BYON4228 is a pan-allelic antagonistic SIRPα antibody that potentiates destruction of antibody-opsonized tumor cells and lacks binding to SIRPγ on T cells.

BACKGROUND: Preclinical studies have firmly established the CD47-signal-regulatory protein (SIRP)α axis as a myeloid immune checkpoint in cancer, and this is corroborated by available evidence from the first clinical studies with CD47 blockers. However, CD47 is ubiquitously expressed and mediates functional interactions with other ligands as well, and therefore targeting of the primarily myeloid cell-restricted inhibitory immunoreceptor SIRPα may represent a better strategy. METHOD: We generated BYON4228, a novel SIRPα-directed antibody. An extensive preclinical characterization was performed, including direct comparisons to previously reported anti-SIRPα antibodies. RESULTS: BYON4228 is an antibody directed against SIRPα that recognizes both allelic variants of SIRPα in the human population, thereby maximizing its potential clinical applicability. Notably, BYON4228 does not recognize the closely related T-cell expressed SIRPγ that mediates interactions with CD47 as well, which are known to be instrumental in T-cell extravasation and activation. BYON4228 binds to the N-terminal Ig-like domain of SIRPα and its epitope largely overlaps with the CD47-binding site. BYON4228 blocks binding of CD47 to SIRPα and inhibits signaling through the CD47-SIRPα axis. Functional studies show that BYON4228 potentiates macrophage-mediated and neutrophil-mediated killing of hematologic and solid cancer cells in vitro in the presence of a variety of tumor-targeting antibodies, including trastuzumab, rituximab, daratumumab and cetuximab. The silenced Fc region of BYON4228 precludes immune cell-mediated elimination of SIRPα-positive myeloid cells, implying anticipated preservation of myeloid immune effector cells in patients. The unique profile of BYON4228 clearly distinguishes it from previously reported antibodies representative of agents in clinical development, which either lack recognition of one of the two SIRPα polymorphic variants (HEFLB), or cross-react with SIRPγ and inhibit CD47-SIRPγ interactions (SIRPAB-11-K322A, 1H9), and/or have functional Fc regions thereby displaying myeloid cell depletion activity (SIRPAB-11-K322A). In vivo, BYON4228 increases the antitumor activity of rituximab in a B-cell Raji xenograft model in human SIRPαBIT transgenic mice. Finally, BYON4228 shows a favorable safety profile in cynomolgus monkeys. CONCLUSIONS: Collectively, this defines BYON4228 as a preclinically highly differentiating pan-allelic SIRPα antibody without T-cell SIRPγ recognition that promotes the destruction of antibody-opsonized cancer cells. Clinical studies are planned to start in 2023.

A cullin-RING ubiquitin ligase targets exogenous α-synuclein and inhibits Lewy body-like pathology.

Parkinson's disease (PD) is a neurological disorder characterized by the progressive accumulation of neuronal α-synuclein (αSyn) inclusions called Lewy bodies. It is believed that Lewy bodies spread throughout the nervous system due to the cell-to-cell propagation of αSyn via cycles of secretion and uptake. Here, we investigated the internalization and intracellular accumulation of exogenous αSyn, two key steps of Lewy body pathogenesis, amplification and spreading. We found that stable αSyn fibrils substantially accumulate in different cell lines upon internalization, whereas αSyn monomers, oligomers, and dissociable fibrils do not. Our data indicate that the uptake-mediated accumulation of αSyn in a human-derived neuroblastoma cell line triggered an adaptive response that involved proteins linked to ubiquitin ligases of the S-phase kinase-associated protein 1 (SKP1), cullin-1 (Cul1), and F-box domain-containing protein (SCF) family. We found that SKP1, Cul1, and the F-box/LRR repeat protein 5 (FBXL5) colocalized and physically interacted with internalized αSyn in cultured cells. Moreover, the SCF containing the F-box protein FBXL5 (SCFFBXL5) catalyzed αSyn ubiquitination in reconstitution experiments in vitro using recombinant proteins and in cultured cells. In the human brain, SKP1 and Cul1 were recruited into Lewy bodies from brainstem and neocortex of patients with PD and related neurological disorders. In both transgenic and nontransgenic mice, intracerebral administration of exogenous αSyn fibrils triggered a Lewy body-like pathology, which was amplified by SKP1 or FBXL5 loss of function. Our data thus indicate that SCFFXBL5 regulates αSyn in vivo and that SCF ligases may constitute targets for the treatment of PD and other α-synucleinopathies.

