A longitudinal proteomic assessment of peptide degradation and loss under acidic storage conditions.

Sample preparation prior to analysis by liquid chromatography electrospray ionization mass spectrometry (LC-ESI-MS) usually involves the storage of frozen peptide samples in an acidic environment for variable time periods. Questions arose in our laboratory regarding the stability of peptides in acid under medium- to long-term storage. Thus, a 10-month longitudinal study was designed to assess the effect on storage of tryptic peptides at -20 and -80°C under acidic conditions. Our conclusion and proposal from this evaluation is that the optimal storage conditions of peptide samples in acid for proteomic experiments is at -80°C and, ideally, as separate aliquots.

Interactome of two diverse RNA granules links mRNA localization to translational repression in neurons.

Transport of RNAs to dendrites occurs in neuronal RNA granules, which allows local synthesis of specific proteins at active synapses on demand, thereby contributing to learning and memory. To gain insight into the machinery controlling dendritic mRNA localization and translation, we established a stringent protocol to biochemically purify RNA granules from rat brain. Here, we identified a specific set of interactors for two RNA-binding proteins that are known components of neuronal RNA granules, Barentsz and Staufen2. First, neuronal RNA granules are much more heterogeneous than previously anticipated, sharing only a third of the identified proteins. Second, dendritically localized mRNAs, e.g., Arc and CaMKIIα, associate selectively with distinct RNA granules. Third, our work identifies a series of factors with known roles in RNA localization, translational control, and RNA quality control that are likely to keep localized transcripts in a translationally repressed state, often in distinct types of RNPs.

Affinity purification strategies for proteomic analysis of transcription factor complexes.

Affinity purification (AP) coupled to mass spectrometry (MS) has been successful in elucidating protein molecular networks of mammalian cells. These approaches have dramatically increased the knowledge of the interconnectivity present among proteins and highlighted biological functions within different protein complexes. Despite significant technical improvements reached in the past years, it is still challenging to identify the interaction networks and the subsequent associated functions of nuclear proteins such as transcription factors (TFs). A straightforward and robust methodology is therefore required to obtain unbiased and reproducible interaction data. Here we present a new approach for TF AP-MS, exemplified with the CCAAT/enhancer binding protein alpha (C/EBPalpha). Utilizing the advantages of a double tag and three different MS strategies, we conducted a total of six independent AP-MS strategies to analyze the protein-protein interactions of C/EBPalpha. The resultant data were combined to produce a cohesive C/EBPalpha interactome. Our study describes a new methodology that robustly identifies specific molecular complexes associated with transcription factors. Moreover, it emphasizes the existence of TFs as protein complexes essential for cellular biological functions and not as single, static entities.

Proteomic analysis of seminal plasma from infertile patients with oligoasthenoteratozoospermia due to oxidative stress and comparison with fertile volunteers.

OBJECTIVE: To compare the expression protein profile of seminal plasma from infertile men with oligoasthenoteratozoospermia (OAT) due to oxidative stress with that of healthy, fertile men to determine the proteins indicative of infertility. DESIGN: Experimental study. SETTING: University hospital and research institute. PATIENT(S): Semen samples from 11 healthy, fertile (according to the 1999 World Health Organization criteria) male volunteers and 11 infertile idiopathic oligoasthenoteratozoospermia (iOAT) patients. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Proteomic analysis performed by liquid chromatography mass spectrometry on a hybrid linear trap quadrupole Orbitrap Velos mass spectrometer, carbonylation assay to determine degree of oxidative stress, semiquantitative proteomic analysis, gene ontology, and pathway analysis. RESULT(S): A total of 2,489 proteins were identified from seminal plasma, which represents the highest number of unique proteins identified to date. Twenty-four proteins were determined as ≥ 1.5-fold up-regulated in the infertile iOAT males as compared with the fertile controls; and 27 proteins from iOAT patients only were identified as common across all analyses. Only five of the proteins were shared between these two groups. CONCLUSION(S): A panel of 46 proteins were identified in patients with iOAT that are potential candidates in understanding the etiology of OAT due to oxidative stress.

