PhoStar: Identifying Tandem Mass Spectra of Phosphorylated Peptides before Database Search

Standard proteomics workflows use tandem mass spectrometry followed by sequence database search to analyze complex biological samples. The identification of proteins carrying post-translational modifications, for example, phosphorylation, is typically addressed by allowing variable modifications in the searched sequences. Accounting for these variations exponentially increases the combinatorial space in the database, which leads to increased processing times and more false positive identifications. The here-presented tool PhoStar identifies spectra that originate from phosphorylated peptides before database search using a supervised machine learning approach. The model for the prediction of phosphorylation was trained and validated with an accuracy of 97.6% on a large set of high-confidence spectra collected from publicly available experimental data. Its power was further validated by predicting phosphorylation in the complete NIST human and mouse high collision-dissociation spectral libraries, achieving an accuracy of 98.2 and 97.9%, respectively. We demonstrate the application of PhoStar by using it for spectra filtering before database search. In database search of HeLa samples the peptide search space was reduced by 27–66% while finding at least 97% of total peptide identifications (at 1% FDR) compared with a standard workflow

Optimized Nonlinear Gradients for Reversed-Phase Liquid Chromatography in Shotgun Proteomics

Reversed-phase liquid chromatography has become the preferred method for separating peptides in most of the mass spectrometry-based proteomics workflows of today. In the way the technique is typically applied, the peptides are released from the chromatography column by the gradual addition of an organic buffer according to a linear function. However, when applied to complex peptide mixtures, this approach leads to unequal spreads of the peptides over the chromatography time. To address this, we investigated the use of nonlinear gradients, customized for each setup at hand. We developed an algorithm to generate optimized gradient functions for shotgun proteomics experiments and evaluated it for two data sets consisting each of four replicate runs of a human complex sample. Our results show that the optimized gradients produce a more even spread of the peptides over the chromatography run, while leading to increased numbers of confident peptide identifications. In addition, the list of peptides identified using nonlinear gradients differed considerably from those found with the linear ones, suggesting that such gradients can be a valuable tool for increasing the proteome coverage of mass spectrometry-based experiments

Improved Precision of iTRAQ and TMT Quantification by an Axial Extraction Field in an Orbitrap HCD Cell

Improving analytical precision is a major goal in quantitative differential proteomics as high precision ensures low numbers of outliers, a source of false positives with regard to quantification. In addition, higher precision increases statistical power, i.e., the probability to detect significant differences. With chemical labeling using isobaric tags for relative and absolute quantitation (iTRAQ) or tandem mass tag (TMT) reagents, quantification is based on the extraction of reporter ions from tandem mass spectrometry (MS/MS) spectra. We compared the performance of two versions of the LTQ Orbitrap higher energy collisional dissociation (HCD) cell with and without an axial electric field with regard to reporter ion quantification. The HCD cell with the axial electric field was designed to push fragment ions into the C-trap and this version is mounted in current Orbitrap XL ETD and Orbitrap Velos instruments. Our goal was to evaluate whether the purported improvement in ion transmission had a measurable impact on the precision of MS/MS based quantification using peptide labeling with isobaric tags. We show that the axial electric field led to an increased percentage of HCD spectra in which the complete set of reporter ions was detected and, even more important, to a reduction in overall variance, i.e., improved analytical precision of the acquired data. Notably, adequate precision of HCD-based quantification was maintained even for low precursor ion intensities of a complex biological sample. These findings may help researchers in their design of quantitative proteomics studies using isobaric tags and establish HCD-based quantification on the LTQ Orbitrap as a highly precise approach in quantitative proteomics

Phosphorylation of SA2 Is Required for Efficient Resolution of Sister Chromatid Arms during Prometaphase and Metaphase

<div><p>(A) HeLa cells expressing SA2-WT-myc or SA2–12xA-myc were spread on glass slides and chromosomes were stained with Giemsa. Representative cells from SA2 WT-myc or SA2–12xA-myc cell lines after induction with 2 μg/ml doxycycline are shown. Scale bar 10 μm.</p> <p>(B) HeLa cells were induced to express SA2-WT-myc or SA2–12xA-myc by different amounts of doxycycline as indicated, and processed as in (A). More than 50 cells in prometaphase or metaphase were selected randomly from each sample. The distance between sister chromatids was determined for five chromosomes in each cell and averaged. Light gray bars indicate average values that have been measured in one or two cells, and darker gray bars indicate average values that have been measured in three or more cells. Diamonds indicate the average distance for all cells in a given sample.</p> <p>(C) Representative immunofluorescence image of normal anaphase in a cell expressing SA2–12xA-myc. The cell was not extracted prior to fixation, so the soluble pool of SA2–12xA-myc is revealed by myc-staining.</p></div