TDP-43 extracted from frontotemporal lobar degeneration subject brains displays distinct aggregate assemblies and neurotoxic effects reflecting disease progression rates.

Accumulation of abnormally phosphorylated TDP-43 (pTDP-43) is the main pathology in affected neurons of people with amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Morphological diversity and neuroanatomical distribution of pTDP-43 accumulations allowed classification of FTLD cases into at least four subtypes, which are correlated with clinical presentations and genetic causes. To understand the molecular basis of this heterogeneity, we developed SarkoSpin, a new method for biochemical isolation of pathological TDP-43. By combining SarkoSpin with mass spectrometry, we revealed proteins beyond TDP-43 that become abnormally insoluble in a disease subtype-specific manner. We show that pTDP-43 extracted from brain forms stable assemblies of distinct densities and morphologies that are associated with disease subtypes. Importantly, biochemically extracted pTDP-43 assemblies showed differential neurotoxicity and seeding that were correlated with disease duration of FTLD subjects. Our data are consistent with the notion that disease heterogeneity could originate from alternate pathological TDP-43 conformations, which are reminiscent of prion strains.

Biology/Disease-Driven Initiative on Protein-Aggregation Diseases of the Human Proteome Project: Goals and Progress to Date.

The Biology/Disease-driven (B/D) working groups of the Human Proteome Project are alliances of research groups aimed at developing or improving proteomic tools to support specific biological or disease-related research areas. Here, we describe the activities and progress to date of the B/D working group focused on protein aggregation diseases (PADs). PADs are characterized by the intra- or extracellular accumulation of aggregated proteins and include devastating diseases such as Parkinson's and Alzheimer's disease and systemic amyloidosis. The PAD B/D working group aims for the development of proteomic assays for the quantification of aggregation-prone proteins involved in PADs to support basic and clinical research on PADs. Because the proteins in PADs undergo aberrant conformational changes, a goal is to quantitatively resolve altered protein structures and aggregation states in complex biological specimens. We have developed protein-extraction protocols and a set of mass spectrometric (MS) methods that enable the detection and quantification of proteins involved in the systemic and localized amyloidosis and the probing of aberrant protein conformational transitions in cell and tissue extracts. In several studies, we have demonstrated the potential of MS-based proteomics approaches for specific and sensitive clinical diagnoses and for the subtyping of PADs. The developed methods have been detailed in both protocol papers and manuscripts describing applications to facilitate implementation by nonspecialized laboratories, and assay coordinates are shared through public repositories and databases. Clinicians actively involved in the PAD working group support the transfer to clinical practice of the developed methods, such as assays to quantify specific disease-related proteins and their fragments in biofluids and multiplexed MS-based methods for the diagnosis and typing of systemic amyloidosis. We believe that the increasing availability of tools to precisely measure proteins involved in PADs will positively impact research on the molecular bases of these diseases and support early disease diagnosis and a more-confident subtyping.

Chemical stresses fail to mimic the unfolded protein response resulting from luminal load with unfolded polypeptides.

