Landscape of submitochondrial protein distribution.

The mitochondrial proteome comprises ~1000 (yeast)-1500 (human) different proteins, which are distributed into four different subcompartments. The sublocalization of these proteins within the organelle in most cases remains poorly defined. Here we describe an integrated approach combining stable isotope labeling, various protein enrichment and extraction strategies and quantitative mass spectrometry to produce a quantitative map of submitochondrial protein distribution in S. cerevisiae. This quantitative landscape enables a proteome-wide classification of 986 proteins into soluble, peripheral, and integral mitochondrial membrane proteins, and the assignment of 818 proteins into the four subcompartments: outer membrane, inner membrane, intermembrane space, or matrix. We also identified 206 proteins that were not previously annotated as localized to mitochondria. Furthermore, the protease Prd1, misannotated as intermembrane space protein, could be re-assigned and characterized as a presequence peptide degrading enzyme in the matrix.Protein localization plays an important role in the regulation of cellular physiology. Here the authors use an integrated proteomics approach to localize proteins to the mitochondria and provide a detailed map of their specific localization within the organelle.

Temporal quantitative phosphoproteomics of ADP stimulation reveals novel central nodes in platelet activation and inhibition.

Adenosine diphosphate (ADP) enhances platelet activation by virtually any other stimulant to complete aggregation. It binds specifically to the G-protein-coupled membrane receptors P2Y1 and P2Y12, stimulating intracellular signaling cascades, leading to integrin αIIbß3 activation, a process antagonized by endothelial prostacyclin. P2Y12 inhibitors are among the most successful antiplatelet drugs, however, show remarkable variability in efficacy. We reasoned whether a more detailed molecular understanding of ADP-induced protein phosphorylation could identify (1) critical hubs in platelet signaling toward aggregation and (2) novel molecular targets for antiplatelet treatment strategies. We applied quantitative temporal phosphoproteomics to study ADP-mediated signaling at unprecedented molecular resolution. Furthermore, to mimic the antagonistic efficacy of endothelial-derived prostacyclin, we determined how Iloprost reverses ADP-mediated signaling events. We provide temporal profiles of 4797 phosphopeptides, 608 of which showed significant regulation. Regulated proteins are implicated in well-known activating functions such as degranulation and cytoskeletal reorganization, but also in less well-understood pathways, involving ubiquitin ligases and GTPase exchange factors/GTPase-activating proteins (GEF/GAP). Our data demonstrate that ADP-triggered phosphorylation occurs predominantly within the first 10 seconds, with many short rather than sustained changes. For a set of phosphorylation sites (eg, PDE3ASer312, CALDAG-GEFISer587, ENSASer109), we demonstrate an inverse regulation by ADP and Iloprost, suggesting that these are central modulators of platelet homeostasis. This study demonstrates an extensive spectrum of human platelet protein phosphorylation in response to ADP and Iloprost, which inversely overlap and represent major activating and inhibitory pathways.

Combined Quantification of the Global Proteome, Phosphoproteome, and Proteolytic Cleavage to Characterize Altered Platelet Functions in the Human Scott Syndrome.

The Scott syndrome is a very rare and likely underdiagnosed bleeding disorder associated with mutations in the gene encoding anoctamin-6. Platelets from Scott patients are impaired in various Ca2+-dependent responses, including phosphatidylserine exposure, integrin closure, intracellular protein cleavage, and cytoskeleton-dependent morphological changes. Given the central role of anoctamin-6 in the platelet procoagulant response, we used quantitative proteomics to understand the underlying molecular mechanisms and the complex phenotypic changes in Scott platelets compared with control platelets. Therefore, we applied an iTRAQ-based multi-pronged strategy to quantify changes in (1) the global proteome, (2) the phosphoproteome, and (3) proteolytic events between resting and stimulated Scott and control platelets. Our data indicate a limited number of proteins with decreased (70) or increased (64) expression in Scott platelets, among those we confirmed the absence of anoctamin-6 and the strong up-regulation of aquaporin-1 by parallel reaction monitoring. The quantification of 1566 phosphopeptides revealed major differences between Scott and control platelets after stimulation with thrombin/convulxin or ionomycin. In Scott platelets, phosphorylation levels of proteins regulating cytoskeletal or signaling events were increased. Finally, we quantified 1596 N-terminal peptides in activated Scott and control platelets, 180 of which we identified as calpain-regulated, whereas a distinct set of 23 neo-N termini was caspase-regulated. In Scott platelets, calpain-induced cleavage of cytoskeleton-linked and signaling proteins was downregulated, in accordance with an increased phosphorylation state. Thus, multipronged proteomic profiling of Scott platelets provides detailed insight into their protection against detrimental Ca2+-dependent changes that are normally associated with phosphatidylserine exposure.

