MALDI versus ESI: The Impact of the Ion Source on Peptide Identification

For mass spectrometry-based proteomic analyses, electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) are the commonly used ionization techniques. To investigate the influence of the ion source on peptide detection in large-scale proteomics, an optimized GeLC/MS workflow was developed and applied either with ESI/MS or with MALDI/MS for the proteomic analysis of different human cell lines of pancreatic origin. Statistical analysis of the resulting data set with more than 72 000 peptides emphasized the complementary character of the two methods, as the percentage of peptides identified with both approaches was as low as 39%. Significant differences between the resulting peptide sets were observed with respect to amino acid composition, charge-related parameters, hydrophobicity, and modifications of the detected peptides and could be linked to factors governing the respective ion yields in ESI and MALDI

Engraftment and retransplantation of AML cells in NSG mice conserves genetic alterations of the primary sample.

<p>Primary AML patient samples and matched PDX cells, reisolated out of the BM (CD45 chimerism 80–99%) after first passage in NSG mice (PDX-0) or after 1 or 2 re-transplantation cycles (PDX-1/-2), were characterized by targeted resequencing of 43 AML-related genes (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120925#pone.0120925.s009" target="_blank">S1 Table</a>). Plots depict variant allele frequencies for each driver gene mutation found within the sample. a/b/c/d/f: PDX cells of three to five mice injected in parallel were analyzed. *: primary cells were frozen and thawed before injection. <i>BCOR</i> (BCL-6 corepressor); <i>CEBPA</i> (CCAAT/enhancer binding protein alpha); <i>DNMT3A</i> (DNA (cytosine-5)-methyltransferase 3 alpha); <i>FLT3</i> (Fms-related tyrosine kinase 3); ITD (internal tandem duplication); <i>KRAS</i> (Kirsten rat sarcoma viral oncogene homolog); <i>NPM1</i> (nucleophosmin-1); <i>NRAS</i> (neuroblastoma RAS viral oncogene homolog); <i>SRSF2</i> (serine/arginine-rich splicing factor 2); <i>TET2</i> (tet methylcytosine dioxygenase 2); <i>TP53</i> (tumor protein p53). Raw data is depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120925#pone.0120925.s010" target="_blank">S2 Table</a>.</p

BLI is highly sensitive and reliable in single mice.

<p><b>(A)</b> 1x10⁵ t-PDX AML-372 cells were injected into two mice. At indicated days after cell injection, mice were monitored by BLI. Images of one representative mouse are shown. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120925#pone.0120925.s005" target="_blank">S5A Fig.</a> for further images. <b>(B)</b> BLI signals from the kinetic in <b>A</b> were quantified in both animals (diamonds); cells positive for both hCD45 and hCD33 in PB were analyzed in parallel (circles). <b>(C)</b> t-PDX AML-372 cells were injected into three mice per group at absolute numbers indicated; 1 and 8 days after cell injection, mice were monitored by BLI; images are shown of one representative mouse per group.</p

Boletín de Segovia: Número 30 - 1918 marzo 11

Copia digital. Madrid : Ministerio de Cultura. Subdirección General de Coordinación Bibliotecaria, 200

Engraftment of primary AML cells in NSG mice predicts reengraftment capacity.

<p>10⁷ fresh primary AML cells were injected and successfully engrafted in NSG mice; shown are characteristics of the first engraftment regarding passaging time (time period from cell injection until clinical signs of leukemia or latest between 20 and 25 weeks) <b>(A)</b>; percentage of cells positive for both hCD45 and hCD33 at time of sacrifice within mouse PB <b>(B)</b> and within BM (black cubes) or spleen (grey circles) <b>(C)</b>. Each mark visualizes data obtained from a single mouse. Open cubes indicate 0% human cells. Dotted line discriminates samples that reengrafted in secondary recipients from samples that did not. Please refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120925#pone.0120925.s001" target="_blank">S1A Fig.</a> for exemplary FACS plots.</p

PDX AML cells allow genetic engineering without altering molecular sample characteristics.

<p><b>(A)</b> Scheme of the process of generating transgenic PDX (t-PDX) AML cells. PDX cells were transduced after first or second retransplantation cycle. <b>(B)</b> Scheme of the vector constructs. <b>(C)</b> Transduction rate in t-PDX AML cells after lentiviral transduction and cell amplification in mice was measured by FACS analysis of fluorochrome or NGFR expression. Each mark visualizes data obtained from a single transduction. Open mark: no transgenic cells were detectable. <b>(D)</b> Enrichment of transgenic cells using flow cytometry was measured using mCherry expression after cell amplification in mice. <b>(E)</b> Genetic engineering does not alter immunophenotype; primary cells, untransduced PDX cells after fourth retransplantation and enriched transgenic t-PDX cells were analyzed by multicolor flow cytometry; specific fluorescence intensity is depicted. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120925#pone.0120925.s003" target="_blank">S3C Fig.</a> for exemplary FACS plots of AML-372. Raw data is depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120925#pone.0120925.s011" target="_blank">S3 Table</a>. <b>(F)</b> Genetic engineering does not markedly alter AML-specific mutations; genomic DNA was isolated out of primary cells, untransduced PDX cells and enriched transgenic t-PDX cells; VAF of mutations was profiled by targeted resequencing. <i>BCOR</i> (BCL-6 corepressor); <i>KRAS</i> (Kirsten rat sarcoma viral oncogene homolog); <i>NRAS</i> (neuroblastoma RAS viral oncogene homolog); <i>TP53</i> (tumor protein p53). Raw data is depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120925#pone.0120925.s010" target="_blank">S2 Table</a>.</p

Additional file 5: Figure S3. of The sialyl-glycolipid stage-specific embryonic antigen 4 marks a subpopulation of chemotherapy-resistant breast cancer cells with mesenchymal features

Correlation among CD24-, CD44-, and SSEA4-expressing subpopulations. To address the correlation between CD24, CD44, and SSEA4 expression, we performed costaining of these markers on residual tumor nodules after AC chemotherapy and untreated tumors of three independent models: HBCx-6 (a), HBCx-10 (b), and HBCx-14 (c). Regulation of the three markers did not correlate among the treatment cycles. (PNG 1664 kb

Additional file 1: of The sialyl-glycolipid stage-specific embryonic antigen 4 marks a subpopulation of chemotherapy-resistant breast cancer cells with mesenchymal features

Supplemental experimental procedures. Detailed description of materials and methods. (DOCX 62 kb

Additional file 9: Table S3. of The sialyl-glycolipid stage-specific embryonic antigen 4 marks a subpopulation of chemotherapy-resistant breast cancer cells with mesenchymal features

Results of microarray analysis. Results of gene and miRNA expression profiling, Gene Ontology analysis, and miRNA target prediction. (XLSX 118 kb

Additional file 12: Figure S8. of The sialyl-glycolipid stage-specific embryonic antigen 4 marks a subpopulation of chemotherapy-resistant breast cancer cells with mesenchymal features

SSEA4-positive breast cancer cells show decreased expression of miRNAs inhibiting EMT inducers. Expression ratios of the 12 miRNAs targeting the key mesenchymal regulator and indicator genes ZEB1, ZEB2, fibronectin 1, Snail1, Snail2, and Twist. Each bar represents the log2 expression ratio of the SSEA4-positive fraction relative to the SSEA4-negative fraction for the respective tumor model. (TIFF 201 kb