The replicative helicase MCM recruits cohesin acetyltransferase ESCO2 to mediate centromeric sister chromatid cohesion.

Chromosome segregation depends on sister chromatid cohesion which is established by cohesin during DNA replication. Cohesive cohesin complexes become acetylated to prevent their precocious release by WAPL before cells have reached mitosis. To obtain insight into how DNA replication, cohesion establishment and cohesin acetylation are coordinated, we analysed the interaction partners of 55 human proteins implicated in these processes by mass spectrometry. This proteomic screen revealed that on chromatin the cohesin acetyltransferase ESCO2 associates with the MCM2-7 subcomplex of the replicative Cdc45-MCM-GINS helicase. The analysis of ESCO2 mutants defective in MCM binding indicates that these interactions are required for proper recruitment of ESCO2 to chromatin, cohesin acetylation during DNA replication, and centromeric cohesion. We propose that MCM binding enables ESCO2 to travel with replisomes to acetylate cohesive cohesin complexes in the vicinity of replication forks so that these complexes can be protected from precocious release by WAPL Our results also indicate that ESCO1 and ESCO2 have distinct functions in maintaining cohesion between chromosome arms and centromeres, respectively.

apQuant: Accurate Label-Free Quantification by Quality Filtering.

Label-free quantification of shotgun proteomics data is a frequently used strategy, offering high dynamic range, sensitivity, and the ability to compare a high number of samples without additional labeling effort. Here, we present a bioinformatics approach that significantly improves label-free quantification results. We employ Percolator to assess the quality of quantified peptides. This allows to extract accurate and reliable quantitative results based on false discovery rate. Benchmarking our approach on previously published public data shows that it considerably outperforms currently available algorithms. apQuant is available free of charge as a node for Proteome Discoverer.

Spectral Clustering Improves Label-Free Quantification of Low-Abundant Proteins.

Label-free quantification has become a common-practice in many mass spectrometry-based proteomics experiments. In recent years, we and others have shown that spectral clustering can considerably improve the analysis of (primarily large-scale) proteomics data sets. Here we show that spectral clustering can be used to infer additional peptide-spectrum matches and improve the quality of label-free quantitative proteomics data in data sets also containing only tens of MS runs. We analyzed four well-known public benchmark data sets that represent different experimental settings using spectral counting and peak intensity based label-free quantification. In both approaches, the additionally inferred peptide-spectrum matches through our spectra-cluster algorithm improved the detectability of low abundant proteins while increasing the accuracy of the derived quantitative data, without increasing the data sets' noise. Additionally, we developed a Proteome Discoverer node for our spectra-cluster algorithm which allows anyone to rebuild our proposed pipeline using the free version of Proteome Discoverer.

Improved Sensitivity in Low-Input Proteomics Using Micropillar Array-Based Chromatography.

Capitalizing on the massive increase in sample concentrations which are produced by extremely low elution volumes, nanoliquid chromatography-electrospray ionization-tandem mass spectrometry (nano-LC-ESI-MS/MS) is currently one of the most sensitive analytical technologies for the comprehensive characterization of complex protein samples. However, despite tremendous technological improvements made in the production and the packing of monodisperse spherical particles for nanoflow high-pressure liquid chromatography (HPLC), current state-of-the-art systems still suffer from limits in operation at the maximum potential of the technology. With the recent introduction of the µPAC system, which provides perfectly ordered micropillar array based chromatographic support materials, completely new chromatographic concepts for optimization toward the needs of ultrasensitive proteomics become available. Here we report on a series of benchmarking experiments comparing the performance of a commercially available 50 cm micropillar array column to a widely used nanoflow HPLC column for the proteomics analysis of 10 ng of tryptic HeLa cell digest. Comparative analysis of LC-MS/MS-data corroborated that micropillar array cartridges provide outstanding chromatographic performance, excellent retention time stability, and increased sensitivity in the analysis of low-input proteomics samples and thus repeatedly yielded almost twice as many unique peptide and unique protein group identifications when compared to conventional nanoflow HPLC columns.

SIRT2 regulates nuclear envelope reassembly through ANKLE2 deacetylation.

Sirtuin 2 (SIRT2) is an NAD-dependent deacetylase known to regulate microtubule dynamics and cell cycle progression. SIRT2 has also been implicated in the pathology of cancer, neurodegenerative diseases and progeria. Here, we show that SIRT2 depletion or overexpression causes nuclear envelope reassembly defects. We link this phenotype to the recently identified regulator of nuclear envelope reassembly ANKLE2. ANKLE2 acetylation at K302 and phosphorylation at S662 are dynamically regulated throughout the cell cycle by SIRT2 and are essential for normal nuclear envelope reassembly. The function of SIRT2 therefore extends beyond the regulation of microtubules to include the regulation of nuclear envelope dynamics.