Quantitative Profiling for Substrates of the Mitochondrial Presequence Processing Protease Reveals a Set of Nonsubstrate Proteins Increased upon Proteotoxic Stress.

The majority of mitochondrial preproteins are targeted via N-terminal presequences that are cleaved upon import into the organelle. The essential mitochondrial processing protease (MPP) is assumed to cleave the majority of incoming precursors; however, only a small fraction of mitochondrial precursors have been experimentally analyzed limiting the information on MPP recognition and substrate specificity. Here we present the first systematic approach for identification of authentic MPP substrate proteins using a temperature-sensitive mutant of the MPP subunit Mas1. Inactivation of MPP at nonpermissive temperature leads to accumulation of immature precursors in mitochondria, which were measured by quantitative N-terminal ChaFRADIC. This led to the identification of 66 novel MPP substrates. Deduction of the cleaved presequences determines arginine in position -2 of the cleavage site as a main factor for MPP recognition. Interestingly, a set of nonprocessed proteins was also increased in mas1 mutant mitochondria. Additionally, mas1 mitochondria respond to temperature elevation with an increase in membrane potential and oxygen consumption. These changes might indicate that mas1 cells exert a response to balance the proteotoxic stress induced by MPP dysfunction.

Amyloid-ß peptide induces mitochondrial dysfunction by inhibition of preprotein maturation.

Most mitochondrial proteins possess N-terminal presequences that are required for targeting and import into the organelle. Upon import, presequences are cleaved off by matrix processing peptidases and subsequently degraded by the peptidasome Cym1/PreP, which also degrades Amyloid-beta peptides (Aß). Here we find that impaired turnover of presequence peptides results in feedback inhibition of presequence processing enzymes. Moreover, Aß inhibits degradation of presequence peptides by PreP, resulting in accumulation of mitochondrial preproteins and processing intermediates. Dysfunctional preprotein maturation leads to rapid protein degradation and an imbalanced organellar proteome. Our findings reveal a general mechanism by which Aß peptide can induce the multiple diverse mitochondrial dysfunctions accompanying Alzheimer's disease.

What can proteomics tell us about platelets?

More than 130 years ago, it was recognized that platelets are key mediators of hemostasis. Nowadays, it is established that platelets participate in additional physiological processes and contribute to the genesis and progression of cardiovascular diseases. Recent data indicate that the platelet proteome, defined as the complete set of expressed proteins, comprises >5000 proteins and is highly similar between different healthy individuals. Owing to their anucleate nature, platelets have limited protein synthesis. By implication, in patients experiencing platelet disorders, platelet (dys)function is almost completely attributable to alterations in protein expression and dynamic differences in post-translational modifications. Modern platelet proteomics approaches can reveal (1) quantitative changes in the abundance of thousands of proteins, (2) post-translational modifications, (3) protein-protein interactions, and (4) protein localization, while requiring only small blood donations in the range of a few milliliters. Consequently, platelet proteomics will represent an invaluable tool for characterizing the fundamental processes that affect platelet homeostasis and thus determine the roles of platelets in health and disease. In this article we provide a critical overview on the achievements, the current possibilities, and the future perspectives of platelet proteomics to study patients experiencing cardiovascular, inflammatory, and bleeding disorders.

Method and platform standardization in MRM-based quantitative plasma proteomics.

