mitochondrial homeostasis, and stress response. Collectively, our results indicate that TCDD exposure alters the adult zebrafish heart in a way that could result in cardiac hypertrophy and heart failure and suggests a potential mechanism for the diastolic dysfunction observed in TCDD-exposed embryos.

732 AN IN-DEPTH ANALYSIS OF THE PEDIATRIC URINARY PROTEOME

You have accessJournal of UrologyPediatrics: Basic Research1 Apr 2012732 AN IN-DEPTH ANALYSIS OF THE PEDIATRIC URINARY PROTEOME John W. Froehlich, Ali R. Vaezzadeh, Marc Kirchner, Andrew C. Briscoe, Oliver Hofmann, Winston Hide, Hanno Steen, and Richard S. Lee John W. FroehlichJohn W. Froehlich Boston, MA More articles by this author , Ali R. VaezzadehAli R. Vaezzadeh Boston, MA More articles by this author , Marc KirchnerMarc Kirchner Boston, MA More articles by this author , Andrew C. BriscoeAndrew C. Briscoe Boston, MA More articles by this author , Oliver HofmannOliver Hofmann Boston, MA More articles by this author , Winston HideWinston Hide Boston, MA More articles by this author , Hanno SteenHanno Steen Boston, MA More articles by this author , and Richard S. LeeRichard S. Lee Boston, MA More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2012.02.817AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Urine is a valuable body fluid for clinical analysis, and may be obtained noninvasively in large amounts. Many researchers are currently using urinary proteomics to identify biomarkers for specific diseases. Despite efforts to identify biomarkers in diseased samples, detailed proteomic analysis of non-diseased pediatric urine samples is still lagging behind. Furthermore, although age-specific changes are known to occur in rats, similar variations between pediatric and adult urines have not been determined. This study compares healthy male adult and infant urines, to establish which proteins are enriched or specific in infant's urine, and which proteins are present independent of age. METHODS Urine samples and demographic data were obtained from a urine specimen registry using an IRB approved protocol. Cellular debris was removed and 100 microgram protein aliquots were separated by PAGE. Proteins were digested with trypsin, extracted from the gel and purified before analysis by mass spectrometry using an LTQ-FT mass spectrometer (Thermo). MS data were searched against the uniprot KB database (homo sapiens). Peptides were filtered to ensure a 1% false discovery rate. A spectral counting approach was employed, using normalized spectral counts to minimize interpersonal variability. Statistically significant proteins (p<0.05) were identified, and analyzed by genome ontology to find overrepresented terms (p<0.0001) associated exclusively with adult and infant urines. RESULTS A total of 672 proteins were commonly identified in both cohorts, out of 1321 total protein identifications. Between our data and contemporary adult proteomic studies, 222 proteins were identified in infants alone. The infant-specific proteome was overrepresented in cellular metabolic processes, gene expression, and RNA processing. In addition, among commonly identified proteins, several proteins associated with glycolysis and ROS were enriched in the infant. These include pyruvate kinase isozymes M1/M2, Phosphoglycerate kinase 1, Aldose-1 epimerase, Retinal dehydrogenase 1, Fructose-1,6-bisphosphatase 1 and L-lactate dehydrogenase B chain. CONCLUSIONS We demonstrated that the male urinary proteome is highly age dependent, and hypothesize that the differences reflect differing growth and metabolic states between the adult and infant cohorts. These results highlight the importance of understanding the variation among non-diseased samples to better understand normal progression of the urinary proteome. © 2012 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 187Issue 4SApril 2012Page: e300-e301 Advertisement Copyright & Permissions© 2012 by American Urological Association Education and Research, Inc.MetricsAuthor Information John W. Froehlich Boston, MA More articles by this author Ali R. Vaezzadeh Boston, MA More articles by this author Marc Kirchner Boston, MA More articles by this author Andrew C. Briscoe Boston, MA More articles by this author Oliver Hofmann Boston, MA More articles by this author Winston Hide Boston, MA More articles by this author Hanno Steen Boston, MA More articles by this author Richard S. Lee Boston, MA More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

