Martijn W. H. Pinkse

Phosphorylation Dynamics during Early Differentiation of Human Embryonic Stem Cells

Pluripotent stem cells self-renew indefinitely and possess characteristic protein-protein networks that remodel during differentiation. How this occurs is poorly understood. Using quantitative mass spectrometry, we analyzed the (phospho)proteome of human embryonic stem cells (hESCs) during differentiation induced by bone morphogenetic protein (BMP) and removal of hESC growth factors. Of 5222 proteins identified, 1399 were phosphorylated on 3067 residues. Approximately 50% of these phosphosites were regulated within 1 hr of differentiation induction, revealing a complex interplay of phosphorylation networks spanning different signaling pathways and kinase activities. Among the phosphorylated proteins was the pluripotency-associated protein SOX2, which was SUMOylated as a result of phosphorylation. Using the data to predict kinase-substrate relationships, we reconstructed the hESC kinome; CDK1/2 emerged as central in controlling self-renewal and lineage specification. The findings provide new insights into how hESCs exit the pluripotent state and present the hESC (phospho)proteome resource as a complement to existing pluripotency network databases.

A Quest for Human and Mouse Embryonic Stem Cell-specific Proteins

Embryonic stem cells (ESCs) are of immense interest as they can proliferate indefinitely in vitro and give rise to any adult cell type, serving as a potentially unlimited source for tissue replacement in regenerative medicine. Extensive analyses of numerous human and mouse ESC lines have shown generic similarities and differences at both the transcriptional and functional level. However, comprehensive proteome analyses are missing or are restricted to mouse ESCs. Here we have used an extensive proteomic approach to search for ESC-specific proteins by analyzing the differential protein expression profiles of human and mouse ESCs and their differentiated derivatives. The data sets comprise 1,775 non-redundant proteins identified in human ESCs, 1,532 in differentiated human ESCs, 1,871 in mouse ESCs, and 1,552 in differentiated mouse ESCs with a false positive rate of <0.2%. Comparison of the data sets distinguished 191 proteins exclusively identified in both human and mouse ESCs but not in their differentiated derivatives. Besides well known ESC benchmarks, this subset included many uncharacterized proteins, some of which may be novel ESC-specific markers. To complement the mass spectrometric approach, differential expression of a selection of these proteins was confirmed by Western blotting, immunofluorescence confocal microscopy, and fluorescence-activated cell sorting. Additionally two other independently isolated and cultured human ESC lines as well as their differentiated derivatives were monitored for differential expression of selected proteins. Some of these proteins were identified exclusively in ESCs of all three human lines and may thus serve as generic ESC markers. Our wide scale proteomic approach enabled us to screen thousands of proteins rapidly and select putative ESC-associated proteins for further analysis. Validation by three independent conventional protein analysis techniques shows that our methodology is robust, provides an excellent tool to characterize ESCs at the protein level, and may disclose novel ESC-specific benchmarks.

An experimental correction for arginine-to-proline conversion artifacts in SILAC-based quantitative proteomics

An experimental correction for arginine-to-proline conversion artifacts in SILAC-based quantitative proteomics

Online Automated in Vivo Zebrafish Phosphoproteomics: From Large-Scale Analysis Down to a Single Embryo

In the developing embryo, as in many other biological processes, complex signaling pathways are under tight control of reversible phosphorylation, guiding cell proliferation, differentiation, and growth. Therefore the large-scale identification of signaling proteins and their post-translational modifications is crucial to understand the proteome biology of the developing zebrafish embryo. Here, we used an automated, robust, and sensitive online TiO 2-based LC-MS/MS setup to enrich for phosphorylated peptides from 1 day old zebrafish embryos. We identified, with high confidence, 1067 endogenous phosphorylation sites in a sample taken from 60 embryos (approximately 180 microg), 321 from 10 embryos, and 47 phosphorylation sites from a single embryo, illustrating the sensitivity of the method. This data set, representing by far the largest for zebrafish, was further exploited by searching for serine/threonine or tyrosine kinase motifs using Scansite. For one-third of the identified phosphopeptides a potential kinase motif could be predicted, where it appeared that Cdk5 kinase, p38MAPK, PKA, and Casein Kinase 2 substrates were the most predominant motifs present, underpinning the importance of these kinases in signaling pathways in embryonic development. The phosphopeptide data set was further interrogated using alignments with phosphopeptides identified in recent large-scale phosphoproteomics screens in human and mouse samples. These alignments revealed conservation of phosphorylation sites in several proteins suggesting preserved function in embryonic development.