The stress sensors ATF6, IRE1, and PERK monitor deviations from homeostatic conditions in the endoplasmic reticulum (ER), a protein biogenesis compartment of eukaryotic cells. Their activation elicits unfolded protein responses (UPR) to re-establish proteostasis. UPR have been extensively investigated in cells exposed to chemicals that activate ER stress sensors by perturbing calcium, N-glycans, or redox homeostasis. Cell responses to variations in luminal load with unfolded proteins are, in contrast, poorly characterized. Here, we compared gene and protein expression profiles in HEK293 cells challenged with ER stress-inducing drugs or expressing model polypeptides. Drug titration to limit up-regulation of the endogenous ER stress reporters heat shock protein family A (Hsp70) member 5 (BiP/HSPA5) and homocysteine-inducible ER protein with ubiquitin-like domain 1 (HERP/HERPUD1) to levels comparable with luminal accumulation of unfolded proteins substantially reduced the amplitude of both transcriptional and translational responses. However, these drug-induced changes remained pleiotropic and failed to recapitulate responses to ER load with unfolded proteins. These required unfolded protein association with BiP and induced a much smaller subset of genes participating in a chaperone complex that binds unfolded peptide chains. In conclusion, UPR resulting from ER load with unfolded proteins proceed via a well-defined and fine-tuned pathway, whereas even mild chemical stresses caused by compounds often used to stimulate UPR induce cellular responses largely unrelated to the UPR or ER-mediated protein secretion.

Systematic proteome and proteostasis profiling in human Trisomy 21 fibroblast cells.

Down syndrome (DS) is mostly caused by a trisomy of the entire Chromosome 21 (Trisomy 21, T21). Here, we use SWATH mass spectrometry to quantify protein abundance and protein turnover in fibroblasts from a monozygotic twin pair discordant for T21, and to profile protein expression in 11 unrelated DS individuals and matched controls. The integration of the steady-state and turnover proteomic data indicates that protein-specific degradation of members of stoichiometric complexes is a major determinant of T21 gene dosage outcome, both within and between individuals. This effect is not apparent from genomic and transcriptomic data. The data also reveal that T21 results in extensive proteome remodeling, affecting proteins encoded by all chromosomes. Finally, we find broad, organelle-specific post-transcriptional effects such as significant downregulation of the mitochondrial proteome contributing to T21 hallmarks. Overall, we provide a valuable proteomic resource to understand the origin of DS phenotypic manifestations.

Measuring protein structural changes on a proteome-wide scale using limited proteolysis-coupled mass spectrometry.

Protein structural changes induced by external perturbations or internal cues can profoundly influence protein activity and thus modulate cellular physiology. A number of biophysical approaches are available to probe protein structural changes, but these are not applicable to a whole proteome in a biological extract. Limited proteolysis-coupled mass spectrometry (LiP-MS) is a recently developed proteomics approach that enables the identification of protein structural changes directly in their complex biological context on a proteome-wide scale. After perturbations of interest, proteome extracts are subjected to a double-protease digestion step with a nonspecific protease applied under native conditions, followed by complete digestion with the sequence-specific protease trypsin under denaturing conditions. This sequential treatment generates structure-specific peptides amenable to bottom-up MS analysis. Next, a proteomics workflow involving shotgun or targeted MS and label-free quantification is applied to measure structure-dependent proteolytic patterns directly in the proteome extract. Possible applications of LiP-MS include discovery of perturbation-induced protein structural alterations, identification of drug targets, detection of disease-associated protein structural states, and analysis of protein aggregates directly in biological samples. The approach also enables identification of the specific protein regions involved in the structural transition or affected by the binding event. Sample preparation takes approximately 2 d, followed by one to several days of MS and data analysis time, depending on the number of samples analyzed. Scientists with basic biochemistry training can implement the sample preparation steps. MS measurement and data analysis require a background in proteomics.

Lysine acetylome profiling uncovers novel histone deacetylase substrate proteins in Arabidopsis.

