Methods of quantitative proteomics and their application to plant organelle characterization

Many cell biologists wish to know the subcellular localization of proteins of interest. Proteomics methods have the potential to describe the entire protein content of organelles. However, practical limitations in organelle isolation and analysis of low abundance proteins have meant that organelle proteomics has had, until recently, only limited success. Some examples of quantitative proteomic methods and their use in the study of plant organelle proteomes are discussed here. It is concluded that 2D-difference gel electrophoresis (2D-DIGE) as well as differential isotope tagging strategies coupled to non-gel-based LC-MS are proving useful in this area of research.     

Sub-cellular localization of membrane proteins

In eukaryotes, numerous complex sub-cellular structures exist. The majority of these are delineated by membranes. Many proteins are trafficked to these in order to be able to carry out their correct physiological function. Assigning the sub-cellular location of a protein is of paramount importance to biologists in the elucidation of its role and in the refinement of knowledge of cellular processes by tracing certain activities to specific organelles. Membrane proteins are a key set of proteins as these form part of the boundary of the organelles and represent many important functions such as transporters, receptors, and trafficking. They are, however, some of the most challenging proteins to work with due to poor solubility, a wide concentration range within the cell and inaccessibility to many of the tools employed in proteomics studies. This review focuses on membrane proteins with particular emphasis on sub-cellular localization in terms of methodologies that can be used to determine the accurate location of membrane proteins to organelles. We also discuss what is known about the membrane protein cohorts of major organelles.     

Quantitative proteomic approach to study subcellular localization of membrane proteins

As proteins within cells are spatially organized according to their role, knowledge about protein localization gives insight into protein function. Here, we describe the LOPIT technique (localization of organelle proteins by isotope tagging) developed for the simultaneous and confident determination of the steady-state distribution of hundreds of integral membrane proteins within organelles. The technique uses a partial membrane fractionation strategy in conjunction with quantitative proteomics. Localization of proteins is achieved by measuring their distribution pattern across the density gradient using amine-reactive isotope tagging and comparing these patterns with those of known organelle residents. LOPIT relies on the assumption that proteins belonging to the same organelle will co-fractionate. Multivariate statistical tools are then used to group proteins according to the similarities in their distributions, and hence localization without complete centrifugal separation is achieved. The protocol requires approximately 3 weeks to complete and can be applied in a high-throughput manner to material from many varied sources.     

Identification of endosomal epidermal growth factor receptor signaling targets by functional organelle proteomics

Epidermal growth factor (EGF) receptor (EGFR) signal transduction is organized by scaffold and adaptor proteins, which have specific subcellular distribution. On a way from the plasma membrane to the lysosome EGFRs are still in their active state and can signal from distinct subcellular locations. To identify organelle-specific targets of EGF receptor signaling on endosomes a combination of subcellular fractionation, two-dimensional DIGE, fluorescence labeling of phosphoproteins, and MALDI-TOF/TOF mass spectrometry was applied. All together 23 EGF-regulated (phospho)proteins were identified as being differentially associated with endosomal fractions by functional organelle proteomics; among them were proteins known to be involved in endosomal trafficking and cytoskeleton rearrangement (Alix, myosin-9, myosin regulatory light chain, Trap1, moesin, cytokeratin 8, septins 2 and 11, and CapZbeta). Interestingly R-Ras, a small GTPase of the Ras family that regulates cell survival and integrin activity, was associated with endosomes in a ligand-dependent manner. EGF-dependent association of R-Ras with late endosomes was confirmed by confocal laser scanning immunofluorescence microscopy and Western blotting of endosomal fractions. EGFR tyrosine kinase inhibitor gefitinib was used to confirm EGF-dependent regulation of all identified proteins. EGF-dependent association of signaling molecules, such as R-Ras, with late endosomes suggests signaling specification through intracellular organelles.     