Global analysis of muscle-specific kinase signaling by quantitative phosphoproteomics.

The development of the neuromuscular synapse depends on signaling processes that involve protein phosphorylation as a crucial regulatory event. Muscle-specific kinase (MuSK) is the key signaling molecule at the neuromuscular synapse whose activity is required for the formation of a mature and functional synapse. However, the signaling cascade downstream of MuSK and the regulation of the different components are still poorly understood. In this study we used a quantitative phosphoproteomics approach to study the phosphorylation events and their temporal regulation downstream of MuSK. We identified a total of 10,183 phosphopeptides, of which 203 were significantly up- or down-regulated. Regulated phosphopeptides were classified into four different clusters according to their temporal profiles. Within these clusters we found an overrepresentation of specific protein classes associated with different cellular functions. In particular, we found an enrichment of regulated phosphoproteins involved in posttranscriptional mechanisms and in cytoskeletal organization. These findings provide novel insights into the complex signaling network downstream of MuSK and form the basis for future mechanistic studies.

Comprehensive Cross-Linking Mass Spectrometry Reveals Parallel Orientation and Flexible Conformations of Plant HOP2-MND1.

The HOP2-MND1 heterodimer is essential for meiotic homologous recombination in plants and other eukaryotes and promotes the repair of DNA double-strand breaks. We investigated the conformational flexibility of HOP2-MND1, important for understanding the mechanistic details of the heterodimer, with chemical cross-linking in combination with mass spectrometry (XL-MS). The final XL-MS workflow encompassed the use of complementary cross-linkers, quenching, digestion, size exclusion enrichment, and HCD-based LC-MS/MS detection prior to data evaluation. We applied two different homobifunctional amine-reactive cross-linkers (DSS and BS(2)G) and one zero-length heterobifunctional cross-linker (EDC). Cross-linked peptides of four biological replicates were analyzed prior to 3D structure prediction by protein threading and protein-protein docking for cross-link-guided molecular modeling. Miniaturization of the size-exclusion enrichment step reduced the required starting material, led to a high amount of cross-linked peptides, and allowed the analysis of replicates. The major interaction site of HOP2-MND1 was identified in the central coiled-coil domains, and an open colinear parallel arrangement of HOP2 and MND1 within the complex was predicted. Moreover, flexibility of the C-terminal capping helices of both complex partners was observed, suggesting the coexistence of a closed complex conformation in solution.

Anion-exchange chromatography of phosphopeptides: weak anion exchange versus strong anion exchange and anion-exchange chromatography versus electrostatic repulsion-hydrophilic interaction chromatography.

Most phosphoproteomics experiments rely on prefractionation of tryptic digests before online liquid chromatography-mass spectrometry. This study compares the potential and limitations of electrostatic repulsion-hydrophilic interaction chromatography (ERLIC) and anion-exchange chromatography (AEX). At a pH higher than 5, phosphopeptides have two negative charges per residue and are well-retained in AEX. However, peptides with one or two phosphate groups are not separated from peptides with multiple Asp or Glu residues, interfering with the identification of phosphopeptides. At a pH of 2, phosphate residues have just a single negative charge but Asp and Glu are uncharged. This facilitates the separation of phosphopeptides from unmodified acidic peptides. Singly phosphorylated peptides are retained weakly under these conditions, due to electrostatic repulsion, unless hydrophilic interaction is superimposed in the ERLIC mode. Weak anion-exchange (WAX) and strong anion-exchange (SAX) columns were compared, with both peptide standards and a HeLa cell tryptic digest. The SAX column exhibited greater retention at pH 6 than did the WAX column. However, only about 60% as many phosphopeptides were identified with SAX at pH 6 than via ERLIC at pH 2. In one ERLIC run, 12 467 phosphopeptides were identified, including 4233 with more than one phosphate. We conclude that chromatography of phosphopeptides is best performed at low pH in the ERLIC mode. Under those conditions, the performances of the SAX and WAX materials were comparable. The data have been deposited with the ProteomeXchange with identifier PXD001333.

Quantitative phospho-proteomics to investigate the polo-like kinase 1-dependent phospho-proteome.

Polo-like kinase 1 (PLK1) is a key regulator of mitotic progression and cell division, and small molecule inhibitors of PLK1 are undergoing clinical trials to evaluate their utility in cancer therapy. Despite this importance, current knowledge about the identity of PLK1 substrates is limited. Here we present the results of a proteome-wide analysis of PLK1-regulated phosphorylation sites in mitotic human cells. We compared phosphorylation sites in HeLa cells that were or were not treated with the PLK1-inhibitor BI 4834, by labeling peptides via methyl esterification, fractionation of peptides by strong cation exchange chromatography, and phosphopeptide enrichment via immobilized metal affinity chromatography. Analysis by quantitative mass spectrometry identified 4070 unique mitotic phosphorylation sites on 2069 proteins. Of these, 401 proteins contained one or multiple phosphorylation sites whose abundance was decreased by PLK1 inhibition. These include proteins implicated in PLK1-regulated processes such as DNA damage, mitotic spindle formation, spindle assembly checkpoint signaling, and chromosome segregation, but also numerous proteins that were not suspected to be regulated by PLK1. Analysis of amino acid sequence motifs among phosphorylation sites down-regulated under PLK1 inhibition in this data set identified two potential novel variants of the PLK1 consensus motif.