Oleate Hydratase Catalyzes the Hydration of a Nonactivated Carbon-Carbon Bond

The hydration of oleic acid into 10-hydroxystearic acid was originally described for a Pseudomonas cell extract almost half a century ago. In the intervening years, the enzyme has never been characterized in any detail. We report here the isolation and characterization of oleate hydratase (EC 4.2.1.53) from Elizabethkingia meningoseptica.

Endogenous Phosphotyrosine Signaling in Zebrafish Embryos

In the developing embryo, cell growth, differentiation, and migration are strictly regulated by complex signaling pathways. One of the most important cell signaling mechanisms is protein phosphorylation on tyrosine residues, which is tightly controlled by protein-tyrosine kinases and protein-tyrosine phosphatases. Here we investigated endogenous phosphotyrosine signaling in developing zebrafish embryos. Tyrosine phosphorylated proteins were immunoaffinity-purified from zebrafish embryos at 3 and 5 days postfertilization and identified by multidimensional LC-MS. Among the identified proteins were tyrosine kinases, including Src family kinases, Eph receptor kinases, and focal adhesion kinases, as well as the adaptor proteins paxillin, p130Cas, and Crk. We identified several known and some unknown in vivo tyrosine phosphorylation sites in these proteins. Whereas most immunoaffinity-purified proteins were detected at both developmental stages, significant differences in abundance and/or phosphorylation state were also observed. In addition, multiplex in vitro kinase assays were performed by incubating a microarray of peptide substrates with the lysates of the two developmental stages. Many of the in vivo observations were confirmed by this on-chip in vitro kinase assay. Our experiments are the first to show that global tyrosine phosphorylation-mediated signaling can be studied at endogenous levels in complex multicellular organisms.

Sample Preparation Techniques for the Untargeted LC-MS-Based Discovery of Peptides in Complex Biological Matrices

Although big progress has been made in sample pretreatment over the last years, there are still considerable limitations when it comes to overcoming complexity and dynamic range problems associated with peptide analyses from biological matrices. Being the little brother of proteomics, peptidomics is a relatively new field of research aiming at the direct analysis of the small proteins, called peptides, many of which are not amenable for typical trypsin-based analytics. In this paper, we present an overview of different techniques and methods currently used for reducing a samples complexity and for concentrating low abundant compounds to enable successful peptidome analysis. We focus on techniques which can be employed prior to liquid chromatography coupled to mass spectrometry for peptide detection and identification and indicate their advantages as well as their shortcomings when it comes to the untargeted analysis of native peptides from complex biological matrices.

Archaeal (Per)Chlorate Reduction at High Temperature: An Interplay of Biotic and Abiotic Reactions

Archaea Powered by Rocket Fuel Perchlorate is a ubiquitous chlorine-based compound that forms naturally in the atmosphere. It is only present in large deposits in a few locations, such as the Atacama Desert in Chile, which has been assumed to be because perchlorate-reducing bacteria normally degrade it into chloride and oxygen. Liebensteiner et al. (p. 85; see the Perspective by Nerenberg), however, found that an archeon, Archaeoglobus fulgidus, can also reduce perchlorate. The archaeal perchlorate reduction pathway shares limited similarity with bacterial perchlorate reduction: Instead of producing chloride and oxygen, enzymatically produced chlorite reacts with sulfide to produce oxidized sulfur compounds. Because hyperthermophilic anaerobic archaea similar to A. fulgidus are thought to have been among the first complex organisms to evolve on Earth, they may have started creating oxidized conditions in some habitats before the emergence of oxygen-generating photosynthesis. Reduction of perchlorate by a hyperthermophilic archaeon suggests early evolution of enzymes using chlorine oxyanions. [Also see Perspective by Nerenberg] Perchlorate and chlorate anions [(per)chlorate] exist in the environment from natural and anthropogenic sources, where they can serve as electron acceptors for bacteria. We performed growth experiments combined with genomic and proteomic analyses of the hyperthermophile Archaeoglobus fulgidus that show (per)chlorate reduction also extends into the archaeal domain of life. The (per)chlorate reduction pathway in A. fulgidus relies on molybdo-enzymes that have similarity with bacterial enzymes; however, chlorite is not enzymatically split into chloride and oxygen. Evidence suggests that it is eliminated by an interplay of abiotic and biotic redox reactions involving sulfur compounds. Biological (per)chlorate reduction by ancient archaea at high temperature may have prevented accumulation of perchlorate in early terrestrial environments and consequently given rise to oxidizing conditions on Earth before the rise of oxygenic photosynthesis.