Interlaboratory reproducibility of large-scale human protein-complex analysis by standardized AP-MS.

The characterization of all protein complexes of human cells under defined physiological conditions using affinity purification-mass spectrometry (AP-MS) is a highly desirable step in the quest to understand the phenotypic effects of genomic information. However, such a challenging goal has not yet been achieved, as it requires reproducibility of the experimental workflow and high data consistency across different studies and laboratories. We systematically investigated the reproducibility of a standardized AP-MS workflow by performing a rigorous interlaboratory comparative analysis of the interactomes of 32 human kinases. We show that it is possible to achieve high interlaboratory reproducibility of this standardized workflow despite differences in mass spectrometry configurations and subtle sample preparation-related variations and that combination of independent data sets improves the approach sensitivity, resulting in even more-detailed networks. Our analysis demonstrates the feasibility of obtaining a high-quality map of the human protein interactome with a multilaboratory project.

Mass spectrometric characterization of recombinant rat 5-hydroxytryptamine receptor 1A (5-HT(1A) R) expressed in tsA201 human embryonic kidney cells.

The 5-hydroxytryptamine 1A receptor (serotonin 1A receptor; 5-HT(1A) R) is involved in a large series of brain functions, and roles in anxiety, depression, and cognition have been reported. So far, published information on mass spectrometrical characterization of 5-HT(1A) R is limited to the presence of two 5-HT(1A) R peptides in rat's whole brain as observed by in-solution digestion followed by LC-MS/MS. Knowledge about the protein sequence and PTMs, however, would have implications for generation of specific antibodies and designing studies on the 5-HT(1A) R at the protein level. A rat recombinant 5-HT(1A) R was extracted from the tsA201 cell line, run using several gel-based principles with subsequent in-gel digestion with several proteases, chymotrypsin, trypsin, AspN, proteinase K, and pepsin followed by nano-LC-ESI-MS/MS analysis on a high capacity ion trap and an LTQ Orbitrap Velos. Using two search engines, Mascot and Modiro™, the recombinant 5-HT(1A) R was identified showing 94.55% sequence coverage. A single phosphorylation at S301 was identified and verified by phosphatase treatment and a series of amino acid substitutions were detected. Characterization of 5-HT(1A) R, a key player of brain functions and neurotransmission, was shown and may enable generation of specific antibodies, design of future, and interpretation of previous studies in the rat at the protein level.

Functional dissection of the TBK1 molecular network.

TANK-binding kinase 1 (TBK1) and inducible IκB-kinase (IKK-i) are central regulators of type-I interferon induction. They are associated with three adaptor proteins called TANK, Sintbad (or TBKBP1) and NAP1 (or TBKBP2, AZI2) whose functional relationship to TBK1 and IKK-i is poorly understood. We performed a systematic affinity purification-mass spectrometry approach to derive a comprehensive TBK1/IKK-i molecular network. The most salient feature of the network is the mutual exclusive interaction of the adaptors with the kinases, suggesting distinct alternative complexes. Immunofluorescence data indicated that the individual adaptors reside in different subcellular locations. TANK, Sintbad and NAP1 competed for binding of TBK1. The binding site for all three adaptors was mapped to the C-terminal coiled-coil 2 region of TBK1. Point mutants that affect binding of individual adaptors were used to reconstitute TBK1/IKK-i-deficient cells and dissect the functional relevance of the individual kinase-adaptor edges within the network. Using a microarray-derived gene expression signature of TBK1 in response virus infection or poly(I∶C) stimulation, we found that TBK1 activation was strictly dependent on the integrity of the TBK1/TANK interaction.

Initial characterization of the human central proteome.