TurboID-based proteomic profiling of meiotic chromosome axes in Arabidopsis thaliana.

During meiotic prophase I, sister chromatids are arranged in a loop-base array along a proteinaceous structure, called the meiotic chromosome axis. This structure is essential for synapsis and meiotic recombination progression and hence formation of genetically diverse gametes. Proteomic studies in plants aiming to unravel the composition and regulation of meiotic axes are constrained by limited meiotic cells embedded in floral organs. Here we report TurboID (TbID)-based proximity labelling (PL) in meiotic cells of Arabidopsis thaliana. TbID fusion to the two meiotic chromosome axis proteins ASY1 and ASY3 enabled the identification of their proximate 'interactomes' based on affinity purification coupled with mass spectrometry. We identified 39 ASY1 and/or ASY3 proximate candidates covering most known chromosome axis-related proteins. Functional studies of selected candidates demonstrate that not only known meiotic candidates but also new meiotic proteins were uncovered. Hence, TbID-based PL in meiotic cells enables the identification of chromosome axis proximate proteins in A. thaliana.

Structural basis for regulation of apoptosis and autophagy by the BIRC6/SMAC complex.

Inhibitor of apoptosis proteins (IAPs) bind to pro-apoptotic proteases, keeping them inactive and preventing cell death. The atypical ubiquitin ligase BIRC6 is the only essential IAP, additionally functioning as a suppressor of autophagy. We performed a structure-function analysis of BIRC6 in complex with caspase-9, HTRA2, SMAC, and LC3B, which are critical apoptosis and autophagy proteins. Cryo-electron microscopy structures showed that BIRC6 forms a megadalton crescent shape that arcs around a spacious cavity containing receptor sites for client proteins. Multivalent binding of SMAC obstructs client binding, impeding ubiquitination of both autophagy and apoptotic substrates. On the basis of these data, we discuss how the BIRC6/SMAC complex can act as a stress-induced hub to regulate apoptosis and autophagy drivers.

QIKS--Quantitative identification of kinase substrates.

Signaling networks regulate cellular responses to external stimuli through post-translational modifications such as protein phosphorylation. Phosphoproteomics facilitate the large-scale identification of kinase substrates. Yet, the characterization of critical connections within these networks and the identification of respective kinases remain the major analytical challenge. To address this problem, we present a novel approach for the identification of direct kinase substrates using chemical genetics in combination with quantitative phosphoproteomics. Quantitative identification of kinase substrates (QIKS) is a novel-screening platform developed for the proteome-wide substrate-analysis of specific kinases. Here, we aimed to identify substrates of mitogen-activated protein kinase/Erk kinase (Mek1), an essential kinase in the mitogen-activated protein kinase cascade. An ATP analog-sensitive mutant of Mek1 (Mek1-as) was incubated with a cell extract from Mek1 deficient cells. Phosphorylated proteins were analyzed by LC-MS/MS of IMAC-enriched phosphopeptides, labeled differentially for relative quantification. The identification of extracellular regulated kinase 1/2 as the sole cytoplasmic substrates of MEK1 validates the applicability of this approach and suggests that QIKS could be used to identify substrates of a wide variety of kinases.

Probing the phosphoproteome of HeLa cells using nanocast metal oxide microspheres for phosphopeptide enrichment.

Metal oxide affinity chromatography (MOAC) has become a prominent method to enrich phosphopeptides prior to their analysis by liquid chromatography-mass spectrometry. To overcome limitations in material design, we have previously reported the use of nanocasting as a means to generate metal oxide spheres with tailored properties. Here, we report on the application of two oxides, tin dioxide (stannia) and titanium dioxide (titania), for the analysis of the HeLa phosphoproteome. In combination with nanoflow LC-MS/MS analysis on a linear ion trap-Fourier transform ion cyclotron resonance instrument, we identified 619 phosphopeptides using the new stannia material, and 896 phosphopeptides using titania prepared in house. We also compared the newly developed materials to commercial titania material using an established enrichment protocol. Both titania materials yielded a comparable total number of phosphopeptides, but the overlap of the two data sets was less than one-third. Although fewer peptides were identified using stannia, the complementarity of SnO(2)-based MOAC could be shown as more than 140 phosphopeptides were exclusively identified by this material.

