Stable isotope labeling by amino acids in cell culture (SILAC) has become a versatile tool for quantitative, mass spectrometry (MS)-based proteomics. Here, we completely label mice with a diet containing either the natural or the (13)C(6)-substituted version of lysine. Mice were labeled over four generations with the heavy diet, and development, growth, and behavior were not affected. MS analysis of incorporation levels allowed for the determination of incorporation rates of proteins from blood cells and organs. The F2 generation was completely labeled in all organs tested. SILAC analysis from various organs lacking expression of beta1 integrin, beta-Parvin, or the integrin tail-binding protein Kindlin-3 confirmed their absence and disclosed a structural defect of the red blood cell membrane skeleton in Kindlin-3-deficient erythrocytes. The SILAC-mouse approach is a versatile tool by which to quantitatively compare proteomes from knockout mice and thereby determine protein functions under complex in vivo conditions.

Mitochondria, through oxidative phosphorylation, are the primary source of energy production in all tissues under aerobic conditions. Although critical to life, energy production is not the only function of mitochondria, and the composition of this organelle is tailored to meet the specific needs of each cell type. As an organelle, the mitochondrion has been a popular subject for proteomic analysis, but quantitative proteomic methods have yet to be applied to tease apart subtle differences among mitochondria from different tissues or muscle types. Here we used mass spectrometry-based proteomics to analyze mitochondrial proteins extracted from rat skeletal muscle, heart, and liver tissues. Based on 689 proteins identified with high confidence, mitochondria from the different tissues are qualitatively quite similar. However, striking differences emerged from the quantitative comparison of protein abundance between the tissues. Furthermore we applied similar methods to analyze mitochondrial matrix and intermembrane space proteins extracted from the same mitochondrial source, providing evidence for the submitochondrial localization of a number of proteins in skeletal muscle and liver. Several proteins not previously thought to reside in mitochondria were identified, and their presence in this organelle was confirmed by protein correlation profiling. Hierarchical clustering of microarray expression data provided further evidence that some of the novel mitochondrial candidates identified in the proteomic survey might be associated with mitochondria. These data reveal several important distinctions between mitochondrial and submitochondrial proteomes from skeletal muscle, heart, and liver Different mammalian tissues have distinct energy needs, and mitochondria morphology can vary widely, although the structure is not exclusively linked to respiration (1). Morphology and structure of mitochondria in mammalian skeletal muscle, heart, and liver are very different (2). In skeletal muscle mitochondria are distributed between sarcomeres and tightly embedded in the microfilaments (F-actin) and microtubules. Indeed the intimate association of mitochondria with the myofilaments minimizes diffusion distance and facilitates conversion of chemical energy to mechanical work. Mammalian skeletal muscle is a highly specialized tissue composed of fibers with a diverse range of properties. The aerobic type I fibers contain many mitochondria and rely on oxidative phosphorylation for ATP. Type IIa fibers are very rich in small mitochondria and are characterized by a high capacity for oxidative ATP generation. These mitochondria can only oxidize glycolytic products unlike those in type I fibers that can also utilize fatty acids and ketones. Type IIb fibers rely upon anaerobic glycolysis, contain few mitochondria, and have high levels of glycogen (3). Given these functional differences it is reasonable to assume that mitochondrial and mitochondria-related proteins are present in different amounts depending on the specific energy requirement...

Mitochondria are functionally specialized in different tissues, and a detailed understanding of this specialization is important to elucidate mitochondrial involvement in normal physiology and disease. In adaptive thermogenesis, brown fat converts mitochondrial energy to heat, whereas tissue-specific functions of mitochondria in white fat are less characterized. Here we apply high-resolution quantitative mass spectrometry to directly and accurately compare the in vivo mouse mitochondrial proteomes of brown and white adipocytes. Their proteomes are substantially different qualitatively and quantitatively and are furthermore characterized by tissue-specific protein isoforms, which are modulated by cold exposure. At transcript and proteome levels, brown fat mitochondria are more similar to their counterparts in muscle. Conversely, white fat mitochondria not only selectively express proteins that support anabolic functions but also degrade xenobiotics, revealing a protective function of this tissue. In vivo comparison of organellar proteomes can thus directly address functional questions in metabolism.

High accuracy mass spectrometry has proven to be a powerful technology for the large scale identification of serine/threonine/tyrosine phosphorylation in the living cell. However, despite many described phosphoproteomes, there has been no comparative study of the extent of phosphorylation and its evolutionary conservation in all domains of life. Here we analyze the results of phosphoproteomics studies performed with the same technology in a diverse set of organisms. For the most ancient organisms, the prokaryotes, only a few hundred proteins have been found to be phosphorylated. Applying the same technology to eukaryotic species resulted in the detection of thousands of phosphorylation events. Evolutionary analysis shows that prokaryotic phosphoproteins are preferentially conserved in all living organisms, whereassite specific phosphorylation is not. Eukaryotic phosphosites are generally more conserved than their nonphosphorylated counterparts (with similar structural constraints) throughout the eukaryotic domain. Yeast and Caenorhabditis elegans are two exceptions, indicating that the majority of phosphorylation events evolved after the divergence of higher eukaryotes from yeast and reflecting the unusually large number of nematode-specific kinases. Mitochondria present an interesting intermediate link between the prokaryotic and eukaryotic domains. Applying the same technology to this organelle yielded 174 phosphorylation sites mapped to 74 proteins. Thus, the mitochondrial phosphoproteome is similarly sparse as the prokaryotic phosphoproteomes. As expected from the endosymbiotic theory, phosphorylated as well as nonphosphorylated mitochondrial proteins are significantly conserved in prokaryotes. However, mitochondrial phosphorylation sites are not conserved throughout prokaryotes, consistent with the notion that serine/threonine phosphorylation in prokaryotes occurred relatively recently in evolution. Thus, the phosphoproteome reflects major events in the evolution of life. Molecular & Cellular Proteomics 9:2642-2653, 2010.Reversible protein phosphorylation on serines, threonines, and tyrosines plays a crucial role in regulating processes in all living organisms ranging from prokaryotes to eukaryotes (1). Traditionally, phosphorylation has been detected in single, purified proteins using in vitro assays. Recent advances in mass spectrometry (MS)-based proteomics now allow the identification of in vivo phosphorylation sites with high accuracy (2-7). On-line databases such as PhosphoSite (8), Phospho.ELM (9), and PHOSIDA 1 (10) have collected and organized thousands of identified phosphosites. These databases as well as dedicated analysis environments such as NetworKIN (11,12) offer and use contextual information including structural features, potential kinases, and conservation. They constitute resources that should allow the derivation of general patterns for phosphorylation events. Specifically, the recent availability of data for archaeal, prokaryotic, and diverse eukaryotic phosphoproteomes in these databa...