Proteomics of industrial fungi: trends and insights for biotechnology

Filamentous fungi are widely known for their industrial applications, namely, the production of food-processing enzymes and metabolites such as antibiotics and organic acids. In the past decade, the full genome sequencing of filamentous fungi increased the potential to predict encoded proteins enormously, namely, hydrolytic enzymes or proteins involved in the biosynthesis of metabolites of interest. The integration of genome sequence information with possible phenotypes requires, however, the knowledge of all the proteins in the cell in a system-wise manner, given by proteomics. This review summarises the progress of proteomics and its importance for the study of biotechnological processes in filamentous fungi. A major step forward in proteomics was to couple protein separation with high-resolution mass spectrometry, allowing accurate protein quantification. Despite the fact that most fungal proteomic studies have been focused on proteins from mycelial extracts, many proteins are related to processes which are compartmentalised in the fungal cell, e.g. β-lactam antibiotic production in the microbody. For the study of such processes, a targeted approach is required, e.g. by organelle proteomics. Typical workflows for sample preparation in fungal organelle proteomics are discussed, including homogenisation and sub-cellular fractionation. Finally, examples are presented of fungal organelle proteomic studies, which have enlarged the knowledge on areas of interest to biotechnology, such as protein secretion, energy production or antibiotic biosynthesis.     

Localization of organelle proteins by isotope tagging (LOPIT)

We describe a proteomics method for determining the subcellular localization of membrane proteins. Organelles are partially separated using centrifugation through self-generating density gradients. Proteins from each organelle co-fractionate and therefore exhibit similar distributions in the gradient. Protein distributions can be determined through a series of pair-wise comparisons of gradient fractions, using cleavable ICAT to enable relative quantitation of protein levels by MS. The localization of novel proteins is determined using multivariate data analysis techniques to match their distributions to those of proteins that are known to reside in specific organelles. Using this approach, we have simultaneously demonstrated the localization of membrane proteins in both the endoplasmic reticulum and the Golgi apparatus in Arabidopsis. Localization of organelle proteins by isotope tagging is a new tool for high-throughput protein localization, which is applicable to a wide range of research areas such as the study of organelle function and protein trafficking.     

The Organelle Proteome of the DT40 Lymphocyte Cell Line

A major challenge in eukaryotic cell biology is to understand the roles of individual proteins and the subcellular compartments in which they reside. Here, we use the localization of organelle proteins by isotope tagging technique to complete the first proteomic analysis of the major organelles of the DT40 lymphocyte cell line. This cell line is emerging as an important research tool because of the ease with which gene knockouts can be generated. We identify 1090 proteins through the analysis of preparations enriched for integral membrane or soluble and peripherally associated proteins and localize 223 proteins to the endoplasmic reticulum, Golgi, lysosome, mitochondrion, or plasma membrane by matching their density gradient distributions to those of known organelle residents. A striking finding is that within the secretory and endocytic pathway a high proportion of proteins are not uniquely localized to a single organelle, emphasizing the dynamic steady-state nature of intracellular compartments in eukaryotic cells.The chicken pre-B cell line DT40 exhibits a remarkably high ratio of targeted to random integration for transfected DNA constructs. This property is unusual in vertebrate cell lines and enables targeted gene disruption experiments to be carried out with relative ease (1). Consequently, DT40 has become a major research tool for the molecular dissection of a wide range of cellular and biochemical mechanisms in a vertebrate context, including membrane traffic, signal transduction, and cell cycle (2).Proteins in eukaryotic cells are organized according to their functions within a dynamic network of membranes. Localization is therefore paramount in assigning functions to uncharacterized proteins and understanding the processes occurring in subcellular compartments. An increased knowledge of the protein localization within the DT40 cell line would be of great value. Traditional localization methods such as immunofluorescence microscopy are typically low throughput and are more suitably applied to the study of specific proteins of interest rather than the cataloguing of large numbers of proteins. Recent developments in proteomics have made it possible to analyze the protein composition of organelles using a variety of different approaches. Several groups have utilized label-free quantitative proteomics in the high throughput assignment of proteins to subcellular compartments. In one approach, protein correlation profiling, proteins from enriched organelle fractions are quantified by peptide ion intensity measurements (3, 4). Other similar methods employ quantitation by spectral counting, recording the number of ions detected per protein (5, 6). Localization of organelle proteins by isotope tagging (LOPIT)¹ is a complementary approach, which employs isotope labeling for quantitation (7–9). Rather than processing each sample separately as in label-free techniques, differentially labeled fractions are pooled early in the LOPIT protocol. This has the important advantage of reducing the points at which variation might be introduced into the data.LOPIT begins with the partial separation of organelles by density gradient centrifugation and relies on the assumption that proteins from each organelle co-fractionate. Protein profiles along the gradient are quantified by the use of isotopically coded tags in conjunction with two-dimensional liquid chromatography of peptides and tandem mass spectrometry. Multivariate statistical techniques are then used to assign localizations to proteins by comparing their gradient profiles to those of established organelle markers in an unbiased manner. The major strength of such an approach is that it enables residents of different subcellular compartments to be resolved even if their gradient distributions overlap, and genuine organelle constituents can be readily distinguished from contaminants.Here we use LOPIT to produce the first proteomic analysis of the major organelles of DT40. We have reproducibly identified 1090 proteins through the parallel analysis of preparations enriched for integral membrane or soluble and peripherally associated proteins. We use the distributions of 102 known organelle resident proteins as a basis to assign a further 223 proteins to five organelles: 79 to the endoplasmic reticulum (ER), 42 to the Golgi, 2 to the lysosome, 31 to the mitochondrion, and 69 to the plasma membrane (PM). We also demonstrate the resolution of components of the vesicular transport machinery. A striking finding is that a high proportion of identified proteins are not localized to a single organelle. This indicates that at steady state a substantial fraction of proteins are in transit between compartments, emphasizing the dynamic nature of intracellular organelles in eukaryotic cells. Our results represent the first application of LOPIT to a vertebrate system, provide the first organelle proteomic analysis of any lymphocyte cell line, and establish a major resource for the DT40 community.     

