Selective Metal-Site-Guided Arylation of Proteins

We describe palladium-mediated S-arylation that exploits natural metal-binding motifs to ensure high site selectivity for a proximal reactive residue. This allows the chemical identification not only of proteins that bind metals but also the environment of the metal-binding site itself through proteomic analysis of arylation sites. The transformation is easy to perform under standard conditions, does not require the isolation of a reactive Ar–Pd complex, is broad in scope, and is applicable in cell lysates as well as to covalent inhibition/modulation of metal-dependent enzymatic activity

Characterization of Biases in Phosphopeptide Enrichment by Ti⁴⁺-Immobilized Metal Affinity Chromatography and TiO₂ Using a Massive Synthetic Library and Human Cell Digests

Outcomes of comparative evaluations of enrichment methods for phosphopeptides depend highly on the experimental protocols used, the operator, the source of the affinity matrix, and the samples analyzed. Here, we attempt such a comparative study exploring a very large synthetic library containing thousands of serine, threonine, and tyrosine phosphorylated peptides, being present in roughly equal abundance, along with their nonphosphorylated counterparts, and use an optimized protocol for enrichment by TiO₂ and Ti⁴⁺-immobilized metal affinity chromatography (IMAC) by a single operator. Surprisingly, our data reveal that there are minimal differences between enrichment of phosphopeptides by TiO₂ and Ti⁴⁺-IMAC when considering biochemical and biophysical parameters such as peptide length, sequence surrounding the site, hydrophobicity, and nature of the amino acid phosphorylated. Similar results were obtained when evaluating a tryptic digest of a cellular lysate, representing a more natural source of phosphopeptides. All the data presented are available via ProteomeXchange with the identifier PXD000759

A high-resolution systems-wide screen for substrates of the SCFTrCP ubiquitin E3 ligase

<p><em>presented in: HUPO World Congress: The proteome quest to understand biology and disease in Madrid, Spain, 2013</em></p> <p> </p> <p>Cellular proteins are degraded by the ubiquitin-proteasome system (UPS) in a precise and timely fashion. Such precision is conferred by the high substrate specificity of ubiquitin ligases, the largest family of enzymes in mammals. Therefore, reliable assays aimed at the identification of substrates of ubiquitin ligases are crucial, not only to unravel the molecular mechanisms by which the UPS controls protein degradation, but also for drug discovery purposes since many established UPS substrates are oncoproteins or tumor suppressors. Here, we develop a combined bioinformatics and affinity purification-mass spectrometry (AP-MS) workflow for identifying in a systems-wide manner bone fide substrates of SCFβTrCP, a member of the SCF family of ubiquitin ligases. These ubiquitin ligases are trademarked by a multi-subunit architecture typically comprising the invariable subunits Rbx1, Cul1, and Skp1 and one of 69 F-box proteins. SCFβTrCP binds, via its WD40 repeats, the DpSGXX(X)pS di-phosphorylated motif in its substrates. Our combined workflow recovers 27 previously reported SCFβTrCP substrates, of which 22 are confidently verified by two independent statistical protocols, confirming the reliability of this approach. Besides known substrates, we identify 221 proteins that, besides harboring the DpSGXX(X)pS motif, also interact specifically with the WD40 repeats. From this list, we highlight several putative novel SCFβTrCP substrates with their putative degron motifs as well as phosphorylation and ubiquitylation sites. Thus, we demonstrate that the integration of structural information, AP-MS and degron motif mining constitutes a generic, specific and effective screen for the identification of substrates of ubiquitin ligases.</p

