Improved Proteome and Phosphoproteome Analysis on a Cation Exchanger by a Combined Acid and Salt Gradient

Currently used elution methods for strong cation exchange (SCX) chromatography are based on two principles: salt and pH gradient. In this paper, we report the first observation of peptide elution by acid gradient. The degree of peptide separation using C18-SCX StageTip was greatly improved by our acid and salt-based elution method compared with a salt-based elution method. This development enabled us to identify over 22 000 phosphopeptides from 2 mg of protein without labor-intensive sample preparation. Our method is simple, robust, scalable, and low-cost and can be easily implemented without any special equipment or techniques

Real-Time Motion Analysis Reveals Cell Directionality as an Indicator of Breast Cancer Progression

<div><p>Cancer cells alter their migratory properties during tumor progression to invade surrounding tissues and metastasize to distant sites. However, it remains unclear how migratory behaviors differ between tumor cells of different malignancy and whether these migratory behaviors can be utilized to assess the malignant potential of tumor cells. Here, we analyzed the migratory behaviors of cell lines representing different stages of breast cancer progression using conventional migration assays or time-lapse imaging and particle image velocimetry (PIV) to capture migration dynamics. We find that the number of migrating cells in transwell assays, and the distance and speed of migration in unconstrained 2D assays, show no correlation with malignant potential. However, the directionality of cell motion during 2D migration nicely distinguishes benign and tumorigenic cell lines, with tumorigenic cell lines harboring less directed, more random motion. Furthermore, the migratory behaviors of epithelial sheets observed under basal conditions and in response to stimulation with epidermal growth factor (EGF) or lysophosphatitic acid (LPA) are distinct for each cell line with regard to cell speed, directionality, and spatiotemporal motion patterns. Surprisingly, treatment with LPA promotes a more cohesive, directional sheet movement in lung colony forming MCF10CA1a cells compared to basal conditions or EGF stimulation, implying that the LPA signaling pathway may alter the invasive potential of MCF10CA1a cells. Together, our findings identify cell directionality as a promising indicator for assessing the tumorigenic potential of breast cancer cell lines and show that LPA induces more cohesive motility in a subset of metastatic breast cancer cells.</p> </div

Cell lines of the MCF10A series show distinct migration speed and directionality.

<p>(<b>A</b>) PIV analysis enables the mapping of velocity fields associated with the underlying epithelial sheet motions captured by phase time-lapse imaging (scale bar  = 100 µm). Spatial profiles of directionality and speed are depicted with white vectors and a heat map, respectively (right panel). (<b>B</b>) Left: Aggregate speed distributions, determined over all times and space, were compiled from 5–6 independent experiments for each cell line. Right: Quantification of the mean of the average speed (mean ±95% CI) for each cell line; M1–M2 (benign, black circles) and M3–M4 (tumorigenic, red triangles). (<b>C</b>) Left: Rose plots depicting aggregate directionality distributions were compiled over all times and space for each cell line (n = 5–6). Right: Variability of the direction of motion was quantified by the coefficient of variation (CV) and reported as mean ±95% CI. Statistical significance: * p<0.05, ** p<0.01, *** p<0.001 (Tukey-Kramer test, n = 5–6). All comparisons were made with M1 cells unless indicated by pairing-brackets.</p

Cell lines of the MCF10A series show distinct migration properties.

<p>(<b>A</b>) Migration potential of M1–M2 (benign, black circles) and M3–M4 (tumorigenic, red triangles) cell lines after 4 h was assessed with the transwell assay using collagen IV coated membranes and no biased stimulation (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058859#pone.0058859.s001" target="_blank">Fig. S1A</a>). (<b>B</b>) Phase images of the M1–M4 cell lines after 0 and 12 h of unconstrained migration. The dash vertical line indicates the initial location of the sheet edge. Scale bar  = 100 µm. (<b>C</b>) Quantification of the net displacement (during the 3–12 h time frame) is presented as in panel A. (<b>D</b>) M1–M4 cells were first pretreated with 25 µg/mL Mitomycin C for 20 min and then allowed to migrate into open space under conditions identical to panel B (black bars). The net displacement (mean ± SD) is shown compared to control (w/o drug) conditions (white bars), n = 2. For panels A and C results are presented as mean ±95% CI of 6–7 independent experiments. Statistical significance: * p<0.05, ** p<0.01, *** p<0.001, ****p<0.0001 (Tukey-Kramer test, n = 6–7). All comparisons were made with M1 cells unless indicated by pairing-brackets.</p

Cell lines of the MCF10A series show distinct responses to EGF and LPA.

<p>(<b>A–D</b>) M1–M4 cells were stimulated with 5 ng/mL EGF (red) or 1 µM LPA (blue) and perturbations of average speed and of directionality (angle distributions and CV) compared to controls (black) were assessed (mean ± SD). Rose plots depict controls (unfilled, black bars) and 5 ng/mL EGF (filled, red bars) or 1 µM LPA (filled, blue bars). Statistical significance: * p<0.05, ** p<0.01, *** p<0.001 (Tukey-Kramer test, n = 3 for all conditions except M2 with EGF where n = 2). All comparisons were made with M1 cells unless indicated by pairing-brackets.</p

The migratory phenotype of MDA-MB 231T is similar to that of M4 cells.

