Relative protein quantification by isobaric SILAC with immonium ion splitting (ISIS)

Metabolic labeling techniques have recently become popular tools for the quantitative profiling of proteomes. Classical stable isotope labeling with amino acids in cell cultures (SILAC) uses pairs of heavy/light isotopic forms of amino acids to introduce predictable mass differences in protein samples to be compared. After proteolysis, pairs of cognate precursor peptides can be correlated, and their intensities can be used for mass spectrometry-based relative protein quantification. We present an alternative SILAC approach by which two cell cultures are grown in media containing isobaric forms of amino acids, labeled either with 13C on the carbonyl (C-1) carbon or 15N on backbone nitrogen. Labeled peptides from both samples have the same nominal mass and nearly identical MS/MS spectra but generate upon fragmentation distinct immonium ions separated by 1 amu. When labeled protein samples are mixed, the intensities of these immonium ions can be used for the relative quantification of the parent proteins. We validated the labeling of cellular proteins with valine, isoleucine, and leucine with coverage of 97% of all tryptic peptides. We improved the sensitivity for the detection of the quantification ions on a pulsing instrument by using a specific fast scan event. The analysis of a protein mixture with a known heavy/light ratio showed reliable quantification. Finally the application of the technique to the analysis of two melanoma cell lines yielded quantitative data consistent with those obtained by a classical two-dimensional DIGE analysis of the same samples. Our method combines the features of the SILAC technique with the advantages of isobaric labeling schemes like iTRAQ. We discuss advantages and disadvantages of isobaric SILAC with immonium ion splitting as well as possible ways to improve it.     

Identification of cyclosporin binding sites in rat liver plasma membranes, isolated hepatocytes, and hepatoma cells by photoaffinity labeling using [3H]cyclosporin-diaziridine

[3H]Cyclosporin diaziridine, a new photoaffinity label, enters rat liver cells in the dark. Photoaffinity labeling of isolated rat liver-cell plasma membranes with this probe modifies several polypeptides with molecular mass of 200, 85, 54, 50, 34 kDa. The major labeled protein of 85 kDa represents 2% of the total plasma membrane protein. A 50 kDa protein is heavily labeled in freshly isolated rat hepatocytes at low temperature and after short incubation in the dark. The 85 kDa protein becomes substituted after longer preincubation periods at temperatures above 10 degrees C. This suggests a localisation at the cytoplasmic side of the membrane. Several controls point to a specific interaction with the above mentioned proteins. Comparison of [3H]cyclosporin-diaziridine- and isothiocyanatobenzamido[3H] cholic acid-labeled membrane proteins reveals identity of binding proteins with the exception of the 85 kDa protein. However, the interaction of bile acids with the 85 kDa protein became apparent at higher concentrations as demonstrated by the differential photoaffinity labeling experiments. In the cytosol of rat liver cells, further [3H]cyclosporin-diaziridine binding proteins could be identified. In particular, a 17 kDa polypeptide was found which appears similar to cyclophilin, a protein known to be present in T-lymphocytes (R. Handschumacher et al. (1984) Science 226, 544-547: Cyclophilin. A specific cytosolic binding protein for cyclosporin A). Proteins with molecular mass of 90, 56, 30, 24, 20 kDa are labeled in AS-30D ascites hepatoma cells and those with molecular mass of 200, 150, 80, 70, 42, 25 kDa in Ehrlich ascites tumor cells.     

Difference in mass analysis using labeled lysines (DIMAL-K): a new, efficient proteomic quantification method applied to the analysis of astrocytic secretomes

Here we describe an original strategy for unbiased quantification of protein expression called difference in mass analysis using labeled lysine (K) (DIMAL-K). DIMAL-K is based on the differential predigestion labeling of lysine residues in complex protein mixtures. The method is relevant for proteomic analysis by two-dimensional electrophoresis and MALDI-TOF mass spectrometry. Protein labeling on lysine residues uses two closely related chemical reagents, S-methyl thioacetimidate and S-methyl thiopropionimidate. Using protein standards, we demonstrated that 1) the chemical labeling was quantitative, specific, and rapid; 2) the differentially labeled proteins co-migrated on two-dimensional gels; and 3) the identification by mass fingerprinting and the relative quantification of the proteins were possible from a single MALDI-TOF mass spectrum. The power of the method was tested by comparing and quantifying the secretion of proteins in normal and proinflammatory astrocytic secretomes (20 microg). We showed that DIMAL-K was more sensitive and accurate than densitometric image analysis and allowed the detection and quantification of novel proteins.     

