Acetylation of proteins on lysine residues is a dynamic posttranslational modification that is known to play a key role in regulating transcription and other DNA-dependent nuclear processes. However, the extent of this modification in diverse cellular proteins remains largely unknown, presenting a major bottleneck for lysine-acetylation biology. Here we report the first proteomic survey of this modification, identifying 388 acetylation sites in 195 proteins among proteins derived from HeLa cells and mouse liver mitochondria. In addition to regulators of chromatin-based cellular processes, nonnuclear localized proteins with diverse functions were identified. Most strikingly, acetyllysine was found in more than 20% of mitochondrial proteins, including many longevity regulators and metabolism enzymes. Our study reveals previously unappreciated roles for lysine acetylation in the regulation of diverse cellular pathways outside of the nucleus. The combined data sets offer a rich source for further characterization of the contribution of this modification to cellular physiology and human diseases.

The positively charged lysine residue plays an important role in protein folding and functions. Neutralization of the charge often has a profound impact on the substrate proteins. Accordingly all the known post-translational modifications at lysine have pivotal roles in cell physiology and pathology. Here we report the discovery of two novel, in vivo lysine modifications in histones, lysine propionylation and butyrylation. We confirmed, by in vitro labeling and peptide mapping by mass spectrometry, that two previously known acetyltransferases, p300 and CREB-binding protein, could catalyze lysine propionylation and lysine butyrylation in histones.

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Early cancers are avascular and hence, profoundly acidic. Pre-malignant cells must adapt to acidosis to thrive in this hostile microenvironment. Here, we investigate MCF-7 cells that are adapted to grow in acidic conditions using SILAC proteomics and we reveal a significant upregulation of lysosomal proteins. Prominent among these is LAMP2 that functions to protect lysosomal membranes from acid proteolysis. LAMP2 upregulation by acidosis is confirmed both in vitro and in vivo. Furthermore, we show that the depletion of LAMP2 is sufficient to increase acidosis-mediated toxicity. In breast cancer patient samples, there is a high correlation of LAMP2 mRNA and protein expression with progression. We also observe that LAMP2 is located at the plasma membrane in clinical samples and this redistribution is acid-induced in vitro. Our findings suggest a potential adaptive mechanism, wherein cells chronically exposed to an acidic environment translocate lysosomal proteins to their surface, thus protecting the plasmalemma from acid-induced hydrolysis.

Global analysis of protein phosphorylation will provide insight into mechanisms by which this dynamic post-translational modification modulates diverse cellular processes. Mass spectrometry-based phosphoproteomics is a potentially powerful approach for global profiling and quantification of protein phosphorylation. Such studies usually involve selective isolation of phosphorylated peptides and their subsequent fragmentation in a mass spectrometer to assign the sequence and localize phosphorylation sites. Three strategies have been described for enriching phosphopeptides based on antibodies (1, 2), chemical derivatization (3, 4), or ionic interactions (e.g. IMAC and strong ion exchange chromatography) (5-9). These methods have achieved limited success in enriching phosphopeptides for proteomics studies. Among these approaches, IMAC is the most convenient and holds much potential for the efficient isolation of phosphopeptides (10 -13). The method has been used for several global analyses of protein phosphorylation in model organisms and cellular organelles (5-9). Nevertheless further refinement of extant IMAC protocols is required to achieve high reproducibility and efficiency (14).Poor fragmentation of phosphopeptides in the mass spectrometer represents the second major challenge for phosphoproteomics. The availability of a relatively low energy fragmentation pathway via ␤-elimination of the phosphate moiety limits fragmentation at peptide bonds that would be informative for identifying the sequence and site(s) of phosphorylation of the peptide. Mass spectrometers under development, such as instruments using electron transfer dissociation (15), might address this problem. However, such mass spectrometers are not currently commercially available. In addition, fragmentation of doubly charged and singly charged peptides in electron transfer dissociation mass spectrometry is compromised. In summary, despite extensive effort in the past several years, efficient proteomics of protein phosphorylation remains a daunting challenge.Here we report analysis of the phosphoproteome of mitochondria using an improved method that integrates an optimized batchwise IMAC protocol for isolation of phosphopeptides and MS3 1 for fragmentation of phosphopeptides. The improved IMAC procedure allowed recovery of ϳ77% of phosphopeptides while retaining few unphosphorylated peptides. MS3 addressed the poor fragmentation of phosphopeptides by generating highly informative fragmentation patterns that allow peptide identification. Analysis of mitochondrial phosphorylation revealed 84 phosphorylation sites from 62 proteins. Most identified phosphorylation sites have not been reported before, providing novel information about mitochondrial regulatory mechanisms. The optimized batchwise IMAC protocol in combination with MS3 offers a relatively simple and more efficient approach for proteomics of protein phosphorylation.EXPERIMENTAL PROCEDURES