733 TISSUE ENGINEERING OF AN ORAL MUCOSA EQUIVALENT BY EVALUATION OF DIFFERENT BIOMATRICES

You have accessJournal of UrologyPediatrics: Basic Research1 Apr 2012733 TISSUE ENGINEERING OF AN ORAL MUCOSA EQUIVALENT BY EVALUATION OF DIFFERENT BIOMATRICES Daniela Flesch, Elena Frerick-Ochs, Celine Pfeifer, Ronald E. Unger, Annette Schröder, Raimund Stein, Joachim W. Thüroff, and Walburgis Brenner Daniela FleschDaniela Flesch Mainz, Germany More articles by this author , Elena Frerick-OchsElena Frerick-Ochs Mainz, Germany More articles by this author , Celine PfeiferCeline Pfeifer Mainz, Germany More articles by this author , Ronald E. UngerRonald E. Unger Mainz, Germany More articles by this author , Annette SchröderAnnette Schröder Mainz, Germany More articles by this author , Raimund SteinRaimund Stein Mainz, Germany More articles by this author , Joachim W. ThüroffJoachim W. Thüroff Mainz, Germany More articles by this author , and Walburgis BrennerWalburgis Brenner Mainz, Germany More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2012.02.818AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Oral mucosa has been used for urethral reconstruction in case of hypospadia for more than a decade. Harvesting large specimens of oral mucosa when reconstructing large sections of the urethra is technically challenging. Besides, it causes great discomfort, pain and secondary scarring to the patient. Therefore alternative techniques are required. One of these techniques is the construction of a tissue engineered oral mucosa, composed of a biomatrix seeded with primary cells. METHODS From human oral mucosa specimens, fibroblasts and epithelial cells were isolated and cultured separately. These primary cells were seeded on two collagen matrices (BioGide, BioGidePro) and on SIS (small intestinal mucosa). For coculture, fibroblasts were seeded on one side of the matrix and epithelial cells on the other side. After 21 days of cultivation the seeded matrices were analysed by fluorescent-vitality dyeing and immunohistochemically staining of Cytokeratin 13, Collagen IV and Tenascin. To achieve prevascularisation of the oral mucosa equivalent microvascular endothelial cells (HDMECs) were seeded together with fibroblasts on the porous side of the matrix. To analyse the triculture system a PECAM staining was performed and examined via confocal laser scanning microscope. Additionally the triculture was stained immunohistochemical. RESULTS Cocultures of the three matrices (BG, BGP, and SIS) showed typical characteristics of native oral mucosa. Staining with Cytokeratin-13 confirmed the formation of a multilayer epithelium on the cell occlusive site of the matrix. Between epithelium and matrix a basal membrane could be detected by immunohistochemical staining of collagen IV. Additionally fibroblast infiltration into the matrix could be observed. Fibroblasts produced new extracellular matrix, detected by Tenascin staining. In tricultures, a capillary-like network could be demonstrated. Fluorescent Laser analysis showed crosslinked capillary-like tubes on all tested matrices. An immunohistochemical staining of CD31 confirmed that the observed capillary-like structures contained a lumen. CONCLUSIONS Prevascularisation of a tissue engineered oral mucosa was successful, implicating advantages for the clinical use. All three matrices are applicable to construct an oral mucosa equivalent for hypospadia repair. © 2012 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 187Issue 4SApril 2012Page: e301 Advertisement Copyright & Permissions© 2012 by American Urological Association Education and Research, Inc.MetricsAuthor Information Daniela Flesch Mainz, Germany More articles by this author Elena Frerick-Ochs Mainz, Germany More articles by this author Celine Pfeifer Mainz, Germany More articles by this author Ronald E. Unger Mainz, Germany More articles by this author Annette Schröder Mainz, Germany More articles by this author Raimund Stein Mainz, Germany More articles by this author Joachim W. Thüroff Mainz, Germany More articles by this author Walburgis Brenner Mainz, Germany More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Proteomics-based Compositional Analysis of Complex Cellulase–Hemicellulase Mixtures

Efficient deconstruction of cellulosic biomass to fermentable sugars for fuel and chemical production is accomplished by a complex mixture of cellulases, hemicellulases, and accessory enzymes (e.g., >50 extracellular proteins). Cellulolytic enzyme mixtures, produced industrially mostly using fungi like Trichoderma reesei, are poorly characterized in terms of their protein composition and its correlation to hydrolytic activity on cellulosic biomass. The secretomes of commercial glycosyl hydrolase-producing microbes was explored using a proteomics approach with high-throughput quantification using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Here, we show that proteomics-based spectral counting approach is a reasonably accurate and rapid analytical technique that can be used to determine protein composition of complex glycosyl hydrolase mixtures that also correlates with the specific activity of individual enzymes present within the mixture. For example, a strong linear correlation was seen between Avicelase activity and total cellobiohydrolase content. Reliable, quantitative and cheaper analytical methods that provide insight into the cellulosic biomass degrading fungal and bacterial secretomes would lead to further improvements toward commercialization of plant biomass-derived fuels and chemicals.