An efficient and cost-effective isotope labeling protocol for proteins expressed in shape Escherichia coli

A cost-effective protocol for uniform ¹⁵N and/or¹³ C isotope labeling of bacterially expressed proteins is presented. Unlike most standard protocols, cells are initially grown in a medium containing nutrients at natural abundance and isotopically labeled nutrients are only supplied at the later stages of growth and during protein expression. This permits the accumulation of a large cell mass without the need to employ expensive isotopically labeled nutrients. The abrupt decrease in oxygen consumption that occurs upon complete exhaustion of essential nutrients is used to precisely time the switch between unlabeled and labeled nutrients. Application of the protocol is demonstrated for wild-type and a mutant of the N-terminal zinc-binding domain of HIV-1 integrase.     

Photoaffinity labeling and fatty acid permeation in 3T3-L1 adipocytes

Long chain fatty acid uptake was investigated in 3T3-L1 cells. Differentiation of these cells from fibroblasts to adipocytes was accompanied by an 8.5-fold increase in the rate of oleate uptake. This was saturable in adipocytes with apparent Kt and Vmax values of 78 nM and 16 nmol/min/mg cell protein, respectively. A number of proteins in various subcellular fractions of differentiated cells were labeled with the photoreactive fatty acid 11-m-diazirinophenoxy[11-3H]undecanoate. A 15-kDa cytoplasmic protein was induced upon differentiation to adipocytes. This protein was labeled with the photoreactive fatty acid in cytoplasm isolated from differentiated adipocytes, but not in cytoplasm from undifferentiated, fibroblastic cells. Furthermore, a high affinity fatty acid binding protein of 22 kDa was identified in plasma membranes of undifferentiated cells, and its level of labeling increased 2-fold upon differentiation. These results indicate the usefulness of the photoreactive fatty acid in identifying cellular fatty acid binding proteins, and its potential to elucidate the spatial and temporal distribution of fatty acids in intact cells.     

Beyond quantitative proteomics: signal enhancement of the a1 ion as a mass tag for peptide sequencing using dimethyl labeling

Stable isotope-based dimethyl labeling that produces a dimethyl labeled terminal amine or a monomethylated proline N-terminus by reductive methylation (Anal. Chem. 2003, 75, 6843-6852) was reported as a promising strategy for global quantitative proteomics because of the simplicity of the process and its fast and complete reaction. This labeling strategy provides a signal enhancement for the produced a1 ions, which are usually hard to detect among most of the nonderivatized fragments. To assist peptide sequencing, in this study, the enhanced a1 ion produced under either collision induced dissociation (CID) or post source decay (PSD) modes was further characterized and applied as a mass tag for fingerprinting the identity of N-terminal amino acid. On the basis of the analysis of standard peptides, tryptic digests of hemoglobin and cell lysates, it was proved that such signal enhancement occurred to a1 ions derived from all 20 of the amino acids residues and this phenomenon was explained based the formation of stable quaternary immoniun ions. Accurate determination of a1 ions was shown to increase the chance for peptide de novo sequencing and also provided higher confidence in the scores obtained when identifying a protein through database searching. In addition, the a1 ion was further demonstrated to be used as a universal tag for precursor ion scan in a Q-TOF instrument, leading to a greater number of peptide ions sequenced. Combined with the capability for differential quantitation, the stable isotope-based dimethyl labeling increases the usefulness of the labeling method for MS-based proteomics.     