Histone deacetylases have central functions in regulating stress defenses and development in plants. However, the knowledge about the deacetylase functions is largely limited to histones, although these enzymes were found in diverse subcellular compartments. In this study, we determined the proteome-wide signatures of the RPD3/HDA1 class of histone deacetylases in Arabidopsis Relative quantification of the changes in the lysine acetylation levels was determined on a proteome-wide scale after treatment of Arabidopsis leaves with deacetylase inhibitors apicidin and trichostatin A. We identified 91 new acetylated candidate proteins other than histones, which are potential substrates of the RPD3/HDA1-like histone deacetylases in Arabidopsis, of which at least 30 of these proteins function in nucleic acid binding. Furthermore, our analysis revealed that histone deacetylase 14 (HDA14) is the first organellar-localized RPD3/HDA1 class protein found to reside in the chloroplasts and that the majority of its protein targets have functions in photosynthesis. Finally, the analysis of HDA14 loss-of-function mutants revealed that the activation state of RuBisCO is controlled by lysine acetylation of RuBisCO activase under low-light conditions.

Dimethyl-Labeling-Based Quantification of the Lysine Acetylome and Proteome of Plants.

Photorespiratory enzymes in different cellular compartments have been reported to be posttranslational modified by phosphorylation, disulfide formation, S-nitrosylation, glutathionylation, and lysine acetylation. However, not much is known yet about the function of these modifications to regulate the activities, localizations, or interactions of the proteins in this metabolic pathway. Hence, it will be of great importance to study these modifications and their temporal and conditional occurrence in more detail. Here, we focus on the analysis of lysine acetylation as a relatively newly discovered modification on plant metabolic enzymes. The acetylation of lysine residues within proteins is a highly conserved and reversible posttranslational modification which occurs in all living organisms. First discovered on histones and implied in the regulation of gene expression, lysine acetylation also occurs on a diverse set of cellular proteins in different subcellular compartments and is particularly abundant on metabolic enzymes. Upon lysine acetylation, the function of proteins can be modulated due to the loss of the positive charge of the lysine residue. Lysine acetylation was also discovered on proteins involved in photosynthesis and novel tools are needed to study the regulation of this modification in dependence on the environmental conditions, tissues, or plant genotype. This chapter describes a method for the identification and relative quantification of lysine-acetylated proteins in plant tissues using a dimethyl labeling technique combined with an anti-acetyl lysine antibody enrichment strategy. Here, we describe the protein purification, labeling of trypsinated peptides, as well as immuno-enrichment of lysine-acetylated peptides followed by liquid chromatography tandem mass spectrometry (LC-MS/MS) data acquisition and analysis.

Cell-wide analysis of protein thermal unfolding reveals determinants of thermostability.

Temperature-induced cell death is thought to be due to protein denaturation, but the determinants of thermal sensitivity of proteomes remain largely uncharacterized. We developed a structural proteomic strategy to measure protein thermostability on a proteome-wide scale and with domain-level resolution. We applied it to Escherichia coli, Saccharomyces cerevisiae, Thermus thermophilus, and human cells, yielding thermostability data for more than 8000 proteins. Our results (i) indicate that temperature-induced cellular collapse is due to the loss of a subset of proteins with key functions, (ii) shed light on the evolutionary conservation of protein and domain stability, and (iii) suggest that natively disordered proteins in a cell are less prevalent than predicted and (iv) that highly expressed proteins are stable because they are designed to tolerate translational errors that would lead to the accumulation of toxic misfolded species.

Proteomics beyond large-scale protein expression analysis.

Proteomics is commonly referred to as the application of high-throughput approaches to protein expression analysis. Typical results of proteomics studies are inventories of the protein content of a sample or lists of differentially expressed proteins across multiple conditions. Recently, however, an explosion of novel proteomics workflows has significantly expanded proteomics beyond the analysis of protein expression. Targeted proteomics methods, for example, enable the analysis of the fine dynamics of protein systems, such as a specific pathway or a network of interacting proteins, and the determination of protein complex stoichiometries. Structural proteomics tools allow extraction of restraints for structural modeling and identification of structurally altered proteins on a proteome-wide scale. Other variations of the proteomic workflow can be applied to the large-scale analysis of protein activity, location, degradation and turnover. These exciting developments provide new tools for multi-level 'omics' analysis and for the modeling of biological networks in the context of systems biology studies.