Phosphorylation of SA2 Is Required for Cohesin Dissociation from Chromosome Arms during Prometaphase

<div><p>(A) Logarithmically proliferating HeLa cells expressing SA2-WT-myc or SA2–12xA-myc were extracted prior to fixation, and stained with myc antibodies. In the upper set of images, kinetochores were labeled with human CREST serum, and DNA was counterstained with DAPI. In the lower set of images, only SA2-myc staining is shown.</p> <p>(B) SA2-myc expression was induced by different amounts of doxycycline (Dox.), and cells were arrested in prometaphase by nocodazole (Noc.) treatment for 3 or 10 h. Cells were spun on glass slides, extracted by detergent, fixed, and processed for immunostaining as in (A). Scale bars 10 μm.</p></div

The Presence of SA2–12xA on Chromosome Arms Correlates with Cohesion between Sister Chromatid Arms

<div><p>(A) Cells were cultured in the absence or presence of different amounts of doxycycline as indicated. After arrest in nocodazole for 3 h, cells were fixed, spread on glass slides, and stained with Giemsa (photomicrographs, above). Single chromosomes (indicated by a box) are shown at higher magnification in the lower right corners. The number of cells with chromosome arms that had opened (arms open) or that were connected (arms closed) was scored as indicated (bar graphs, below). Scale bar 10 μm.</p> <p>(B) Whole-cell extracts were prepared from HeLa cells expressing SA2–12xA-myc after treatment with increasing amounts of doxycycline (0, 0.2, and 2.0 μg/ml). The ratio of exogenous SA2–12xA-myc to endogenous SA2 was visualized by immunoblotting with antibodies to SA2. The position of molecular weight markers is indicated on the right.</p></div

Identification of Mitosis-Specific Phosphorylation Sites on Human Cohesin

<div><p>(A and B) Cohesin was immunoprecipitated by antibody 447 (which recognizes SA1 and SA2) from extracts prepared from HeLa cells that were either arrested in S-phase by hydroxyurea (HU) or in mitosis by nocodazole (Noc). Cohesin was eluted by buffer of low pH and analyzed by (A) silver staining and (B) immunoblotting with antibodies to cohesin subunits and phosphorylated threonine (P-Thr).</p> <p>(C) Schematic representation of the phosphorylation sites on Scc1 and SA2 that were identified by mass spectrometry, and of the mutant versions of the proteins that have been generated. The star indicates a phosphorylation site that was found in both interphase and mitotic Scc1. All SA2 constructs used for in vitro experiments lack the 69 N-terminal amino acids. SA2-WT-myc and SA2–12xA-myc cell lines contain the entire open reading frame of 1,231 amino acids.</p></div

Deep and Precise Quantification of the Mouse Synaptosomal Proteome Reveals Substantial Remodeling during Postnatal Maturation

During postnatal murine maturation, behavioral patterns emerge and become shaped by experience-dependent adaptations. During the same period, the morphology of dendritic spines, the morphological correlates of excitatory synapses, is known to change, and there is evidence of concurrent alterations of the synaptosomal protein machinery. To obtain comprehensive and quantitative insights in the developmental regulation of the proteome of synapses, we prepared cortical synaptosomal fractions from a total of 16 individual juvenile and adult mouse brains (age 3 or 8 weeks, respectively). We then applied peptide-based iTRAQ labeling (four pools of 4 animals) and high-resolution two-dimensional peptide fractionation (99 SCX fractions and 3 h reversed-phase gradients) using a hybrid CID–HCD acquisition method on a Velos Orbitrap mass spectrometer to identify a comprehensive set of synaptic proteins and to quantify changes in protein expression. We obtained a data set tracking expression levels of 3500 proteins mapping to 3427 NCBI GeneIDs during development with complete quantification data available for 3422 GeneIDs, which, to the best of our knowledge, constitutes the deepest coverage of the synaptosome proteome to date. The inclusion of biological replicates in a single mass spectrometry analysis demonstrated both high reproducibility of our synaptosome preparation method as well as high precision of our quantitative data (correlation coefficient <i>R</i> = 0.87 for the biological replicates). To evaluate the validity of our data, the developmental regulation of eight proteins identified in our analysis was confirmed independently using western blotting. A gene ontology analysis confirmed the synaptosomal nature of a large fraction of identified proteins. Of note, the set of the most strongly regulated proteins revealed candidates involved in neurological processes in health and disease states. This highlights the fact that developmentally regulated proteins can play additional roles in neurological disease processes. All data have been deposited to the ProteomeXchange with identifier PXD000552