Nicotinamide Cofactors Suppress Active-Site Labeling of Aldehyde Dehydrogenases

Active site labeling by (re)­activity-based probes is a powerful chemical proteomic tool to globally map active sites in native proteomes without using substrates. Active site labeling is usually taken as a readout for the active state of the enzyme because labeling reflects the availability and reactivity of active sites, which are hallmarks for enzyme activities. Here, we show that this relationship holds tightly, but we also reveal an important exception to this rule. Labeling of <i>Arabidopsis</i> ALDH3H1 with a chloroacetamide probe occurs at the catalytic Cys, and labeling is suppressed upon nitrosylation and oxidation, and upon treatment with other Cys modifiers. These experiments display a consistent and strong correlation between active site labeling and enzymatic activity. Surprisingly, however, labeling is suppressed by the cofactor NAD⁺, and this property is shared with other members of the ALDH superfamily and also detected for unrelated GAPDH enzymes with an unrelated hydantoin-based probe in crude extracts of plant cell cultures. Suppression requires cofactor binding to its binding pocket. Labeling is also suppressed by ALDH modulators that bind at the substrate entrance tunnel, confirming that labeling occurs through the substrate-binding cavity. Our data indicate that cofactor binding adjusts the catalytic Cys into a conformation that reduces the reactivity toward chloroacetamide probes

Ultra Acidic Strong Cation Exchange Enabling the Efficient Enrichment of Basic Phosphopeptides

We present a straightforward method to enrich phosphopeptides with multiple basic residues, an under-represented class in common enrichment strategies. Our method is based on a two-dimensional strong cation exchange (SCX) strategy, operating at two different acidic pHs, enabling both separation and enrichment of different classes of phosphopeptides. The principle of enrichment is based on the change of net charge of phosphorylated peptides under strong acidic conditions in the second SCX, whereas the net charge of regular peptides remains unchanged, thus enabling separation based on net charge. Application of our tandem SCX approach to a modest amount of human cells allowed the identification of over 10 000 unique “basic” phosphopeptides of which many represent putative targets of basophilic kinases

Universal Quantitative Kinase Assay Based on Diagonal SCX Chromatography and Stable Isotope Dimethyl Labeling Provides High-definition Kinase Consensus Motifs for PKA and Human Mps1

In order to understand cellular signaling, a clear understanding of kinase–substrate relationships is essential. Some of these relationships are defined by consensus recognition motifs present in substrates making them amendable for phosphorylation by designated kinases. Here, we explore a method that is based on two sequential steps of strong cation exchange chromatography combined with differential stable isotope labeling, to define kinase consensus motifs with high accuracy. We demonstrate the value of our method by evaluating the motifs of two very distinct kinases: cAMP regulated protein kinase A (PKA) and human monopolar spindle 1 (Mps1) kinase, also known as TTK. PKA is a well-studied basophilic kinase with a relatively well-defined motif and numerous known substrates <i>in vitro</i> and <i>in vivo</i>. Mps1, a kinase involved in chromosome segregation, has been less well characterized. Its substrate specificity is unclear and here we show that Mps1 is an acidophilic kinase with a striking tendency for phosphorylation of threonines. The final outcomes of our work are high-definition kinase consensus motifs for PKA and Mps1. Our generic method, which makes use of proteolytic cell lysates as a source for peptide-substrate libraries, can be implemented for any kinase present in the kinome

Universal Quantitative Kinase Assay Based on Diagonal SCX Chromatography and Stable Isotope Dimethyl Labeling Provides High-definition Kinase Consensus Motifs for PKA and Human Mps1

In order to understand cellular signaling, a clear understanding of kinase–substrate relationships is essential. Some of these relationships are defined by consensus recognition motifs present in substrates making them amendable for phosphorylation by designated kinases. Here, we explore a method that is based on two sequential steps of strong cation exchange chromatography combined with differential stable isotope labeling, to define kinase consensus motifs with high accuracy. We demonstrate the value of our method by evaluating the motifs of two very distinct kinases: cAMP regulated protein kinase A (PKA) and human monopolar spindle 1 (Mps1) kinase, also known as TTK. PKA is a well-studied basophilic kinase with a relatively well-defined motif and numerous known substrates <i>in vitro</i> and <i>in vivo</i>. Mps1, a kinase involved in chromosome segregation, has been less well characterized. Its substrate specificity is unclear and here we show that Mps1 is an acidophilic kinase with a striking tendency for phosphorylation of threonines. The final outcomes of our work are high-definition kinase consensus motifs for PKA and Mps1. Our generic method, which makes use of proteolytic cell lysates as a source for peptide-substrate libraries, can be implemented for any kinase present in the kinome