Global analysis of protein structural changes in complex proteomes.

Changes in protein conformation can affect protein function, but methods to probe these structural changes on a global scale in cells have been lacking. To enable large-scale analyses of protein conformational changes directly in their biological matrices, we present a method that couples limited proteolysis with a targeted proteomics workflow. Using our method, we assessed the structural features of more than 1,000 yeast proteins simultaneously and detected altered conformations for ~300 proteins upon a change of nutrients. We find that some branches of carbon metabolism are transcriptionally regulated whereas others are regulated by enzyme conformational changes. We detect structural changes in aggregation-prone proteins and show the functional relevance of one of these proteins to the metabolic switch. This approach enables probing of both subtle and pronounced structural changes of proteins on a large scale.

The mitochondrial lysine acetylome of Arabidopsis.

Posttranslational modifications are essential regulators of protein functions as they can modify enzyme activities or protein-molecule interactions by changing the charge state or chemical properties of their target amino acid. The acetyl moiety of the central energy metabolite acetyl-CoA can be transferred to the ε-amino group of lysine, a process known as lysine acetylation which is implicated in the regulation of key metabolic enzymes in various organisms. Since plant mitochondria are of great importance for plant growth and development and as they house key enzymes of oxidative phosphorylation and photorespiration, it is essential to investigate the occurrence of lysine acetylation in this organelle. Here we characterised the plant mitochondrial acetylome of Arabidopsis mitochondria by LC-MS/MS analysis. In total 120 lysine-acetylated mitochondrial proteins containing 243 acetylated sites were identified. These proteins were mapped into functional categories showing that many proteins with essential functions from the tricaboxylic cycle and the respiratory chain are lysine-acetylated, as well as proteins involved in photorespiration, amino acid and protein metabolism, and redox regulation. Immuno-detection of mitochondrial proteins revealed that many lysine-acetylated proteins reside in native protein complexes. Furthermore, in vitro experiments demonstrated that lysine acetylation can occur non-enzymatically in Arabidopsis mitochondria at physiological matrix pH.

The Arabidopsis class II sirtuin is a lysine deacetylase and interacts with mitochondrial energy metabolism.

The posttranslational regulation of proteins by lysine (Lys) acetylation has recently emerged to occur not only on histones, but also on organellar proteins in plants and animals. In particular, the catalytic activities of metabolic enzymes have been shown to be regulated by Lys acetylation. The Arabidopsis (Arabidopsis thaliana) genome encodes two predicted sirtuin-type Lys deacetylases, of which only Silent Information Regulator2 homolog (SRT2) contains a predicted presequence for mitochondrial targeting. Here, we have investigated the function of SRT2 in Arabidopsis. We demonstrate that SRT2 functions as a Lys deacetylase in vitro and in vivo. We show that SRT2 resides predominantly at the inner mitochondrial membrane and interacts with a small number of protein complexes mainly involved in energy metabolism and metabolite transport. Several of these protein complexes, such as the ATP synthase and the ATP/ADP carriers, show an increase in Lys acetylation in srt2 loss-of-function mutants. The srt2 plants display no growth phenotype but rather a metabolic phenotype with altered levels in sugars, amino acids, and ADP contents. Furthermore, coupling of respiration to ATP synthesis is decreased in these lines, while the ADP uptake into mitochondria is significantly increased. Our results indicate that SRT2 is important in fine-tuning mitochondrial energy metabolism.

Quantification of the N-glycosylated secretome by super-SILAC during breast cancer progression and in human blood samples.

