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