Effect of 2MEGA labeling on membrane proteome analysis using LC-ESI QTOF MS

One of the challenges associated with large-scale proteome analysis using tandem mass spectrometry (MS/MS) and automated database searching is to reduce the number of false positive identifications without sacrificing the number of true positives found. In this work, a systematic investigation of the effect of 2MEGA labeling (N-terminal dimethylation after lysine guanidination) on the proteome analysis of a membrane fraction of an Escherichia coli cell extract by 2-dimensional liquid chromatography MS/MS is presented. By a large-scale comparison of MS/MS spectra of native peptides with those from the 2MEGA-labeled peptides, the labeled peptides were found to undergo facile fragmentation with enhanced a1 or a1-related (a(1)-17 and a(1)-45) ions derived from all N-terminal amino acids in the MS/MS spectra; these ions are usually difficult to detect in the MS/MS spectra of nonderivatized peptides. The 2MEGA labeling alleviated the biased detection of arginine-terminated peptides that is often observed in MALDI and ESI MS experiments. 2MEGA labeling was found not only to increase the number of peptides and proteins identified but also to generate enhanced a1 or a1-related ions as a constraint to reduce the number of false positive identifications. In total, 640 proteins were identified from the E. coli membrane fraction, with each protein identified based on peptide mass and sequence match of one or more peptides using MASCOT database search algorithm from the MS/MS spectra generated by a quadrupole time-of-flight mass spectrometer. Among them, the subcellular locations of 336 proteins are presently known, including 258 membrane and membrane-associated proteins (76.8%). Among the classified proteins, there was a dramatic increase in the total number of integral membrane proteins identified in the 2MEGA-labeled sample (153 proteins) versus the unlabeled sample (77 proteins).     

Phosphatidylinositol 3,4,5-trisphosphate activity probes for the labeling and proteomic characterization of protein binding partners

Phosphatidylinositol polyphosphate lipids, such as phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P?], regulate critical biological processes, many of which are aberrant in disease. These lipids often act as site-specific ligands in interactions that enforce membrane association of protein binding partners. Herein, we describe the development of bifunctional activity probes corresponding to the headgroup of PI(3,4,5)P? that are effective for identifying and characterizing protein binding partners from complex samples, namely cancer cell extracts. These probes contain both a photoaffinity tag for covalent labeling of target proteins and a secondary handle for subsequent detection or manipulation of labeled proteins. Probes bearing different secondary tags were exploited, either by direct attachment of a fluorescent dye for optical detection or by using an alkyne that can be derivatized after protein labeling via click chemistry. First, we describe the design and modular synthetic strategy used to generate multiple probes with different reporter tags of use for characterizing probe-labeled proteins. Next, we report initial labeling studies using purified protein, the PH domain of Akt, in which probes were found to label this target, as judged by in-gel detection. Furthermore, protein labeling was abrogated by controls including competition with an unlabeled PI(3,4,5)P? headgroup analogue as well as through protein denaturation, indicating specific labeling. In addition, probes featuring linkers of different lengths between the PI(3,4,5)P? headgroup and photoaffinity tag led to variations in protein labeling, indicating that a shorter linker was more effective in this case. Finally, proteomic labeling studies were performed using cell extracts; labeled proteins were observed by in-gel detection and characterized using postlabeling with biotin, affinity chromatography, and identification via tandem mass spectrometry. These studies yielded a total of 265 proteins, including both known and novel candidate PI(3,4,5)P?-binding proteins.     

Lactate Dependent Protein Synthesis in Round Spermatids from Rat Testes

Lactate (20 mM) markedly increased protein labeling of round spermatids (steps 1–8) from rat testes. The stimulatory effect of lactate on protein labeling was also observed to some degree in spermatocytes and late spermatids (steps 13–16), but not in Leydig cells and 7 day-old testis cell suspznsions. In the lactate-treated spermatids, 51 % of the labeled proteins was found in the water-soluble fraction (the 105,000 × g 1 hr supernatant), whereas only 21%, in the control cells. The labeled proteins did not break down for at least 90 minutes in the presence of lactate. Actinomycin D (20 μg/ml) had no effect on [³H]leucine incorporation into the water-soluble proteins of spermatids, while labeling of the water-insoluble proteins (the 105,000 × g 1 hr pellet) was decreased by 23%.
These findings suggest that the round spermatids might be the most susceptible for the lactate-induced stimulation of protein synthesis among various types of testicular cells, and that lactate increases the synthesis of water-soluble proteins which may be regulated in the translational level of protein synthesis.     