Molecular motors and developmental asymmetry

The generation of distinct cell fates can require movement of specific molecules or organelles to particular locations within the cell. These subcellular movements are often the jobs of motor proteins. Seemingly disparate developmental processes--determination of right and left in vertebrates, setting up the axes of polarity in insect embryos, mating-type switching in yeast, and coordinated organelle movements in Drosophila--converge in their dependence on motor proteins. The extent of possible regulatory complexity is only beginning to emerge.     

Protein import into the photosynthetic organelles of Paulinella chromatophora and its implications for primary plastid endosymbiosis

The rhizarian amoeba Paulinella chromatophora harbors two photosynthetically active organelles of cyanobacterial origin that have been acquired independently of classic primary plastids. Because their acquisition did take place relatively recently, they are expected to provide new insight into the ancient cyanobacterial primary endosymbiosis. During the process of Paulinella endosymbiont-to-organelle transformation, more than 30 genes have been transferred from the organelle to the host nuclear genome via endosymbiotic gene transfer (EGT). The article discusses step-by-step protein import of EGT-derived proteins into Paulinella photosynthetic organelles with the emphasis on the nature of their targeting signals and the final passage of proteins through the inner organelle membrane. The latter most probably involves a simplified Tic translocon composed of Tic21- and Tic32-like proteins as well as a Hsp70-based motor responsible for pulling of imported proteins into the organelle matrix. Our results indicate that although protein translocation across the inner membrane of Paulinella photosynthetic organelles seems to resemble the one in classic primary plastids, the transport through the outer membrane does not. The differences could result from distinct integration pathways of Paulinella photosynthetic organelles and primary plastids with their respective host cells.     

The proteome of lysosomes

Lysosomes are organelles of eukaryotic cells that are critically involved in the degradation of macromolecules mainly delivered by endocytosis and autophagocytosis. Degradation is achieved by more than 60 hydrolases sequestered by a single phospholipid bilayer. The lysosomal membrane facilitates interaction and fusion with other compartments and harbours transport proteins catalysing the export of catabolites, thereby allowing their recycling. Lysosomal proteins have been addressed in various proteomic studies that are compared in this review regarding the source of material, the organelle/protein purification scheme, the proteomic methodology applied and the proteins identified. Distinguishing true constituents of an organelle from co-purifying contaminants is a central issue in subcellular proteomics, with additional implications for lysosomes as being the site of degradation of many cellular and extracellular proteins. Although many of the lysosomal hydrolases were identified by classical biochemical approaches, the knowledge about the protein composition of the lysosomal membrane has remained fragmentary for a long time. Using proteomics many novel lysosomal candidate proteins have been discovered and it can be expected that their functional characterisation will help to understand functions of lysosomes at a molecular level that have been characterised only phenomenologically so far and to generally deepen our understanding of this indispensable organelle.     

Subcellular fractionation methods and strategies for proteomics

Developments in subcellular fractionation strategies have provided the means to profile and analyze the protein composition of organelles and cellular structures by proteomics. Here, we review the application of classical (e.g. density gradient centrifugation) and emerging sophisticated techniques (fluorescent-assisted organelle sorting) in the fractionation, and statistical/bioinformatics tools for the prediction of protein localization in subcellular proteomics. We also review the validation methods currently used (such as microscopy, RNA interference and multiple reaction monitoring) and discuss the importance of verification of the results obtained in subcellular proteomics. Finally, the numerous challenges facing subcellular proteomics including the dynamics of organelles are being examined. However, complementary approaches such as modern statistics, bioinformatics and large-scale integrative analysis are beginning to emerge as powerful tools to proteomics for analyzing subcellular organelles and structures.     