There exists a growing demand in the proteomics community to standardize experimental methods and liquid chromatography-mass spectrometry (LC/MS) platforms in order to enable the acquisition of more precise and accurate quantitative data. This necessity is heightened by the evolving trend of verifying and validating candidate disease biomarkers in complex biofluids, such as blood plasma, through targeted multiple reaction monitoring (MRM)-based approaches with stable isotope-labeled standards (SIS). Considering the lack of performance standards for quantitative plasma proteomics, we previously developed two reference kits to evaluate the MRM with SIS peptide approach using undepleted and non-enriched human plasma. The first kit tests the effectiveness of the LC/MRM-MS platform (kit #1), while the second evaluates the performance of an entire analytical workflow (kit #2). Here, these kits have been refined for practical use and then evaluated through intra- and inter-laboratory testing on 6 common LC/MS platforms. For an identical panel of 22 plasma proteins, similar concentrations were determined, regardless of the kit, instrument platform, and laboratory of analysis. These results demonstrate the value of the kit and reinforce the utility of standardized methods and protocols. BIOLOGICAL SIGNIFICANCE: The proteomics community needs standardized experimental protocols and quality control methods in order to improve the reproducibility of MS-based quantitative data. This need is heightened by the evolving trend for MRM-based validation of proposed disease biomarkers in complex biofluids such as blood plasma. We have developed two kits to assist in the inter- and intra-laboratory quality control of MRM experiments: the first kit tests the effectiveness of the LC/MRM-MS platform (kit #1), while the second evaluates the performance of an entire analytical workflow (kit #2). In this paper, we report the use of these kits in intra- and inter-laboratory testing on 6 common LC/MS platforms. This article is part of a Special Issue entitled: Standardization and Quality Control in Proteomics.

Cytomegalovirus downregulates IRE1 to repress the unfolded protein response.

During viral infection, a massive demand for viral glycoproteins can overwhelm the capacity of the protein folding and quality control machinery, leading to an accumulation of unfolded proteins in the endoplasmic reticulum (ER). To restore ER homeostasis, cells initiate the unfolded protein response (UPR) by activating three ER-to-nucleus signaling pathways, of which the inositol-requiring enzyme 1 (IRE1)-dependent pathway is the most conserved. To reduce ER stress, the UPR decreases protein synthesis, increases degradation of unfolded proteins, and upregulates chaperone expression to enhance protein folding. Cytomegaloviruses, as other viral pathogens, modulate the UPR to their own advantage. However, the molecular mechanisms and the viral proteins responsible for UPR modulation remained to be identified. In this study, we investigated the modulation of IRE1 signaling by murine cytomegalovirus (MCMV) and found that IRE1-mediated mRNA splicing and expression of the X-box binding protein 1 (XBP1) is repressed in infected cells. By affinity purification, we identified the viral M50 protein as an IRE1-interacting protein. M50 expression in transfected or MCMV-infected cells induced a substantial downregulation of IRE1 protein levels. The N-terminal conserved region of M50 was found to be required for interaction with and downregulation of IRE1. Moreover, UL50, the human cytomegalovirus (HCMV) homolog of M50, affected IRE1 in the same way. Thus we concluded that IRE1 downregulation represents a previously undescribed viral strategy to curb the UPR.

Intermembrane space proteome of yeast mitochondria.

The intermembrane space (IMS) represents the smallest subcompartment of mitochondria. Nevertheless, it plays important roles in the transport and modification of proteins, lipids, and metal ions and in the regulation and assembly of the respiratory chain complexes. Moreover, it is involved in many redox processes and coordinates key steps in programmed cell death. A comprehensive profiling of IMS proteins has not been performed so far. We have established a method that uses the proapoptotic protein Bax to release IMS proteins from isolated mitochondria, and we profiled the protein composition of this compartment. Using stable isotope-labeled mitochondria from Saccharomyces cerevisiae, we were able to measure specific Bax-dependent protein release and distinguish between quantitatively released IMS proteins and the background efflux of matrix proteins. From the known 31 soluble IMS proteins, 29 proteins were reproducibly identified, corresponding to a coverage of >90%. In addition, we found 20 novel intermembrane space proteins, out of which 10 had not been localized to mitochondria before. Many of these novel IMS proteins have unknown functions or have been reported to play a role in redox regulation. We confirmed IMS localization for 15 proteins using in organello import, protease accessibility upon osmotic swelling, and Bax-release assays. Moreover, we identified two novel mitochondrial proteins, Ymr244c-a (Coa6) and Ybl107c (Mic23), as substrates of the MIA import pathway that have unusual cysteine motifs and found the protein phosphatase Ptc5 to be a novel substrate of the inner membrane protease (IMP). For Coa6 we discovered a role as a novel assembly factor of the cytochrome c oxidase complex. We present here the first and comprehensive proteome of IMS proteins of yeast mitochondria with 51 proteins in total. The IMS proteome will serve as a valuable source for further studies on the role of the IMS in cell life and death.