Protein C-Terminal Labeling and Biotinylation Using Synthetic Peptide and Split-Intein

Background

Site-specific protein labeling or modification can facilitate the characterization of proteins with respect to their structure, folding, and interaction with other proteins. However, current methods of site-specific protein labeling are few and with limitations, therefore new methods are needed to satisfy the increasing need and sophistications of protein labeling.

Methodology

A method of protein C-terminal labeling was developed using a non-canonical split-intein, through an intein-catalyzed trans-splicing reaction between a protein and a small synthetic peptide carrying the desired labeling groups. As demonstrations of this method, three different proteins were efficiently labeled at their C-termini with two different labels (fluorescein and biotin) either in solution or on a solid surface, and a transferrin receptor protein was labeled on the membrane surface of live mammalian cells. Protein biotinylation and immobilization on a streptavidin-coated surface were also achieved in a cell lysate without prior purification of the target protein.

Conclusions

We have produced a method of site-specific labeling or modification at the C-termini of recombinant proteins. This method compares favorably with previous protein labeling methods and has several unique advantages. It is expected to have many potential applications in protein engineering and research, which include fluorescent labeling for monitoring protein folding, location, and trafficking in cells, and biotinylation for protein immobilization on streptavidin-coated surfaces including protein microchips. The types of chemical labeling may be limited only by the ability of chemical synthesis to produce the small C-intein peptide containing the desired chemical groups.     

Optimization of the isotope-coded affinity tag-labeling procedure for quantitative proteome analysis

The combination of isotope coded affinity tag (ICAT) reagents and tandem mass spectrometry constitutes a new method for quantitative proteomics. It involves the site-specific, covalent labeling of proteins with isotopically normal or heavy ICAT reagents, proteolysis of the combined, labeled protein mixture, followed by the isolation and mass spectrometric analysis of the labeled peptides. The method critically depends on labeling protocols that are specific, quantitative, general, robust, and reproducible. Here we describe the systematic evaluation of important parameters of the labeling protocol and describe optimized labeling conditions. The tested factors include the ICAT reagent concentration, the influence of the protein, SDS, and urea concentrations on the labeling reaction, and the reaction time. We demonstrate that using the optimized conditions specific and quantitative labeling was achieved on standard proteins as well as in complex protein mixtures such as a yeast cell lysate.     

Application of fed-batch fermentation to the preparation of isotopically labeled or selenomethionyl-labeled proteins.

An increasing demand for isotopically labeled samples for spectroscopic and crystallographic studies has led to a corresponding need for effective and efficient methods for producing these samples. The present work is based on the strategy of using an isotopically labeled compound as the growth-limiting nutrient during protein expression in Escherichia coli (DE3) strains. By using dissolved O2 and agitation rate data, the cell growth, feeding of the isotopic label, induction of protein expression, and the harvest of cells can be coordinated in a feedback controlled fermenter in a simple, easily defined manner. This approach is demonstrated for the nutrient-limited production of [U-15N]- and [U-13C, U-15N]-labeled toluene 4-monooxygenase effector protein in E. coli BL21(DE3) with isotopic abundance identical to that of the labeled precursors. For selective labeling, demonstrated with selenomethionine using methionine auxotroph E. coli B834(DE3), approximately 80-85% incorporation was obtained from methionine-dependent growth of the auxotroph followed by selenomethionine feeding and protein induction upon methionine depletion. This selective labeling is accomplished in a single culture, does not require washing or resuspension, minimizes costly incorporation of label into host cell mass prior to induction, and can be easily adapted to selective labeling with other amino acids. Moreover, cell mass yield from these experiments can be readily optimized to provide the desired level of protein for a given investigation from a single growth and purification. This combination provides an efficient, controllable option for isotopic labeling experiments.     

In vivo imaging of retrogradely transported synaptic vesicle proteins in Caenorhabditis elegans neurons

Axonal transport is an essential process that carries cargoes in the anterograde direction to the synapse and in the retrograde direction back to the cell body. We have developed a novel in vivo method to exclusively mark and dynamically track retrogradely moving compartments carrying specific endogenous synaptic vesicle proteins in the Caenorhabditis elegans model. Our method is based on the uptake of a fluorescently labeled anti-green fluorescent protein (GFP) antibody delivered in an animal expressing the synaptic vesicle protein synaptobrevin-1::GFP in neurons. We show that this method largely labels retrogradely moving compartments. Very little labeling is observed upon blocking vesicle exocytosis or if the synapse is physically separated from the cell body. The extent of labeling is also dependent on the dyenin-dynactin complex. These data support the interpretation that the labeling of synaptobrevin-1::GFP largely occurs after vesicle fusion and the major labeling likely takes place at the synapse. Further, we observe that the retrograde compartment carrying synaptobrevin contains synaptotagmin but lacks the endosomal marker RAB-5. This labeling method is very general and can be readily adapted to any transmembrane protein on synaptic vesicles with a GFP tag inside the vesicle and can also be extended to other model systems.     