<p>(<b>A</b>) Phase contrast images of MDA-MB-231T cells moving into open space after 12 h under control (Black), 5 ng/mL EGF (red), and 1 µM LPA (blue) treatments. Bar = 100 µm. (<b>B</b>) Right: The effects of EGF and LPA treatment on average speed (top) and directionality, CV (bottom) determined over all times and space, were compiled from 5–6 independent experiments and reported as mean ± SD. Left: Aggregate directionality profiles for control, EGF and LPA conditions. Statistical significance: *p<0.05 (Tukey-Kramer test, n = 3). (<b>C</b>) Representative spatiotemporal heat plots show speed responses in control, EGF, and LPA treated cells. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058859#pone-0058859-g004" target="_blank">Fig. 4</a> for details.</p

Validation of protein expression using western blot and immunohistochemistry.

<p>(A) Metastases lysates were prepared from three mice per group and evaluated by western blot with anti-vimentin, anti-Hsp90a/b, and anti-Eno1 antibodies. β-actin was used as internal normalization. (B) Representative image of positive vimentin staining is shown exclusively in the metastases lesion and not in the surrounding lung tissue. White dotted lines represent a boundary of tumor and surrounding normal lung tissue. M, metastases; NT, normal lung tissue; PV, pulmonary vein. (C) Randomly selected high power fields were immunostained with vimentin and were quantitated using Zeiss software with the % area occupied by metastasized nodules in 4T1 metastasized tumors. A significant reduction in the number of vimentin positive cells was seen in the SB-431542-treated tumors. Scale bars represent 100 μm; hpf, high power fields.</p

Protein expression profiling of 4T1 metastases tumor using dimethyl labeling with triplex stable isotopes.

<p>(A) Schematic representation of the proteomics approach. Lung-metastasized 4T1 tumor samples were isolated from each group (control and SB-431542-treated tumors). Subsequently, the tumor protein was lysed, and differential protein expression was detected by relative quantification using dimethyl-labeling (light, medium and high) followed by an SCX-LC-MS/MS (LTQ Orbitrap Velos) analysis for each set of sample quadruplicates. Isotope-light was used as the reference sample and contained mixed aliquots of all control and SB-431542-treated tumor samples. Four sets of dimethyl-labeled experiments were performed to compare the protein profiles of 8 metastasized tumor samples. (B) Representative dimethyl labeled-based LC-MS/MS spectrum for one of the peptides from vimentin (P20152) showing RQVQSLTCEVDALK, a double charged peptide.</p

Orthotopic metastasis model demonstrates that a specific inhibitor of TGF-β receptor kinase, SB-431542, decreases lung metastasis but does not significantly alter growth of the primary tumor.

<p>(A) Scheme for the experimental approach using 4T1 metastasis model. 4T1 tumor cells (1×10⁴ cells) were transplanted into the mammary fat pad of Balb/c mice. Mice bearing 4T1 mammary tumors were treated three times weekly with SB-431542 (10 mg/kg body weight) or vehicle (20% DMSO/80% corn oil). At day 10 post-injection of 4T1 cells, primary tumors were surgically excised, and the mice were kept alive to allow the tumor to metastasize to the lung. (B) Graph showing relative primary tumor growth of 4T1 cells over time. Data are presented as mean ± SEM. (ns = not significant, control, n = 14; SB-431542, n = 15, unpaired <i>t</i>-test). (C) The number of gross metastasis (control, n = 14; SB-431542-treated, n = 15) was counted. The administration of SB-431542 markedly reduced both the number and the size of the metastasized tumor compared to the control group. Lungs were collected after 40 days and the lung surface was examined for the metastasis. The number of visible lung metastasis was counted. ** p<0.001.; Mann Whitney <i>U</i>-test. Data are presented as the median ± SEM. (D) Representative gross lung images from control and SB-431542-treated groups are shown from control (top) and SB-431542-treated (bottom) animals, with metastases visible at the lung surface marked by bold black arrows. (E) Representative histological view of lung metastases treated with vehicle, DMSO (left) and SB-431542 (right). H&E staining; black dotted lines demarcate tumor parenchyma (*) from the normal lung tissue.</p

EIF signaling was the most enriched canonical IPA pathway of the down-regulated proteins in SB-431542-treated groups.

<p>(A) Pathway analysis conducted using IPA (<a href="http://www.ingenuitypathway.com" target="_blank">www.ingenuitypathway.com</a>) showed a ranking of the most enriched pathways from the down-regulated proteins in SB-431542-treated groups. (B) Whole cell lysates from each group were evaluated by western blot with the EIF family of proteins: anti-eIF4G1, anti-eIF4E, and anti-Eef2 antibodies. (n = 3/group).β -actin was used as a loading control. (C) Sections were assessed by H&E staining (top panel), and immunohistochemistry was performed using anti-eIF4G1 (middle panel) and anti-eIF4E (bottom panel) antibodies. Both eIF4G1 and eIF4E immunostaining in 4T1 lung metastasis showed the heterogeneity of staining patterns seen among individual metastases. 100× magnification. Tumor sections are boxed with a black dotted line, M, metastases; NT, normal lung tissue. (D) eIF4G1- and eIF4E-positive areas were semiquantified using Image Pro Premier Software in three randomly selected high power fields from each samples. Data are represented as box plots (n = 3/group), ** p<0.001 using an unpaired <i>t</i>-test.</p