BACKGROUND: On the basis of large proteomics datasets measured from seven human cell lines we consider their intersection as an approximation of the human central proteome, which is the set of proteins ubiquitously expressed in all human cells. Composition and properties of the central proteome are investigated through bioinformatics analyses. RESULTS: We experimentally identify a central proteome comprising 1,124 proteins that are ubiquitously and abundantly expressed in human cells using state of the art mass spectrometry and protein identification bioinformatics. The main represented functions are proteostasis, primary metabolism and proliferation. We further characterize the central proteome considering gene structures, conservation, interaction networks, pathways, drug targets, and coordination of biological processes. Among other new findings, we show that the central proteome is encoded by exon-rich genes, indicating an increased regulatory flexibility through alternative splicing to adapt to multiple environments, and that the protein interaction network linking the central proteome is very efficient for synchronizing translation with other biological processes. Surprisingly, at least 10% of the central proteome has no or very limited functional annotation. CONCLUSIONS: Our data and analysis provide a new and deeper description of the human central proteome compared to previous results thereby extending and complementing our knowledge of commonly expressed human proteins. All the data are made publicly available to help other researchers who, for instance, need to compare or link focused datasets to a common background.

Proteomic analysis of human cataract aqueous humour: Comparison of one-dimensional gel LCMS with two-dimensional LCMS of unlabelled and iTRAQ®-labelled specimens.

In this study, we report a comparative and quantitative analysis by mass spectrometry of the protein content of aqueous humour from cataract (control) patients. In addition to protein profiling, the approach is layered with quantitative proteomics using the iTRAQ® methodology. Aqueous humour from ten clinically-matched patients was collected and depleted of albumin and immunoglobulin G. Pairs of patient material were pooled and divided into three aliquots for subsequent analysis by alternative proteomic approaches. Excluding keratin, trypsin, residual albumin and immunoglobulins, a total of 198 protein groups were identified across the entire study. Relative protein quantitation with iTRAQ® revealed that 88% of the proteins had a maximal ±2-fold differential regulation between 3 of the 4 labelled samples, indicating minimal variation. The identified proteins were categorised by gene ontology and one third of the proteins were annotated as extracellular. The major molecular functions of the proteins in aqueous humour are binding (protein, metal ion, heparin, and DNA) and inhibition of proteolytic activity. Complementary to molecular function, the predominant biological processes for the proteins in aqueous humour are assigned to inflammatory and immune responses, and transport.

CD14 is a coreceptor of Toll-like receptors 7 and 9.

Recognition of pathogens by the innate immune system requires proteins that detect conserved molecular patterns. Nucleic acids are recognized by cytoplasmic sensors as well as by endosomal Toll-like receptors (TLRs). It has become evident that TLRs require additional proteins to be activated by their respective ligands. In this study, we show that CD14 (cluster of differentiation 14) constitutively interacts with the MyD88-dependent TLR7 and TLR9. CD14 was necessary for TLR7- and TLR9-dependent induction of proinflammatory cytokines in vitro and for TLR9-dependent innate immune responses in mice. CD14 associated with TLR9 stimulatory DNA in precipitation experiments and confocal imaging. The absence of CD14 led to reduced nucleic acid uptake in macrophages. Additionally, CD14 played a role in the stimulation of TLRs by viruses. Using various types of vesicular stomatitis virus, we showed that CD14 is dispensable for viral uptake but is required for the triggering of TLR-dependent cytokine responses. These data show that CD14 has a dual role in nucleic acid-mediated TLR activation: it promotes the selective uptake of nucleic acids, and it acts as a coreceptor for endosomal TLR activation.

Acid elution and one-dimensional shotgun analysis on an Orbitrap mass spectrometer: an application to drug affinity chromatography.