Abstract P6-04-11: Quantitative Differences among Spectral Counts Based on LC-MS/MS Spectra Accurately Cluster Basal, Her2-Overexpressing and Luminal A Breast Carcinomas

Abstract Characterization of breast cancer subtypes at the protein-level is largely unexplored and is a powerful approach to identify novel biomarkers that may have diagnostic and prognostic utility. Shotgun proteomics analyzes mixtures at the peptide-level to generate MS/MS spectra that are then used to identify the peptides and proteins from which they were derived. We developed a method in our laboratory utilizing SDS-PAGE followed by in-gel digestion coupled with reversed-phase (RP) liquid chromatography-tandem mass spectrometry (RP-LC-MS/MS) for proteomic analysis of LCM-acquired cells. This method was then applied to a clinical sample set consisting of basal, Her2-overexpressing and luminal A frozen tumor tissues as determined by prior microarray studies. Three tumors per group were dissected in triplicate, for a total of 27 samples. Each dissection consisted of approximately 10,000 cells, corresponding to 3-5 μg protein. Following LCM, the thermoplastic membranes containing the captured cells were peeled from the cap, suspended in SDS loading buffer and gently heated for 10 minutes. The solubilized proteins were electrophoresed 2 cm into a 10-20% Tricine gel followed by in-gel trypsin digestion and peptide extraction. Replicate LC-MS/MS analysis of the tryptic peptides from each of the 27 samples was performed on a Thermo LTQ XL ion trap mass spectrometer. The resultant tandem mass spectra were searched against the human IPI database using the Myrimatch search algorithm and the results filtered using IDPicker. A spectral counting approach was applied to the data-dependent data to compare quantitative differences among the identified proteins. A total of 91,646 spectra were confidently identified, corresponding to 1671 protein groups across all biological and technical replicates. An average of 545, 543, and 610 protein groups were identified in the basal, ERBB2, and luminal A subtypes, respectively, from an equivalent of approximately 800 cells. Spectral count differences between datasets were analyzed using both a Quasi-likelihood Poisson regression model, developed in-house, and the Limma package in Bioconductor. Using a False Discovery Rate &amp;lt; 0.05, a total of 90 proteins showed statistically significant differences in expression among the breast tumor subtypes. Hierarchical cluster analysis generated a heat map of the protein expression patterns. Novel proteins as well as proteins corresponding to genes previously shown by gene array studies to be specific to each tumor type, including NES, ERBB2 and LGALS3BP, were represented among the differentially expressed proteins. These results demonstrate the feasability of this method to identify differential expression of protein biomarkers among breast cancer subtypes. These biomarkers should yield new insights into the pathogenesis of breast cancer and could be rapidly adapted into clinical diagnostics to guide therapeutic decisions for individual patients. The possible role in breast cancer for the novel proteins identified is currently under investigation. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P6-04-11.

Abstract 3746: Proteomic analysis of laser capture microdissected cells from basal, Her 2 positive and luminal A human breast tumors