On the Statistical Significance of Compressed Ratios in Isobaric Labeling: A Cross-Platform Comparison

Isobaric labeling is gaining popularity in proteomics due to its multiplexing capacity. However, copeptide fragmentation introduces a bias that undermines its accuracy. Several strategies have been shown to partially and, in some cases, completely solve this issue. However, it is still not clear how ratio compression affects the ability to identify a protein’s change of abundance as statistically significant. Here, by using the “two proteomes” approach (<i>E. coli</i> lysates with fixed 2.5 ratios in the presence or absence of human lysates acting as the background interference) and manipulating isolation width values, we were able to model isobaric data with different levels of accuracy and precision in three types of mass spectrometers: LTQ Orbitrap Velos, Impact, and Q Exactive. We determined the influence of these variables on the statistical significance of the distorted ratios and compared them to the ratios measured without impurities. Our results confirm previous findings− regarding the importance of optimizing acquisition parameters in each instrument in order to minimize interference without compromising precision and identification. We also show that, under these experimental conditions, the inclusion of a second replicate increases statistical sensitivity 2–3-fold and counterbalances to a large extent the issue of ratio compression

Toward a Comprehensive Characterization of a Human Cancer Cell Phosphoproteome

Mass spectrometry (MS)-based phosphoproteomics has achieved extraordinary success in qualitative and quantitative analysis of cellular protein phosphorylation. Considering that an estimated level of phosphorylation in a cell is placed at well above 100 000 sites, there is still much room for improvement. Here, we attempt to extend the depth of phosphoproteome coverage while maintaining realistic aspirations in terms of available material, robustness, and instrument running time. We developed three strategies, where each provided a different balance between these three key parameters. The first strategy simply used enrichment by Ti⁴⁺-IMAC followed by reversed chromatography LC–MS (termed 1D). The second strategy incorporated an additional fractionation step through the use of HILIC (2D). Finally, a third strategy was designed employing first an SCX fractionation, followed by Ti⁴⁺-IMAC enrichment and additional fractionation by HILIC (3D). A preliminary evaluation was performed on the HeLa cell line. Detecting 3700 phosphopeptides in about 2 h, the 1D strategy was found to be the most sensitive but limited in comprehensivity, mainly due to issues with complexity and dynamic range. Overall, the best balance was achieved using the 2D based strategy, identifying close to 17 000 phosphopeptides with less than 1 mg of material in about 48 h. Subsequently, we confirmed the findings with the K562 cell sample. When sufficient material was available, the 3D strategy increased phosphoproteome allowing over 22 000 unique phosphopeptides to be identified. Unfortunately, the 3D strategy required more time and over 1 mg of material before it started to outperform 2D. Ultimately, combining all strategies, we were able to identify over 16 000 and nearly 24 000 unique phosphorylation sites from the cancer cell lines HeLa and K562, respectively. In summary, we demonstrate the need to carry out extensive fractionation for deep mining of the phosphoproteome and provide a guide for appropriate strategies depending on sample amount and/or analysis time

Profiling of Diet-Induced Neuropeptide Changes in Rat Brain by Quantitative Mass Spectrometry

Neuropeptides are intercellular signal transmitters that play key roles in modulation of many behavioral and physiological processes. Neuropeptide signaling in several nuclei in the hypothalamus contributes to the control of food intake. Additionally, food intake regulation involves neuropeptide signaling in the reward circuitry in the striatum. Here, we analyze neuropeptides extracted from hypothalamus and striatum from rats in four differentially treated dietary groups including a high-fat/high-sucrose diet, mimicking diet-induced obesity. We employ high-resolution tandem mass spectrometry using higher-energy collision dissociation and electron transfer dissociation fragmentation for sensitive identification of more than 1700 unique endogenous peptides, including virtually all key neuropeptides known to be involved in food intake regulation. Label-free quantification of differential neuropeptide expression revealed comparable upregulation of orexigenic and anorexigenic neuropeptides in rats that were fed on a high-fat/high-sucrose diet