In this study, the target profile of the promiscuous kinase inhibitor bosutinib from whole cell K562 lysates was investigated by an improved chemical proteomic approach to identify natural binders. By (i) miniaturizing the drug pulldown method, (ii) introducing a 50 microm inner diameter (i.d.) analytical column for peptide separation, (iii) decreasing the inlet flow rate to 100 nL/minute, and (iv) analyzing the samples on an LTQ Orbitrap XL mass spectrometer, it was clearly demonstrated that the entire approach could be successfully down-scaled by a factor of 100, that is, equivalent to 2 x 10(6) K562 cells. The known major targets of bosutinib were still unequivocally identified in addition to 30 targets not previously identified by gel-based mass spectrometry in our laboratory. In total, 70 individual targets were identified by mass spectrometry across this study of which 19 had not been previously reported. The down-scaled technique was made feasible by eluting the proteins that interact with bosutinib with acid and analyzing the proteins by one-dimensional shotgun proteomics. Overall, these improvements should allow utilization of very limited amounts of patient material to generate a comprehensive and comparative profile of protein drug targets.

Charting the molecular network of the drug target Bcr-Abl.

The tyrosine kinase Bcr-Abl causes chronic myeloid leukemia and is the cognate target of tyrosine kinase inhibitors like imatinib. We have charted the protein-protein interaction network of Bcr-Abl by a 2-pronged approach. Using a monoclonal antibody we have first purified endogenous Bcr-Abl protein complexes from the CML K562 cell line and characterized the set of most tightly-associated interactors by MS. Nine interactors were subsequently subjected to tandem affinity purifications/MS analysis to obtain a molecular interaction network of some hundred cellular proteins. The resulting network revealed a high degree of interconnection of 7 "core" components around Bcr-Abl (Grb2, Shc1, Crk-I, c-Cbl, p85, Sts-1, and SHIP-2), and their links to different signaling pathways. Quantitative proteomics analysis showed that tyrosine kinase inhibitors lead to a disruption of this network. Certain components still appear to interact with Bcr-Abl in a phosphotyrosine-independent manner. We propose that Bcr-Abl and other drug targets, rather than being considered as single polypeptides, can be considered as complex protein assemblies that remodel upon drug action.

An orthogonal proteomic-genomic screen identifies AIM2 as a cytoplasmic DNA sensor for the inflammasome.

Cytoplasmic DNA triggers activation of the innate immune system. Although 'downstream' signaling components have been characterized, the DNA-sensing components remain elusive. Here we present a systematic proteomics screen for proteins that associate with DNA, 'crossed' to a screen for transcripts induced by interferon-beta, which identified AIM2 as a candidate cytoplasmic DNA sensor. AIM2 showed specificity for double-stranded DNA. It also recruited the inflammasome adaptor ASC and localized to ASC 'speckles'. A decrease in AIM2 expression produced by RNA-mediated interference impaired DNA-induced maturation of interleukin 1beta in THP-1 human monocytic cells, which indicated that endogenous AIM2 is required for DNA recognition. Reconstitution of unresponsive HEK293 cells with AIM2, ASC, caspase-1 and interleukin 1beta showed that AIM2 was sufficient for inflammasome activation. Our data suggest that AIM2 is a cytoplasmic DNA sensor for the inflammasome.

Chemical proteomic profiles of the BCR-ABL inhibitors imatinib, nilotinib, and dasatinib reveal novel kinase and nonkinase targets.

The BCR-ABL tyrosine kinase inhibitor imatinib represents the current frontline therapy in chronic myeloid leukemia. Because many patients develop imatinib resistance, 2 second-generation drugs, nilotinib and dasatinib, displaying increased potency against BCR-ABL were developed. To predict potential side effects and novel medical uses, we generated comprehensive drug-protein interaction profiles by chemical proteomics for all 3 drugs. Our studies yielded 4 major findings: (1) The interaction profiles of the 3 drugs displayed strong differences and only a small overlap covering the ABL kinases. (2) Dasatinib bound in excess of 30 Tyr and Ser/Thr kinases, including major regulators of the immune system, suggesting that dasatinib might have a particular impact on immune function. (3) Despite the high specificity of nilotinib, the receptor tyrosine kinase DDR1 was identified and validated as an additional major target. (4) The oxidoreductase NQO2 was bound and inhibited by imatinib and nilotinib at physiologically relevant drug concentrations, representing the first nonkinase target of these drugs.