Abstract The analysis of tissue is often confounded by the heterogenous nature, small size of biopsies, and focal presence of tumors. The use of laser capture microdissection (LCM) allows for isolation of nearly pure cell populations enhancing specificity of subsequent analyses. A method developed in our laboratory facilitating the removal of adhered cells utilizing SDS-PAGE followed by in-gel digestion and tandem mass spectrometry was applied to LCM-acquired cells from Basal, Her 2 positive and Luminal A breast tumor tissues. Cells were LCM-acquired from frozen tissue sections corresponding to Basal, Her 2 positive or Luminal A human breast tumor tissue. The sample set consisted of 3 tumors per subgroup with 3 caps per tumor, for a total of 27 caps each containing approximately 10,000 cells corresponding to 3-5 μg protein. The thermoplastic membranes containing the captured cells were peeled from the cap and suspended in SDS loading buffer and gently heated for 10 minutes. The solubilized proteins were electrophoresed 2 cm into a 10-20% Tricine gel followed by in-gel trypsin digestion and peptide extraction. Tryptic peptides were analyzed by reversed-phase (RP) liquid chromatography-tandem mass spectrometry (LC-MS-MS) on a Thermo Orbitrap instrument. Following LC/MS/MS analysis of the LCM digests, tandem spectra were searched against the human IPI database using the search algorithm, Myrimatch, and the search results filtered using IDPicker. A spectral counting approach was applied to the data-dependent data to compare quantitative differences of proteins identified. A total of 91646 spectra were confidently identified which corresponded to 1671 protein groups across all biological and technical replicates. An average of 545, 543, and 610 protein groups were identified in the basal, Her 2 positive, and Luminal A subtypes, respectively, from an equivalent of roughly 800 cells. Spectral count differences between datasets between normal and tumor samples were analyzed using both a Quasi-likelihood Poisson regression model, developed in-house, in addition to using the Limma package in Bioconductor. Using the criteria of a False Discovery Rate (FDR) &amp;lt; 0.05, a total of 90 proteins showed statistically significant differences in expression among the breast tumor subtypes. A hierarchical clustering of these proteins was performed, creating a heat map representing the gene expression patterns of these proteins. As expected, ERBB2 was identified in the Her 2 positive tumor tissue samples; however, this protein was not identified in either the Basal or Luminal A samples. The proteins identified which were found to distinguish each tissue subtype are currently being investigated for their biological relevance in breast cancer pathways and as possible prognostic and predictive biomarkers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3746.

Abstract 4358: Analysis of the cell surface proteome for the identification of candidate diagnostic and therapeutic targets in drug resistant childhood acute lymphoblastic leukemia (ALL)

Abstract Relapse of acute lymphoblastic leukemia (ALL) is a leading cause of cancer-related childhood mortality. Persistence of significant levels of minimal residual disease after almost 6 months of treatment identifies a group of patients with very high risk of relapse (VHR-ALL). Prognostic markers are not available to identify this subgroup at diagnosis. These VHR-ALL patients would qualify for new experimental approaches as part of their initial therapy. Here we present an extensive analysis of the cell surface glycoproteome of ALL cells from 8 VHR-ALL patients and from a corresponding set of ALL patients with good clinical outcome. Given the very limited amount of primary diagnostic material available for research, we have amplified primary human ALL cells in our established xenotransplantation model, using immunodeficient NSG mice. This amplification of primary human material allowed us for the first time to generate normally rare human leukemic cells in numbers sufficient for proteomic studies. We have optimized and extended the mass spectrometry-based Cell Surface Capturing (CSC) technology with complementary enrichment strategies, to increase the protein sequence coverage. Roughly 2500 candidate cell surface proteins were identified. 40% of the candidates were predicted by the TMHMM algorithm to contain a transmembrane domain or a GPI-anchor, also including 174 known CD markers. The immunophenotype analysis of leukemia associated surface markers by flow cytometry at diagnosis was recapitulated in our proteomic dataset in all cases. A semi-quantitative spectral counting approach identified a subset of 200 proteins consistently expressed on most ALL samples and a subset of 60 proteins preferentially detected on the surface of VHR-ALL samples. A subgroup of VHR-ALL, which represented 50% of the analysed cases, was characterized by high expression of members of the vanin (VNN) protein family. Flow cytometry data of VNN-2 from xenografted samples correlated with the semiquantitative estimation of protein levels by the CSC technology. In an independent cohort of 20 ALL samples, we did not detect VNN-2 expression on ALL cells from standard risk patients, while we detected VNN-2 expression in 4 out of 8 cases of high risk or relapsed patients. Anti-VNN-2 antibodies stained the ALL cells homogeneously, indicating that VNN-2 expression could mark the entire population of leukemia cells. Since vanin family proteins have been proposed to mediate interactions between hematopoietic cells and their microenvironment, our findings also provide the basis to evaluate the functional role of vanins in ALL. Our data provide an unprecedented view at the cell surface landscape of the most refractory leukemia cases and identify subsets of cell surface proteins that could be used for diagnosis or therapeutic intervention in this deadly disease. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4358.