The O-linked N-acetylglucosamine (O-GlcNAc) modification of serine/threonine residues is an abundant posttranslational modification present in cytosolic and nuclear proteins. The functions and subproteome of O-GlcNAc modification remain largely undefined. Here we report the application of the tagging-via-substrate (TAS) approach for global identification of O-GlcNAc-modified proteins. The TAS method utilizes an O-GlcNAc azide analogue for metabolic labeling of O-GlcNAc-modified proteins, which can be chemoselectively conjugated for detection and enrichment of the proteins for proteomics studies. Our study led to the identification of 199 putative O-GlcNAcmodified proteins from HeLa cells, among which 23 were confirmed using reciprocal immunoprecipitation. Functional classification shows that proteins with diverse functions are modified by O-GlcNAc, implying that OGlcNAc might be involved in the regulation of multiple cellular pathways.The modification of nuclear and cytoplasmic proteins at serine and threonine residues with O-linked N-acetylglucosamine (OGlcNAc) was first described over two decades ago. 1 The modification was found in various classes of proteins including enzymes, transcription factors, cytoskeletal proteins, signaling proteins, receptors, nuclear pore complex proteins, and kinases. 2 Similar to phosphorylation, the O-GlcNAc modification is dynamic with a turnover rate faster than that of the proteins it modifies. 3 The O-GlcNAc modification has been shown to affect protein-protein interactions, protein-DNA interactions, protein stability and activity, and cell signaling cascades. 4 Disregulation of the OGlcNAc modification has been implicated in the development of disease states including diabetes, cancer, and Alzheimer's. 2,4 Given the potentially broad regulatory influence of the O-GlcNAc modification, a more comprehensive understanding of the targets of O-GlcNAc transferase is needed to elucidate its functional consequences.In this report, we describe the global detection and proteomic analysis of O-GlcNAc-modified proteins in HeLa cells. An affinitytagged version of the O-GlcNAc modification is metabolically incorporated onto proteins using an azide-tagged analogue of N-acetylglucosamine. The azido-GlcNAc-modified proteins thus contain an azide handle for chemoselective conjugation using a biotinylated phosphine reagent. The resulting conjugates were affinity-purified with streptavidin beads and subsequently digested with trypsin and analyzed by nano-HPLC-MS/MS. Using this strategy, we identified 199 azido-GlcNAc-modified proteins in HeLa cells. We subsequently validated the presence of this modification among 10 previously reported and 13 newly identified O-GlcNAcmodified proteins using specific antibodies. Our results reveal that proteins with a wide range of functions are modified by O-GlcNAc, implying its diverse cellular functions.