Evaluation of preferred binding regions on ubiquitin and IgG1 FC for interacting with multimodal cation exchange resins using DEPC labeling/mass spectrometry

There is significant interest in identifying the preferred binding domains of biological products to various chromatographic materials. In this work, we develop a biophysical technique that uses diethyl pyrocarbonate (DEPC) based covalent labeling in concert with enzymatic digestion and mass spectrometry to identify the binding patches for proteins bound to commercially available multimodal (MM) cation exchange chromatography resins. The technique compares the changes in covalent labeling of the protein in solution and in the bound state and uses the differences in this labeling to identify residues that are sterically shielded upon resin binding and, therefore, potentially involved in the resin binding process. Importantly, this approach enables the labeling of many amino acids and can be carried out over a pH range of 5.5–7.5, thus enabling the protein surface mapping at conditions of interest in MM cation exchange systems. The protocol is first developed using the model protein ubiquitin and the results indicate that lysine residues located on the front face of the protein show dramatic changes in DEPC labeling while residues present on other regions have minimal or no reductions. This indicates that the front face of ubiquitin is likely involved in resin binding. In addition, surface property maps indicate that the hypothesized front face binding region consists of overlapping positively charged and hydrophobic patches. The technique is then employed with an IgG1 F_C and the results indicate that residues on the C_H2–C_H3 interface and the hinge are significantly sterically shielded upon binding to the resin. Further, these regions are again associated with significant overlap of positively charged and hydrophobic patches. On the other hand, while, residues on the C_H2 and the front face of the IgG1 F_C also exhibited some changes in DEPC labeling upon binding, these regions have less distinct charged and hydrophobic patches. Importantly, the hypothesized binding patches identified for both ubiquitin and F_C using this approach are shown to be consistent with previously reported NMR studies. In contrast to NMR, this new approach enables the identification of preferred binding regions without the need for isotopically labeled proteins or chemical shift assignments. The technique developed in this work sets the stage for the evaluation of the binding domains of a wide range of biological products to chromatographic surfaces, with important implications for designing biomolecules with improved biomanufacturability properties.     

Preprocessing Significantly Improves the Peptide/Protein Identification Sensitivity of High-resolution Isobarically Labeled Tandem Mass Spectrometry Data