Cells secrete a large number of proteins to communicate with their surroundings. Furthermore, plasma membrane proteins and intracellular proteins can be released into the extracellular space by regulated or non-regulated processes. Here, we profiled the supernatant of 11 cell lines that are representative of different stages of breast cancer development by specifically capturing N-glycosylated peptides using the N-glyco FASP technology. For accurate quantification we developed a super-SILAC mix from several labeled breast cancer cell lines and used it as an internal standard for all samples. In total, 1398 unique N-glycosylation sites were identified and quantified. Enriching for N-glycosylated peptides focused the analysis on classically secreted and membrane proteins. N-glycosylated secretome profiles correctly clustered the different cell lines to their respective cancer stage, suggesting that biologically relevant differences were detected. Five different profiles of glycoprotein dynamics during cancer development were detected, and they contained several proteins with known roles in breast cancer. We then used the super-SILAC mix in plasma, which led to the quantification of a large number of the previously identified N-glycopeptides in this important body fluid. The combination of quantifying the secretome of cancer cell lines and of human plasma with a super-SILAC approach appears to be a promising new approach for finding markers of disease.

Wnt signaling through inhibition of ß-catenin degradation in an intact Axin1 complex.

Degradation of cytosolic ß-catenin by the APC/Axin1 destruction complex represents the key regulated step of the Wnt pathway. It is incompletely understood how the Axin1 complex exerts its Wnt-regulated function. Here, we examine the mechanism of Wnt signaling under endogenous levels of the Axin1 complex. Our results demonstrate that ß-catenin is not only phosphorylated inside the Axin1 complex, but also ubiquinated and degraded via the proteasome, all within an intact Axin1 complex. In disagreement with current views, we find neither a disassembly of the complex nor an inhibition of phosphorylation of Axin1-bound ß-catenin upon Wnt signaling. Similar observations are made in primary intestinal epithelium and in colorectal cancer cell lines carrying activating Wnt pathway mutations. Wnt signaling suppresses ß-catenin ubiquitination normally occurring within the complex, leading to complex saturation by accumulated phospho-ß-catenin. Subsequently, newly synthesized ß-catenin can accumulate in a free cytosolic form and engage nuclear TCF transcription factors.

Targeted quantitative phosphoproteomics approach for the detection of phospho-tyrosine signaling in plants.

Tyrosine (Tyr) phosphorylation plays an essential role in signaling in animal systems. However, a few studies have also reported Tyr phosphorylation in plants, but the relative contribution of tyrosine phosphorylation to plant signal transduction has remained an open question. We present an approach to selectively measure and quantify Tyr phosphorylation in plant cells, which can also be applied to whole plants. We combined a (15)N stable isotope metabolic labeling strategy with an immuno-affinity purification using phospho-tyrosine (pY) specific antibodies. This single enrichment strategy was sufficient to reproducibly identify and quantify pY containing peptides from total plant cell extract in a single LC-MS/MS run. We succeeded in identifying 149 unique pY peptides originating from 135 proteins, including a large set of different protein kinases and several receptor-like kinases. We used flagellin perception by Arabidopsis cells, a model system for pathogen triggered immune (PTI) signaling, to test our approach. We reproducibly quantified 23 pY peptides in 2 inversely labeled biological replicates identifying 11 differentially phosphorylated proteins. These include a set of 3 well-characterized flagellin responsive MAP kinases and 4 novel MAP kinases. With this targeted approach, we elucidate a new level of complexity in flagellin-induced MAP kinase activation.

Evaluating experimental bias and completeness in comparative phosphoproteomics analysis.