Preventing carryover of peptides and proteins in nano LC-MS separations.

Sample carryover is a significant problem that occurs in high-performance liquid chromatography (HPLC) analysis. Carryover effects cannot be tolerated in any high-performance liquid chromatography-mass spectroscopy (HPLC-MS) separation system, and proteomics analysis must be performed in a separation system with virtually no carryover. Several procedures have been tested for effective and fast removal of interfering peptides and proteins originating from previous analyses in the HPLC system. We have developed and optimized a cleaning method for eliminating carryover caused by the autosampler and the trap column. The new washing method uses an injection of trifluoroethanol into the injection path and onto the trap column to remove strongly bound peptides and proteins, and it includes trifluoroethanol as an additional solvent in the chromatographic mobile phase for enhanced cleaning of the separation column. By application of this method, a significant reduction in carryover was achieved without any loss in the amount of proteins and peptides identified by MS.

MTHFD1 interaction with BRD4 links folate metabolism to transcriptional regulation.

The histone acetyl reader bromodomain-containing protein 4 (BRD4) is an important regulator of chromatin structure and transcription, yet factors modulating its activity have remained elusive. Here we describe two complementary screens for genetic and physical interactors of BRD4, which converge on the folate pathway enzyme MTHFD1 (methylenetetrahydrofolate dehydrogenase, cyclohydrolase and formyltetrahydrofolate synthetase 1). We show that a fraction of MTHFD1 resides in the nucleus, where it is recruited to distinct genomic loci by direct interaction with BRD4. Inhibition of either BRD4 or MTHFD1 results in similar changes in nuclear metabolite composition and gene expression; pharmacological inhibitors of the two pathways synergize to impair cancer cell viability in vitro and in vivo. Our finding that MTHFD1 and other metabolic enzymes are chromatin associated suggests a direct role for nuclear metabolism in the control of gene expression.

Author Correction: Structural prediction of protein models using distance restraints derived from cross-linking mass spectrometry data.

In the version of this article initially published online, the authors used incorrectly defined restraints for specifying the distance between residues when using the HADDOCK portal. Following the publication of a Correspondence by the developers of the HADDOCK portal (Nat. Protoc. https://dx.doi.org/10.1038/s41596-018-0017-6, 2018) and a Reply by the authors of the Protocol (Nat. Protoc. https://dx.doi.org/10.1038/s41596-018-0018-5, 2018), the syntax in step 21 has been corrected. In addition, the input files (available as Supplementary Data 5-7) have been replaced.

Structural prediction of protein models using distance restraints derived from cross-linking mass spectrometry data.

This protocol describes a workflow for creating structural models of proteins or protein complexes using distance restraints derived from cross-linking mass spectrometry experiments. The distance restraints are used (i) to adjust preliminary models that are calculated on the basis of a homologous template and primary sequence, and (ii) to select the model that is in best agreement with the experimental data. In the case of protein complexes, the cross-linking data are further used to dock the subunits to one another to generate models of the interacting proteins. Predicting models in such a manner has the potential to indicate multiple conformations and dynamic changes that occur in solution. This modeling protocol is compatible with many cross-linking workflows and uses open-source programs or programs that are free for academic users and do not require expertise in computational modeling. This protocol is an excellent additional application with which to use cross-linking results for building structural models of proteins. The established protocol is expected to take 6-12 d to complete, depending on the size of the proteins and the complexity of the cross-linking data.

Systematic phosphorylation analysis of human mitotic protein complexes.

Progression through mitosis depends on a large number of protein complexes that regulate the major structural and physiological changes necessary for faithful chromosome segregation. Most, if not all, of the mitotic processes are regulated by a set of mitotic protein kinases that control protein activity by phosphorylation. Although many mitotic phosphorylation events have been identified in proteome-scale mass spectrometry studies, information on how these phosphorylation sites are distributed within mitotic protein complexes and which kinases generate these phosphorylation sites is largely lacking. We used systematic protein-affinity purification combined with mass spectrometry to identify 1818 phosphorylation sites in more than 100 mitotic protein complexes. In many complexes, the phosphorylation sites were concentrated on a few subunits, suggesting that these subunits serve as "switchboards" to relay the kinase-regulatory signals within the complexes. Consequent bioinformatic analyses identified potential kinase-substrate relationships for most of these sites. In a subsequent in-depth analysis of key mitotic regulatory complexes with the Aurora kinase B (AURKB) inhibitor Hesperadin and a new Polo-like kinase (PLK1) inhibitor, BI 4834, we determined the kinase dependency for 172 phosphorylation sites on 41 proteins. Combination of the results of the cellular studies with Scansite motif prediction enabled us to identify 14 sites on six proteins as direct candidate substrates of AURKB or PLK1.