Comparison of multidimensional shotgun technologies targeting tissue proteomics

A compelling need exists for the development of technologies that facilitate and accelerate the discovery of novel protein biomarkers with therapeutic and diagnostic potential. Comparisons among shotgun proteome technologies, including capillary isotachophoresis (CITP)-based multidimensional separations and multidimensional LC system, are therefore performed in this study regarding their abilities to address the challenges of protein complexity and relative abundance inherent in glioblastoma multiforme-derived cancer stem cells. Comparisons are conducted using a single processed protein digest with equal sample loading, identical second-dimension separation (RPLC) and MS conditions, and consistent search parameters and cutoff established by the target-decoy determined false-discovery rate. Besides achieving superior overall proteome performance in total peptide, distinct peptide, and distinct protein identifications; analytical reproducibility of the CITP proteome platform coupled with the spectral counting approach are determined by a Pearson R(2) value of 0.98 and a CV of 15% across all proteins quantified. In contrast, extensive fraction overlapping in strong cation exchange greatly limits the ability of multidimensional LC separations for mining deeper into the tissue proteome as evidenced by the poor coverage in various protein functional categories and key protein pathways. The CITP proteomic technology, equipped with selective analyte enrichment and ultrahigh resolving power, is expected to serve as a critical component in the overall toolset required for biomarker discovery via shotgun proteomic analysis of tissue specimens.

In-Depth Profiling of Post-Translational Modifications on the Related Transcription Factor Complexes TFIID and SAGA

The basal transcription factor TFIID and the chromatin-modifying complex SAGA, which have several subunits in common, are crucial for transcription regulation. Here, we describe an in-depth profiling of post-translational modifications (PTMs) on both TFIID and SAGA from yeast. We took a multipronged approach using high-resolution mass spectrometry (LC-MS) in combination with the proteases Trypsin, Chymotrypsin and Glu-C. The cumulative peptide identification data, at a false discovery rate <1%, allowed us to cover most TFIID and SAGA subunit sequences to near completion. Additionally, for TFIID/SAGA subunits, we identified 118/102 unique phosphorylated and 54/61 unique lysine acetylated sites. Especially, several lysine residues on the SAGA subunits Spt7p and Sgf73p were found to be acetylated. Using a spectral counting approach, we found that the shared subunit TAF5p is phosphorylated to a significant greater extent in SAGA than in TFIID. Finally, we were able to map for the first time the cleavage site in Spt7p that is related to formation of the SAGA-like complex SLIK/SALSA. In general, our combination of tandem affinity enrichment, digestion with different proteases, extensive prefractionation and high-resolution LC-MS identifies a large number of PTMs of TFIID and SAGA/SLIK that might aid in future functional studies on these transcription factors.

Differential Membrane Proteome Analysis Reveals Novel Proteins Involved in the Degradation of Aromatic Compounds in Geobacter metallireducens

Aromatic compounds comprise a large class of natural and man-made compounds, many of which are of considerable concern for the environment and human health. In aromatic compound-degrading anaerobic bacteria the central intermediate of aromatic catabolism, benzoyl coenzyme A, is attacked by dearomatizing benzoyl-CoA reductases (BCRs). An ATP-dependent BCR has been characterized in facultative anaerobes. In contrast, a previous analysis of the soluble proteome from the obligately anaerobic model organism Geobacter metallireducens identified genes putatively coding for a completely different dearomatizing BCR. The corresponding BamBCDEFGHI complex is predicted to comprise soluble molybdenum or tungsten, selenocysteine, and FeS cluster-containing components. To elucidate key processes involved in the degradation of aromatic compounds in obligately anaerobic bacteria, differential membrane protein abundance levels from G. metallireducens grown on benzoate and acetate were determined by the MS-based spectral counting approach. A total of 931 proteins were identified by combining one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis with liquid chromatography-tandem mass spectrometry. Several membrane-associated proteins involved in the degradation of aromatic compounds were newly identified including proteins with similarities to modules of NiFe/heme b-containing and energy-converting hydrogenases, cytochrome bd oxidases, dissimilatory nitrate reductases, and a tungstate ATP-binding cassette transporter system. The transcriptional regulation of differentially expressed genes was analyzed by quantitative reverse transcription-PCR; in addition benzoate-induced in vitro activities of hydrogenase and nitrate reductase were determined. The results obtained provide novel insights into the poorly understood degradation of aromatic compounds in obligately anaerobic bacteria.