Phospholipid Synthesis and Transport in Mammalian Cells

Membranes of mammalian subcellular organelles contain defined amounts of specific phospholipids that are required for normal functioning of proteins in the membrane. Despite the wide distribution of most phospholipid classes throughout organelle membranes, the site of synthesis of each phospholipid class is usually restricted to one organelle, commonly the endoplasmic reticulum (ER). Thus, phospholipids must be transported from their sites of synthesis to the membranes of other organelles. In this article, pathways and subcellular sites of phospholipid synthesis in mammalian cells are summarized. A single, unifying mechanism does not explain the inter‐organelle transport of all phospholipids. Thus, mechanisms of phospholipid transport between organelles of mammalian cells via spontaneous membrane diffusion, via cytosolic phospholipid transfer proteins, via vesicles and via membrane contact sites are discussed. As an example of the latter mechanism, phosphatidylserine (PS) is synthesized on a region of the ER (mitochondria‐associated membranes, MAM) and decarboxylated to phosphatidylethanolamine in mitochondria. Some evidence is presented suggesting that PS import into mitochondria occurs via membrane contact sites between MAM and mitochondria. Recent studies suggest that protein complexes can form tethers that link two types of organelles thereby promoting lipid transfer. However, many questions remain about mechanisms of inter‐organelle phospholipid transport in mammalian cells.     

Building the mitochondrial proteome

Mitochondria are essential organelles for cellular homeostasis. A variety of pathologies including cancer, myopathies, diabetes, obesity, aging and neurodegenerative diseases are linked to mitochondrial dysfunction. Therefore, mapping the different components of mitochondria is of particular interest to gain further understanding of such diseases. In recent years, proteomics-based approaches have been developed in attempts to determine the complete set of mitochondrial proteins in yeast, plants and mammals. In addition, proteomics-based methods have been applied not only to the analysis of protein function in the organelle, but also to identify biomarkers for diagnosis and therapeutic targets of specific pathologies associated with mitochondria. Altogether, it is becoming clear that proteomics is a powerful tool not only to identify currently unknown components of the mitochondrion, but also to study the different roles of the organelle in cellular homeostasis.     

Guilt by association: the nuclear envelope proteome and disease

The discovery that many inherited diseases are linked to interacting nuclear envelope proteins has raised the possibility that human genetic studies could be assisted by a fusion with proteomics. Two principles could be applied. In the first, the proteome of an organelle associated with a genetically variable disease is determined. The chromosomal locations of the genes encoding the organellar proteins are then determined. If a related disease is linked to a large chromosomal region that includes a gene identified in the organelle, then that gene has an increased likelihood of causing the disease. Directly sequencing this allele from patient samples might speed identification compared with further genetic linkage studies as has been demonstrated for multiple diseases associated with the nuclear envelope. The second principle is that if an organelle has been implicated in the pathology of a particular disorder, then comparison of the organelle proteome from control and patient cells might highlight differences that could indicate the causative protein. The distinct, tissue-specific pathologies associated with nuclear envelope diseases suggest that many tissues will have a set of disorders linked to this organelle, and there are numerous as yet unmapped or partially mapped syndromes that could benefit from such an approach.     

Application of Proteomic Marker Ensembles to Subcellular Organelle Identification