Imaging of Similar Mass Neuropeptides in Neuronal Tissue by Enhanced Resolution MALDI MS with an Ion Trap – Orbitrap TM Hybrid Instrument

Several mass spectrometry imaging (MSI) procedures are used to localize physiologically active peptides in neuronal tissue from American cockroach (Periplaneta americana) neurosecretory organs. We report how to use this model system to assess, for the first time, the performance of the MALDI LTQ Orbitrap XL mass spectrometer to perform MSI of secretory neuropeptides. The method involves the following steps: (1) rapid dissecting of neurosecretory tissue (i.e., insect neurohemal organ) in isotonic sucrose solution; (2) mounting the tissue on a glass slide; (3) controlled spraying of the air-dried tissue with concentrated MALDI matrix solution; (4) loading specimen into the MALDI source of a MS(n) system equipped with an Orbitrap analyzer; (5) setting-up MSI methods by determining tissue areas of interest, spatial resolution, molecular mass range, and molecular mass resolution; (6) acquiring mass spectra; (7) analyzing data using ImageQuest MSI software to generate (single or composite) images of the distribution of peptide(s) of interest; (8) confirming the identity of selected peptides by MS(2) and/or MS( n ) sequencing directly from imaged tissue sample. The results illustrate that high mass accuracy and high mass resolving power of the Orbitrap analyzer are achievable in analyses directly from tissue, such as in MSI experiments. Moreover the mass spectrometric instrumentation evaluated allows for both peptide localization and peptide identification/sequencing directly from tissue.

In Vivo Stable Isotope Labeling of Fruit Flies Reveals Post-transcriptional Regulation in the Maternal-to-zygotic Transition

An important hallmark in embryonic development is characterized by the maternal-to-zygotic transition (MZT) where zygotic transcription is activated by a maternally controlled environment. Post-transcriptional and translational regulation is critical for this transition and has been investigated in considerable detail at the gene level. We used a proteomics approach using metabolic labeling of Drosophila to quantitatively assess changes in protein expression levels before and after the MZT. By combining stable isotope labeling of fruit flies in vivo with high accuracy quantitative mass spectrometry we could quantify 2,232 proteins of which about half changed in abundance during this process. We show that ∼500 proteins increased in abundance, providing direct evidence of the identity of proteins as a product of embryonic translation. The group of down-regulated proteins is dominated by maternal factors involved in translational control of maternal and zygotic transcripts. Surprisingly a direct comparison of transcript and protein levels showed that the mRNA levels of down-regulated proteins remained relatively constant, indicating a translational control mechanism specifically targeting these proteins. In addition, we found evidence for post-translational processing of cysteine proteinase-1 (Cathepsin L), which became activated during the MZT as evidenced by the loss of its N-terminal propeptide. Poly(A)-binding protein was shown to be processed at its C-terminal tail, thereby losing one of its protein-interacting domains. Altogether this quantitative proteomics study provides a dynamic profile of known and novel proteins of maternal as well as embryonic origin. This provides insight into the production, stability, and modification of individual proteins, whereas discrepancies between transcriptional profiles and protein dynamics indicate novel control mechanisms in genome activation during early fly development.