Integral quantification accuracy estimation for reporter ion-based quantitative proteomics (iQuARI).

With the increasing popularity of comparative studies of complex proteomes, reporter ion-based quantification methods such as iTRAQ and TMT have become commonplace in biological studies. Their appeal derives from simple multiplexing and quantification of several samples at reasonable cost. This advantage yet comes with a known shortcoming: precursors of different species can interfere, thus reducing the quantification accuracy. Recently, two methods were brought to the community alleviating the amount of interference via novel experimental design. Before considering setting up a new workflow, tuning the system, optimizing identification and quantification rates, etc. one legitimately asks: is it really worth the effort, time and money? The question is actually not easy to answer since the interference is heavily sample and system dependent. Moreover, there was to date no method allowing the inline estimation of error rates for reporter quantification. We therefore introduce a method called iQuARI to compute false discovery rates for reporter ion based quantification experiments as easily as Target/Decoy FDR for identification. With it, the scientist can accurately estimate the amount of interference in his sample on his system and eventually consider removing shadows subsequently, a task for which reporter ion quantification might not be the solution of choice.

The first comprehensive and quantitative analysis of human platelet protein composition allows the comparative analysis of structural and functional pathways.

Antiplatelet treatment is of fundamental importance in combatting functions/dysfunction of platelets in the pathogenesis of cardiovascular and inflammatory diseases. Dysfunction of anucleate platelets is likely to be completely attributable to alterations in posttranslational modifications and protein expression. We therefore examined the proteome of platelets highly purified from fresh blood donations, using elaborate protocols to ensure negligible contamination by leukocytes, erythrocytes, and plasma. Using quantitative mass spectrometry, we created the first comprehensive and quantitative human platelet proteome, comprising almost 4000 unique proteins, estimated copy numbers for ∼ 3700 of those, and assessed intersubject (4 donors) as well as intrasubject (3 different blood samples from 1 donor) variations of the proteome. For the first time, our data allow for a systematic and weighted appraisal of protein networks and pathways in human platelets, and indicate the feasibility of differential and comprehensive proteome analyses from small blood donations. Because 85% of the platelet proteome shows no variation between healthy donors, this study represents the starting point for disease-oriented platelet proteomics. In the near future, comprehensive and quantitative comparisons between normal and well-defined dysfunctional platelets, or between platelets obtained from donors at various stages of chronic cardiovascular and inflammatory diseases will be feasible.

Robust workflow for iTRAQ-based peptide and protein quantification.

Quantitative proteomics has become a routinely used technique to globally compare protein content and expression profiles of biological samples, for instance after differential stimulation. In this context, chemical stable isotope-based labeling techniques, such as ICAT and iTRAQ, have been successfully applied in a large variety of studies. Since iTRAQ labels are isobaric, quantitation is conducted on the MS/MS level. Consequently, up to eight samples can be multiplexed and quantified in a single experiment without increasing sample complexity. Here, we present a robust workflow to conduct iTRAQ quantification of biological samples such as human platelets, which comprises (a) an adequate sample preparation procedure, (b) an optimized tryptic digestion protocol, (c) SPE desalting and subsequent peptide labeling using a 4-plex iTRAQ labeling kit, and (d) fractionation of the obtained peptide mixture by strong cation exchange chromatography.

iTRAQ data interpretation.

Quantitative proteomic analysis can help elucidating unexplored biological questions; it, however, relies on highly reproducible experiments and reliable data processing. Among the existing strategies, iTRAQ is known as an easy to use method allowing relative comparison of up to eight multiplexed samples.Once the data is acquired it is important that the final protein quantification reflects the actual amounts in the samples. Data interpretation must thus be achieved with a constant focus on quality. Here, we describe a workflow for processing iTRAQ data in user-friendly environments with emphasis on quality control.

A complex standard for protein identification, designed by evolution.