Unraveling the functional dynamics of phosphorylation networks is a crucial step in understanding the way in which biological networks form a living cell. Recently there has been an enormous increase in the number of measured phosphorylation events. Nevertheless, comparative and integrative analysis of phosphoproteomes is confounded by incomplete coverage and biases introduced by different experimental workflows. As a result, we cannot differentiate whether phosphosites indentified in only one or two samples are the result of condition or species specific phosphorylation, or reflect missing data. Here, we evaluate the impact of incomplete phosphoproteomics datasets on comparative analysis, and we present bioinformatics strategies to quantify the impact of different experimental workflows on measured phosphoproteomes. We show that plotting the saturation in observed phosphosites in replicates provides a reproducible picture of the extent of a particular phosphoproteome. Still, we are still far away from a complete picture of the total human phosphoproteome. The impact of different experimental techniques on the similarity between phosphoproteomes can be estimated by comparing datasets from different experimental pipelines to a common reference. Our results show that comparative analysis is most powerful when datasets have been generated using the same experimental workflow. We show this experimentally by measuring the tyrosine phosphoproteome from Caenorhabditis elegans and comparing it to the tyrosine phosphoproteome of HeLa cells, resulting in an overlap of about 4%. This overlap between very different organisms represents a three-fold increase when compared to dataset of older studies, wherein different workflows were used. The strategies we suggest enable an estimation of the impact of differences in experimental workflows on the overlap between datasets. This will allow us to perform comparative analyses not only on datasets specifically generated for this purpose, but also to extract insights through comparative analysis of the ever-increasing wealth of publically available phosphorylation data.

Zwitterionic hydrophilic interaction liquid chromatography (ZIC-HILIC and ZIC-cHILIC) provide high resolution separation and increase sensitivity in proteome analysis.

The complexity of peptide mixtures that are analyzed in proteomics necessitates fractionation by multidimensional separation approaches prior to mass spectrometric analysis. In this work, we introduce and evaluate hydrophilic interaction liquid chromatography (HILIC) based strategies for the separation of complex peptide mixtures. The two zwitterionic HILIC materials (ZIC-HILIC and ZIC-cHILIC) chosen for this work differ in the spatial orientation of the positive and negative charged groups. Online experiments revealed a pH-independent resolving power for the ZIC-cHILIC resin while ZIC-HILIC showed a decrease in resolving power at an acidic pH. Subsequently, we extensively evaluated the performances of ZIC-HILIC and ZIC-cHILIC as first dimension in an off-line two-dimensional liquid chromatography (2D-LC) strategy in combination with reversed phase (RP), with respect to peptide separation efficiency and how the retention time correlates with a number of peptide physicochemical properties. Both resins allowed the identification of more than 20,000 unique peptides corresponding to over 3500 proteins in each experimental condition from a remarkably low (1.5 µg) amount of starting material of HeLa lysate digestion. The resulting data allows the drawing of a comprehensive picture regarding ZIC- and ZIC-cHILIC peptide separation characteristics. Furthermore, the extent of protein identifications observed from such a level of material demonstrates that HILIC can rival or surpass traditional multidimensional strategies employed in proteomics.

Tyrosine phosphorylation profiling in FGF-2 stimulated human embryonic stem cells.

The role of fibroblast growth factor-2 (FGF-2) in maintaining undifferentiated human embryonic stem cells (hESC) was investigated using a targeted phosphoproteomics approach to specifically profile tyrosine phosphorylation events following FGF-2 stimulation. A cumulative total number of 735 unique tyrosine phosphorylation sites on 430 proteins were identified, by far the largest inventory to date for hESC. Early signaling events in FGF-2 stimulated hESC were quantitatively monitored using stable isotope dimethyl labeling, resulting in temporal tyrosine phosphorylation profiles of 316 unique phosphotyrosine peptides originating from 188 proteins. Apart from the rapid activation of all four FGF receptors, trans-activation of several other receptor tyrosine kinases (RTKs) was observed as well as induced tyrosine phosphorylation of downstream proteins such as PI3-K, MAPK and several Src family members. Both PI3-K and MAPK have been linked to hESC maintenance through FGF-2 mediated signaling. The observed activation of the Src kinase family members by FGF-2 and loss of pluripotent marker expression post Src kinase inhibition may point to the regulation of cytoskeletal and actin depending processes to maintain undifferentiated hESC.