Compartmentalization of biological processes and the associated cellular components is crucial for cell function. Typically, the location of a component is revealed through a co-localization and/or co-purification with an organelle marker. Therefore, the identification of reliable markers is critical for a thorough understanding of cellular function and dysfunction. We fractionated macrophage-like RAW264.7 cells, both in the resting and endotoxin-activated states, into six fractions representing the major organelles/compartments: nuclei, mitochondria, cytoplasm, endoplasmic reticulum, and plasma membrane as well as an additional dense microsomal fraction. The identity of the first five of these fractions was confirmed via the distribution of conventional enzymatic markers. Through a quantitative liquid chromatography/mass spectrometry-based proteomics analysis of the fractions, we identified 50-member ensembles of marker proteins (“marker ensembles”) specific for each of the corresponding organelles/compartments. Our analysis attributed 206 of the 250 marker proteins (∼82%) to organelles that are consistent with the location annotations in the public domain (obtained using DAVID 2008, EntrezGene, Swiss-Prot, and references therein). Moreover, we were able to correct locations for a subset of the remaining proteins, thus proving the superior power of analysis using multiple organelles as compared with an analysis using one specific organelle. The marker ensembles were used to calculate the organelle composition of the six above mentioned subcellular fractions. Knowledge of the precise composition of these fractions can be used to calculate the levels of metabolites in the pure organelles. As a proof of principle, we applied these calculations to known mitochondria-specific lipids (cardiolipins and ubiquinones) and demonstrated their exclusive mitochondrial location. We speculate that the organelle-specific protein ensembles may be used to systematically redefine originally morphologically defined organelles as biochemical entities.One of the basic concepts of cell biology is compartmentalization of the cellular processes within subcellular structures, termed organelles. Organelles were originally identified in the 19th century as the morphological entities that are still reflected in their names (e.g. “nucleus” from the Latin “little nut,” “mitochondria” from the Greek “thread” + “grain,” or “reticulum” from the Latin “little net”). Later, the progress of biochemistry made it possible to assign to the various organelles their specific biological functions. Thus, detailed information about the location of biochemical reactions became crucial for the understanding of their roles in cell function or dysfunction. Current technology allows the location of a cell component (a protein or a metabolite) to be linked directly to a morphologically defined organelle (or even a suborganellar compartment) by using electron microscopy. However, more typically, the location of a component is determined on the basis of its co-localization with a known marker for the organelle or subcellular compartment. This co-localization can be either visualized microscopically (imaging approach) to preserve some degree of morphological information or determined through co-purification of the component and the marker in a subcellular fractionation (biochemical approach).For both the imaging and the biochemical approaches, optimal organelle markers are of the utmost importance. Conventional markers include proteins, DNA (for nucleus), and even physical/chemical parameters (electric potential for mitochondria and acidic pH for lysosomes). Protein markers are assayed using either an interaction with specific antibodies or their enzymatic activities. Unfortunately, the former is typically non-quantitative, whereas the latter, although semiquantitative, is subject to interference from multiple parameters of the environment as well as substrate and product sharing with non-marker proteins. For a biochemical approach, tightness of the anchoring of a marker to the corresponding organelle is also an issue. Moreover, an inherent problem is that most proteins are located in several organelles/compartments, which may result in false localization conclusions.Our goal was to identify specific, reliable, and universal protein markers for major subcellular organelles/compartments. The following principles were chosen as the basis for our approach. First, the search had to be conducted without a preconceived notion of the nature of the markers (e.g. we did not expect to necessarily confirm conventional markers as optimal). Second, the search had to be conducted in all major organelles/compartments simultaneously. Third, the aim was to identify relatively large panels (ensembles) of markers as opposed to the best single marker. The last two principles allowed us to address the problem of multiple locations of potential marker proteins. Some of them can be eliminated as markers; for others, the impact of multiple locations on further analysis can be negated by averaging of the data for large numbers of proteins (derivation of marker ensembles).To meet these goals, we performed a complete “quantitative” proteomics analysis of all major subcellular fractions in a single cell type. Numerous reports have focused on the proteomes of specific organelles or interrelated sets of organelles in various cell types (for reviews, see Refs. 1 and 2). However, a need for an integral systematic study in a single cell type has been evident for some time (2), and the present study is the first step aimed at addressing this need.The marker ensembles that we identified from the proteome data were used to quantify the composition of the subcellular fractions. It is becoming appreciated that a physical association of various organelles makes it next to impossible to completely separate the organelles and obtain pure fractions acceptable for detailed proteomics analysis (e.g. see Ref. 3). Therefore, correlative approaches such as protein correlation profiling (1, 3, 4) and localization of organelle proteins by isotope tagging (5, 6) have been suggested to address this problem. These approaches allowed the assignment of protein locations based on co-localization with known markers in a density gradient (1, 4–6) or in multiple fractions (7). We took this approach a step further and derived a quantitative composition of the fractions based on the distribution of the marker ensembles. Furthermore, this enabled us to calculate levels of various components (lipids and proteins) in pure organelles from experimental data obtained with less than pure fractions.The choice of a particular cell type for this study was somewhat arbitrary, and the resulting marker ensembles were optimal for the cell type for which they were generated; of course, they may have to be adjusted to be adapted for other cell types. We chose macrophage cells partly because this study was an integral part of a larger subcellular lipidomics/proteomics study under the auspices of the Lipid Metabolites and Pathways Strategy (LIPID MAPS Consortium). The macrophage plays a central role in inflammation and innate and adaptive immunity. The macrophage detects and attacks pathogens and orchestrates a host response by sending signals to other cells and tissues; in this process, the macrophage itself transits from a resting to an activated state. These two states differ vastly in function, morphology, and underlying protein expression profiles, and therefore, we aimed to identify marker ensembles that would be invariant with regard to the activation process.In the present study, the activation paradigm was treatment with Kdo₂1-lipid A. This defined, nearly homogeneous reagent is a form of lipopolysaccharide endotoxin that has all the essential biological properties of lipopolysaccharide (8).     