Evaluation of confidence and reproducibility in quantitative proteomics performed by a capillary isoelectric focusing‐based proteomic platform coupled with a spectral counting approach

Multidimensional separations of the peptides resulting from enzymatic digestions of complex protein mixtures prior to MS/MS, namely shotgun proteomics, is increasingly utilized for large-scale identification and quantitation of proteins. Inherent to the performance of proteomic measurements is the resolving power of each of the separations both separately and in combination. By simply raising the number of CIEF fractions, the resulting enhancement in the overall peak capacity of combined CIEF/nano-RPLC separations greatly reduces the complexity of eluted peptides prior to MS detection and sequencing and increases the proteome coverage. The capabilities of the CIEF-based proteome platform coupled with the spectral counting approach to confidently and reproducibly quantify proteins and changes in protein expression levels among samples are evaluated. Analytical reproducibility of relative protein abundance is determined to exhibit a Pearson R(2) value greater than 0.99 and a CV of 14.1%. The platform is demonstrated to be capable of measuring changes in protein expression as low as 1.5-fold, with confidence following multiple testing adjustment.

Correlating the Transcriptome, Proteome, and Metabolome in the Environmental Adaptation of a Hyperthermophile

We have performed a comprehensive characterization of global molecular changes for a model organism Pyrococcus furiosus using transcriptomic (DNA microarray), proteomic, and metabolomic analysis as it undergoes a cold adaptation response from its optimal 95 to 72 degrees C. Metabolic profiling on the same set of samples shows the down-regulation of many metabolites. However, some metabolites are found to be strongly up-regulated. An approach using accurate mass, isotopic pattern, database searching, and retention time is used to putatively identify several metabolites of interest. Many of the up-regulated metabolites are part of an alternative polyamine biosynthesis pathway previously established in a thermophilic bacterium Thermus thermophilus. Arginine, agmatine, spermidine, and branched polyamines N4-aminopropylspermidine and N4-( N-acetylaminopropyl)spermidine were unambiguously identified based on their accurate mass, isotopic pattern, and matching of MS/MS data acquired under identical conditions for the natural metabolite and a high purity standard. Both DNA microarray and semiquantitative proteomic analysis using a label-free spectral counting approach indicate the down-regulation of a large majority of genes with diverse predicted functions related to growth such as transcription, amino acid biosynthesis, and translation. Some genes are, however, found to be up-regulated through the measurement of their relative mRNA and protein levels. The complimentary information obtained by the various "omics" techniques is used to catalogue and correlate the overall molecular changes.

Compositional Protein Analysis of High Density Lipoproteins in Hypercholesterolemia by Shotgun LC-MS/MS and Probabilistic Peptide Scoring

A protein of a biological sample is usually quantified by immunological techniques based on antibodies. Mass spectrometry offers alternative approaches that are not dependent on antibody affinity and avidity, protein isoforms, quaternary structures, or steric hindrance of antibody-antigen recognition in case of multiprotein complexes. One approach is the use of stable isotope-labeled internal standards; another is the direct exploitation of mass spectrometric signals recorded by LC-MS/MS analysis of protein digests. Here we assessed the peptide match score summation index based on probabilistic peptide scores calculated by the PHENYX protein identification engine for absolute protein quantification in accordance with the protein abundance index as proposed by Mann and co-workers (Rappsilber, J., Ryder, U., Lamond, A. I., and Mann, M. (2002) Large-scale proteomic analysis of the human spliceosome. Genome Res. 12, 1231-1245). Using synthetic protein mixtures, we demonstrated that this approach works well, although proteins can have different response factors. Applied to high density lipoproteins (HDLs), this new approach compared favorably to alternative protein quantitation methods like UV detection of protein peaks separated by capillary electrophoresis or quantitation of protein spots on SDS-PAGE. We compared the protein composition of a well defined HDL density class isolated from plasma of seven hypercholesterolemia subjects having low or high HDL cholesterol with HDL from nine normolipidemia subjects. The quantitative protein patterns distinguished individuals according to the corresponding concentration and distribution of cholesterol from serum lipid measurements of the same samples and revealed that hypercholesterolemia in unrelated individuals is the result of different deficiencies. The presented approach is complementary to HDL lipid analysis; does not rely on complicated sample treatment, e.g. chemical reactions, or antibodies; and can be used for projective clinical studies of larger patient groups.