Shotgun proteomic investigations rely on the algorithmic assignment of mass spectra to peptides. The quality of these matches is therefore a cornerstone in the analysis and has been the subject of numerous recent developments. In order to establish the benefits of novel algorithms, they are applied to reference samples of known content. However, these were recently shown to be either too simple to resemble typical real-life samples or as leading to results of lower accuracy as the method itself. Here, we describe how to use the proteome of Pyrococcus furiosus , a hyperthermophile, as a standard to evaluate proteomics identification workflows. Indeed, we prove that the Pyrococcus furiosus proteome provides a valid method for detecting random hits, comparable to the decoy databases currently in popular use, but we also prove that the Pyrococcus furiosus proteome goes squarely beyond the decoy approach by also providing many hundreds of highly reliable true positive hits. Searching the Pyrococcus furiosus proteome can thus be used as a unique test that provides the ability to reliably detect both false positives as well as proteome-scale true positives, allowing the rigorous testing of identification algorithms at the peptide and protein level.

Systematic and quantitative comparison of digest efficiency and specificity reveals the impact of trypsin quality on MS-based proteomics.

Trypsin is the most frequently used proteolytic enzyme in mass spectrometry-based proteomics. Beside its good availability, it also offers some major advantages such as an optimal average peptide length of ~14 amino acids, and typically the presence of at least two defined positive charges at the N-terminus as well as the C-terminal Arg/Lys, rendering tryptic peptides well suited for CID-based LC-MS/MS. Here, we conducted a systematic study of different types of commercially available trypsin in order to qualitatively and quantitatively compare cleavage specificity, efficiency as well as reproducibility and the potential impact on quantitation and proteome coverage. We present a straightforward strategy applied to complex digests of human platelets, comprising (1) digest controls using a monolithic column HPLC-setup, (2) SCX enrichment of semitryptic/nonspecific peptides, (3) targeted MRM analysis of corresponding full cleavage/missed cleavage peptide pairs as well as (4) LC-MS analyses of complete digests with a three-step data interpretation. Thus, differences in digest performance can be readily assessed, rendering these procedures extremely beneficial to quality control not only the trypsin of choice, but also to effectively compare as well as optimize different digestion conditions and to evaluate the reproducibility of a dedicated digest protocol for all kinds of quantitative proteome studies.

Peptide identification quality control.

Identification of large proteomics data sets is routinely performed using sophisticated software tools called search engines. Yet despite the importance of the identification process, its configuration and execution is often performed according to established lab habits, and is mostly unsupervised by detailed quality control. In order to establish easily obtainable quality control criteria that can be broadly applied to the identification process, we here introduce several simple quality control methods. An unbiased quality control of identification parameters will be conducted using target/decoy searches providing significant improvement over identification standards. MASCOT identifications were for instance increased by 13% at a constant level of confidence. The target/decoy approach can however not be universally applied. We therefore also quality control the application of this strategy itself, providing useful and intuitive metrics for evaluating the precision and robustness of the obtained false discovery rate.

Quality control of nano-LC-MS systems using stable isotope-coded peptides.

In analytical sciences, there is a general need for quality control to assess whether a product or a process meets defined requirements. Especially in proteomics, which implies analysis of ten thousands of analytes within a complex mixture, quality control to validate LC-MS performance and method setup is inevitable to achieve day-to-day-, inter-system-, as well as inter-user reproducibility. Thus, results deriving from LC-MS analyses can be benchmarked and the need for system maintenance can be revealed. In particular with the advent of label-free quantification of peptides and proteins, which above all depends on highly stable and reproducible LC separations, HPLC performance has to be appropriately monitored throughout the entire analytical procedure to assure quality and validity of the obtained data. Oftentimes, proteolytic digests of standard proteins are used in this context; however, this approach implies some limitations, such as inadequate batch-to-batch reproducibility, limited (if any) dynamic range and compositional inflexibility. Here, we present an alternative strategy of nano-LC-MS/MS quality control based on a mixture of synthetic peptides covering the entire LC-gradient as well as a dynamic range of more than two orders of magnitude. Thus, (i) reproducibility of LC separation, (ii) MS performance (including limit of detection, identification and quantification), as well as (iii) overall nano-LC-MS system performance and reproducibility can be routinely monitored even in highly complex samples.