Comparison of Golgi apparatus and endoplasmic reticulum proteins from livers of juvenile and aged rats using a novel technique for separation and enrichment of organelles.

The broad dynamic range of protein abundances, which can vary from about 10(6) for cells to 10(10) for tissues in complex proteomes, continues to challenge proteomics research. Proteome analysis, in particular organelle proteomics, using current approaches, requires extensive fractionation, separation, and enrichment. Over the years, organelle separation was achieved through the use of differential and density-gradient ultracentrifugation. However, the traditional fixed-volume process is a time-consuming and labor-intensive method, especially with large quantities of sample. Here, we present a novel tool for subcellular fractionation of biologically complex mixtures: continuous-flow ultracentrifugation of tissue homogenates to obtain both organelle separation and extensive organelle enrichment at the same time. In this study, rat liver tissues from two different age groups (3-8 wk and greater than 1 y old) were homogenized by blending. After removing nuclei, the resulting homogenates were further fractionated at the subcellular level by the use of sucrose gradient continuous-flow ultracentrifugation. Each organelle's enriched fractions were identified by Western blot analysis. To study the possible effects of aging on the endoplasmic reticulum and Golgi apparatus, we compared the organelle protein profiles of the two groups of rat liver tissues using two-dimensional gel electrophoresis, digitized imaging of two-dimensional gel electrophoresis, and mass spectrometry. Significant differences in the protein profiles of both organelles were observed between the two groups of rat tissues. The technique described here for fractionation and enrichment of organelles demonstrated a useful tool for proteomics research, including identification of low-abundance proteins and post-translational modifications.     

Building the mitochondrial proteome

Mitochondria are essential organelles for cellular homeostasis. A variety of pathologies including cancer, myopathies, diabetes, obesity, aging and neurodegenerative diseases are linked to mitochondrial dysfunction. Therefore, mapping the different components of mitochondria is of particular interest to gain further understanding of such diseases. In recent years, proteomics-based approaches have been developed in attempts to determine the complete set of mitochondrial proteins in yeast, plants and mammals. In addition, proteomics-based methods have been applied not only to the analysis of protein function in the organelle, but also to identify biomarkers for diagnosis and therapeutic targets of specific pathologies associated with mitochondria. Altogether, it is becoming clear that proteomics is a powerful tool not only to identify currently unknown components of the mitochondrion, but also to study the different roles of the organelle in cellular homeostasis.     

Subcellular proteomics of Trypanosoma cruzi reservosomes

Reservosomes are the endpoint of the endocytic pathway in Trypanosoma cruzi epimastigotes. These organelles have the particular ability to concentrate proteins and lipids obtained from medium together with the main proteolytic enzymes originated from the secretory pathway, being at the same time a storage organelle and the main site of protein degradation. Subcellular proteomics have been extensively used for profiling organelles in different cell types. Here, we combine cell fractionation and LC‐MS/MS analysis to identify reservosome‐resident proteins. Starting from a purified reservosome fraction, we established a protocol to isolate reservosome membranes. Transmission electron microscopy was applied to confirm the purity of the fractions. To achieve a better coverage of identified proteins we analyzed the fractions separately and combined the results. LC‐MS/MS analysis identified in total 709 T. cruzi‐specific proteins; of these, 456 had predicted function and 253 were classified as hypothetical proteins. We could confirm the presence of most of the proteins validated by previous work and identify new proteins from different classes such as enzymes, proton pumps, transport proteins, and others. The definition of the reservosome protein profile is a good tool to assess their molecular signature, identify molecular markers, and understand their relationship with different organelles.