Characterization of protein isoform diversity in human umbilical vein endothelial cells via long-read proteogenomics

Endothelial cells (ECs) comprise the lumenal lining of all blood vessels and are critical for the functioning of the cardiovascular system. Their phenotypes can be modulated by alternative splicing of RNA to produce distinct protein isoforms. To characterize the RNA and protein isoform landscape within ECs, we applied a long read proteogenomics approach to analyse human umbilical vein endothelial cells (HUVECs). Transcripts delineated from PacBio sequencing serve as the basis for a sample-specific protein database used for downstream mass-spectrometry (MS) analysis to infer protein isoform expression. We detected 53,863 transcript isoforms from 10,426 genes, with 22,195 of those transcripts being novel. Furthermore, the predominant isoform in HUVECs does not correspond with the accepted “reference isoform” 25% of the time, with vascular pathway-related genes among this group. We found 2,597 protein isoforms supported through unique peptides, with an additional 2,280 isoforms nominated upon incorporation of long-read transcript evidence. We characterized a novel alternative acceptor for endothelial-related gene CDH5, suggesting potential changes in its associated signalling pathways. Finally, we identified novel protein isoforms arising from a diversity of RNA splicing mechanisms supported by uniquely mapped novel peptides. Our results represent a high-resolution atlas of known and novel isoforms of potential relevance to endothelial phenotypes and function.</p

Accurate Label-Free Protein Quantitation with High- and Low-Resolution Mass Spectrometers

Label-free quantitation of proteins analyzed by tandem mass spectrometry uses either integrated peak intensity from the parent-ion mass analysis (MS1) or features from fragment-ion analysis (MS2), such as spectral counts or summed fragment-ion intensity. We directly compared MS1 and MS2 quantitation by analyzing human protein standards diluted into <i>Escherichia coli</i> extracts on an Orbitrap mass spectrometer. We found that summed MS2 intensities were nearly as accurate as integrated MS1 intensities, and both outperformed MS2 spectral counting in accuracy and linearity. We compared these results to those obtained from two low-resolution ion-trap mass spectrometers; summed MS2 intensities from LTQ and LTQ Velos instruments were similar in accuracy to those from the Orbitrap. Data from all three instruments are available via ProteomeXchange with identifier PXD000602. Abundance measurements using MS1 or MS2 intensities had limitations, however. While measured protein concentration was on average well-correlated with the known concentration, there was considerable protein-to-protein variation. Moreover, not all human proteins diluted to a mole fraction of 10^–3 or lower were detected, with a strong falloff below 10^–4 mole fraction. These results show that MS1 and MS2 intensities are simple measures of protein abundance that are on average accurate but should be limited to quantitation of proteins of intermediate to higher fractional abundance

Protein synthesis is decreased in rigidity-dependent cells cultured on soft gels.

<p>A549 cells were subjected to SILAC analysis to determine rates of protein synthesis on soft or stiff gels. <b>A.</b> Overview of the SILAC procedure. A549 cells were cultured on soft or stiff gels for 4 days in the presence of “heavy” media, followed by a 24-hour incubation with “light” media. The cells were lysed, cellular proteins were digested with trypsin, and the resulting peptides were analyzed by mass spectrometry. <b>B.</b> Boxplots of heavy to light (H/L) ratios of proteins from A549 cells (left) or mPanc96 cells (right) grown on stiff (19200 Pa) or soft (150 Pa) substrates. H/L ratio distributions are significantly different between stiff and soft for A549 cells but not for mPanc96 cells using two-tailed unpaired t-tests. The boxes contain the data between the 25 and 75 percentile, and the line within the box indicates the median. The dashed line at the top of the graph marks the upper limit, above which the outliers were truncated.</p

BrdU pulse-chase of cell cycle progression.

<p>A549 cells were pulsed with BrdU for 30 minutes following growth on soft or stiff gels for 2 days. <b>A.</b> Labeling cells with BrDU for cell cycle analysis. Cells are pulsed with BrDU for 30 min, and the BrDU-positive population is followed over time as it transitions through the phases of the cell cycle. <b>B.</b> Scatter plot histograms of BrdU-labeled cells on soft (top panel) or stiff (bottom panel) gels, stained for DNA content (X-axis) and BrdU (Y-axis). The times indicated are the times, in hours, after the BrdU pulse. <b>C.</b> Cell cycle progression analysis was performed on the scatter plot histograms from the cells grown on gels for 2 days (left) or 5 days (right).</p

Identification of proteins that are differentially regulated by rigidity.

<p>H/L ratios of proteins identified in both stiff and soft samples plotted against each other for A549 cells (left) and mPanc96 cells (right). H/L ratios from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037231#pone-0037231-g004" target="_blank">Figure 4</a> were quantile normalized and t-tests were performed using estimated variability (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037231#s4" target="_blank">Methods</a>) to identify individual proteins with relatively different synthesis rates between stiff and soft samples. Proteins that are synthesized faster (relatively lower H/L ratio and p-value<0.05) in soft samples (compared to stiff samples) are shown in red, and proteins that are synthesized slower in soft samples are shown in green.</p

Cyclin D1 expression in rigidity-dependent cells growing on soft and stiff gels.

<p>A549 cells and MDA-MB-231 cells were cultured on 150 Pa, 4800 Pa, or 19200 Pa polyacrylamide gels for 2 or 5 days. Cells were lysed and analyzed by western blot for the expression of cyclin D1 (top panel). The expression of GAPDH was analyzed as a loading control (bottom panel). The blot is representative of three experiments.</p

Proteins in A549 cells identified by mass spectrometry whose syntheses are most sensitive to changes in rigidity.

<p>The table lists proteins that exhibited higher heavy/light ratios (and therefore, lower synthesis) on soft gels compared to the mean.</p

Proteins in mPanc96 cells identified by mass spectrometry whose rates of syntheses decrease when cultured on soft gels.

<p>The table lists proteins that exhibited higher heavy/light ratios (and therefore, lower synthesis) on soft gels compared to the mean.</p

Cellular levels of proteins identified by mass spectrometry.

<p><b>A.</b> The levels of actin, tubulin, and phosphofructokinase-1 in A549 cells cultured on soft (150 Pa) or stiff (19200 Pa) gels were analyzed by western blot. Bottom panels show levels of GAPDH as a loading control. <b>B.</b> The levels of actin, tubulin, and PFKP-1 in mPanc96 cells cultured on soft or stiff gels as analyzed by western blot. Bottom panels show levels of GAPDH in cell lysates as a loading control. <b>C.</b> Quantitation of western blot results in (A) and (B). The levels of each protein were normalized to the amount of GAPDH in each sample. The levels of protein in samples prepared from stiff gels were set to 100%, and the levels of protein in the soft samples are displayed as percentage expression of these samples. Results show the mean ± S.E. of at least three experiments. * p<0.05 when compared to the level of actin expression.</p