Previously, it was reported that red blood cells (RBCs) are required to demonstrate participation of nitric oxide (NO) in the regulation of rabbit pulmonary vascular resistance (PVR). RBCs do not synthesize NO; hence, we postulated that ATP, present in millimolar amounts in RBCs, was the mediator, which evoked NO synthesis in the vascular endothelium. First, we found that deformation of RBCs, as occurs on passage across the pulmonary circulation with increasing flow rate, evoked increments in ATP release. Here, ATP (300 nM), administered to isolated, salt solution-perfused (PSS) rabbit lungs, decreased total and upstream (arterial) PVR, a response inhibited by N G -nitro-L-arginine methyl ester (L-NAME, 100 M). In lungs perfused with PSS containing RBCs, L-NAME increased total and upstream PVR. In lungs perfused with PSS containing glibenclamide-treated RBCs, which inhibits ATP release, L-NAME was without effect. Apyrase grade VII (8 U/ml), which degrades ATP to AMP, was without effect on PVR in PSS-perfused lungs. These results are consistent with the hypothesis that ATP, released from RBCs as they traverse the pulmonary circulation, evokes endogenous NO synthesis.adenosine-5Ј triphosphate; red blood cell EXTRACELLULAR ATP has been suggested to play an important role in the regulation of vascular resistance in a number of vascular beds, including the kidney (24, 26), mesentery (5, 28), heart (17, 20), and lung (8,10,11,12,16,31). The spacial relationship between the cell that is the source of extracellular ATP and vascular smooth muscle is an important determinant of the vascular response to ATP. Thus ATP released from nerve terminals adjacent to vascular smooth muscle would be expected to activate purinergic receptors that produce contraction of that muscle (18,21). In contrast, ATP released from formed elements in the circulation such as red blood cells (RBCs) (6,9,31,33) or ATP released from the endothelium itself (27) would interact with purinergic receptors present on the endothelium. The stimulation of such receptors has been shown to result in the synthesis of endothelium-derived relaxing factors, including nitric oxide (NO) (5,8,12,11,16). Thus extracellular ATP released from nerve terminals would be expected to increase vascular resistance, whereas ATP released into the vascular lumen could be an important mechanism for decreasing vascular resistance.We reported previously that 1) ATP is released from rabbit RBCs as they traverse the pulmonary circulation (29) and 2) RBCs that are capable of releasing ATP were a requisite component in the perfusate of isolated rabbit lungs to demonstrate the participation of NO as a determinant of vascular resistance (31, 33). In the work presented here, we present evidence that ATP, in the absence of RBCs, is capable of promoting NO synthesis in the pulmonary circulation of the rabbit. Thus we determined the effect of ATP infused into the circulation of isolated rabbit lungs on vascular resistance. Moreover, we present evidence that the major mechanism by which ATP ac...

Identification of proteins bearing a specific post-translational modification would imply functions of the modification. Proteomic analysis of post-translationally modified proteins is usually challenging due to high complexity and wide dynamic range, as well as unavailability of efficient methods to enrich the proteins of interest. Here, we report a strategy for the detection, isolation, and profiling of O-linked N-acetylglucosamine (O-GlcNAc) modified proteins, which involves three steps: metabolic labeling of cells with an unnatural GlcNAc analogue, peracetylated azido-GlcNAc; chemoselective conjugation of azido-GlcNAc modified proteins via the Staudinger ligation, which is specific between phosphine and azide, using a biotinylated phosphine capture reagent; and detection and affinity purification of the resulting conjugated O-GlcNAc modified proteins. Since the approach relies on a tag (azide) in the substrate, we designated it the tagging-via-substrate (TAS) strategy. A similar strategy was used previously for protein farnesylation, phosphorylation, and sumoylation. Using this approach, we were able to specifically label and subsequently detect azido-GlcNAc modified proteins from the cytosolic lysates of HeLa, 3T3, COS-1, and S2 cell lines, suggesting the azido-substrate could be tolerated by the enzymatic systems among these cells from diverse biological species. We isolated azido-GlcNAc modified proteins from the cytosolic extract of S2 cells and identified 10 previously reported and 41 putative O-GlcNAc modified proteins, by nano-HPLC-MS/MS. Our study demonstrates that the TAS approach is a useful tool for the detection and proteomic analysis of O-GlcNAc modified proteins.