Plk1 Facilitates Cleavage of Human Scc1 by Separase In Vitro

<div><p>(A) Recombinant, ³⁵S-labeled, wild-type and mutant Scc1 (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030069#pbio-0030069-g001" target="_blank">Figure 1</a>C) tagged with 9xmyc at the C terminus were incubated with human separase. Recombinant human GST-Plk1 was added to the reaction mixtures where indicated. Samples were withdrawn from the reactions at the indicated time points and analyzed by SDS-PAGE followed by immunoblotting (anti-myc) and Phosphorimager analysis (³⁵S exposure). Arrows indicate full length Scc1-myc (fl), C- and N-terminal fragments resulting from cleavage at Arg¹⁷² (Ct #1, Nt #1, respectively), and C- and N-terminal fragments resulting from cleavage at Arg⁴⁵⁰ (Ct #2, Nt #2, respectively). The lower parts of the membrane or gels were exposed longer than the upper parts. The enhancement of cleavage at Arg¹⁷² by Plk1 can be seen particularly well by comparing the intensities of the N-terminal fragments (Nt #1). Note that in the autoradiographs a band can be seen (particularly clearly in the lanes representing the zero time points) that has almost the same electrophoretic mobility as cleavage product Ct #1. This band is distinct from Ct #1 because it migrates a slightly shorter distance and because it is also present in the absence of separase. This band was therefore not included in the quantification in (B).</p> <p>(B) Quantification of the abundance of Scc1-myc and the Scc1-myc cleavage fragments in the assay shown in the left autoradiograph of (A). For the quantification, autoradiographs of identical exposure were used. The sum of the intensities of full-length and all cleavage fragments was set to 100%. Signal intensities for N- and C-terminal fragments resulting from cleavage at the same site were summed.</p> <p>(C) Chromatin fractions were prepared from HeLa cells stably expressing either wild-type Scc1-myc or the mutant Scc1-S⁴⁵⁴A-myc, and were incubated in either interphase or mitotic <i>Xenopus</i> egg extract. Mitosis-specific cleavage of Scc1 was detected by immunoblotting with myc antibodies.</p></div

Characterization of HeLa Cell Lines Stably Expressing Wild-Type or Mutant Forms of Human Scc1 and SA2

<div><p>(A) Wild-type Scc1 or SA2, or the indicated mutant proteins (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030069#pbio-0030069-g001" target="_blank">Figure 1</a>C), all tagged with 9xmyc at the C terminus, were stably and inducibly expressed in HeLa tet-on cells. After induction by treatment with 2 μg/ml doxycycline for 1–3 d, cell extracts were prepared from either logarithmically proliferating cells (i, interphase) or from cells arrested in mitosis by nocodazole (m, mitosis), then immunoblotted. In the case of Scc1 cell lines (upper blots), only data from interphase extracts are shown. Exogenous protein was detected by immunoblotting with myc antibodies (lower blots). Since the 9xmyc-tag caused a reduced mobility in SDS-PAGE compared to the endogenous protein, Scc1- and SA2-immunoblots (upper blots) revealed the relative amounts of exogenous and endogenous protein in the different cell lines. The position of molecular weight markers is indicated on the right side.</p> <p>(B) Extracts were prepared from the different cell lines as indicated. Immunoprecipitation was performed using myc antibodies, followed by SDS-PAGE and silver staining. As a control, the cohesin complex was immunoprecipitated from untransfected HeLa tet-on cells using antibodies to SA2.</p> <p>(C) Extracts were prepared from SA2-WT-myc or SA2–12xA-myc expressing cells, and fractionated by sucrose density gradient centrifugation (5%–30% sucrose), followed by immunoblotting with antibodies recognizing the proteins indicated on the right (inp. = input/unfractionated sample of the extract).</p></div