Isobaric labeling techniques coupled with high-resolution mass spectrometry have been widely employed in proteomic workflows requiring relative quantification. For each high-resolution tandem mass spectrum (MS/MS), isobaric labeling techniques can be used not only to quantify the peptide from different samples by reporter ions, but also to identify the peptide it is derived from. Because the ions related to isobaric labeling may act as noise in database searching, the MS/MS spectrum should be preprocessed before peptide or protein identification. In this article, we demonstrate that there are a lot of high-frequency, high-abundance isobaric related ions in the MS/MS spectrum, and removing isobaric related ions combined with deisotoping and deconvolution in MS/MS preprocessing procedures significantly improves the peptide/protein identification sensitivity. The user-friendly software package TurboRaw2MGF (v2.0) has been implemented for converting raw TIC data files to mascot generic format files and can be downloaded for free from https://github.com/shengqh/RCPA.Tools/releases as part of the software suite ProteomicsTools. The data have been deposited to the ProteomeXchange with identifier PXD000994.Mass spectrometry-based proteomics has been widely applied to investigate protein mixtures derived from tissue, cell lysates, or from body fluids (1, 2). Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS)¹ is the most popular strategy for protein/peptide mixtures analysis in shotgun proteomics (3). Large-scale protein/peptide mixtures are separated by liquid chromatography followed by online detection by tandem mass spectrometry. The capabilities of proteomics rely greatly on the performance of the mass spectrometer. With the improvement of MS technology, proteomics has benefited significantly from the high-resolution and excellent mass accuracy (4). In recent years, based on the higher efficiency of higher energy collision dissociation (HCD), a new “high–high” strategy (high-resolution MS as well as MS/MS(tandem MS)) has been applied instead of the “high–low” strategy (high-resolution MS, i.e. in Orbitrap, and low-resolution MS/MS, i.e. in ion trap) to obtain high quality tandem MS/MS data as well as full MS in shotgun proteomics. Both full MS scans and MS/MS scans can be performed, and the whole cycle time of MS detection is very compatible with the chromatographic time scale (5).High-resolution measurement is one of the most important features in mass spectrometric application. In this high–high strategy, high-resolution and accurate spectra will be achieved in tandem MS/MS scans as well as full MS scans, which makes isotopic peaks distinguishable from one another, thus enabling the easy calculation of precise charge states and monoisotopic mass. During an LC-MS/MS experiment, a multiply charged precursor ion (peptide) is usually isolated and fragmented, and then the multiple charge states of the fragment ions are generated and collected. After full extraction of peak lists from original tandem mass spectra, the commonly used search engines (i.e. Mascot (6), Sequest (7)) have no capability to distinguish isotopic peaks and recognize charge states, so all of the product ions are considered as all charge state hypotheses during the database search for protein identification. These multiple charge states of fragment ions and their isotopic cluster peaks can be incorrectly assigned by the search engine, which can cause false peptide identification. To overcome this issue, data preprocessing of the high-resolution MS/MS spectra is required before submitting them for identification. There are usually two major preprocessing steps used for high-resolution MS/MS data: deisotoping and deconvolution (8, 9). Deisotoping of spectra removes all isotopic peaks except monoisotopic peaks from multi-isotopic peaks. Deconvolution of spectra translates multiply charged ions to singly charged ions and also accumulates the intensity of fragment ions by summing up all the intensities from their multiply charged states. After performing these two data-preprocessing steps, the resulting spectra is simpler and cleaner and allows more precise database searching and accurate bioinformatics analysis.With the capacity to analyze multiple samples simultaneously, stable isotope labeling approaches have been widely used in quantitative proteomics. Stable isotope labeling approaches are categorized as metabolic labeling (SILAC, stable isotope labeling by amino acids in cell culture) and chemical labeling (10, 11). The peptides labeled by the SILAC approach are quantified by precursor ions in full MS spectra, whereas peptides that have been isobarically labeled using chemical means are quantified by reporter ions in MS/MS spectra. There are two similar isobaric chemical labeling methods: (1) isobaric tag for relative and absolute quantification (iTRAQ), and (2) tandem mass tag (TMT) (12, 13). These reagents contain an amino-reactive group that specifically reacts with N-terminal amino groups and epilson-amino groups of lysine residues to label digested peptides in a typical shotgun proteomics experiment. There are four different channels of isobaric tags: TMT two-plex, iTRAQ four-plex, TMT six-plex, and iTRAQ eight-plex (12–16). The number before “plex” denotes the number of samples that can be analyzed by the mass spectrum simultaneously. Peptides labeled with different isotopic variants of the tag show identical or similar mass and appear as a single peak in full scans. This single peak may be selected for subsequent MS/MS analysis. In an MS/MS scan, the mass of reporter ions (114 to 117 for iTRAQ four-plex, 113 to 121 for iTRAQ eight-plex, and 126 to 131for TMT six-plex upon CID or HCD activation) are associated with corresponding samples, and the intensities represent the relative abundances of the labeled peptides. Meanwhile, the other ions from the MS/MS spectra can be used for peptide identification. Because of the multiplexing capability, isobaric labeling methods combined with bottom-up proteomics have been widely applied for accurate quantification of proteins on a global scale (14, 17–19). Although mostly associated with peptide labeling, these isobaric labeling methods have also been applied at protein level (20–23).For the proteomic analysis of isobarically labeled peptides/proteins in “high–high” MS strategy, the common consensus is that accurate reporter ions can contribute to more accurate quantification. However, there is no evidence to show how the ions related to isobaric labeling affect the peptide/protein identification and what preprocessing steps should be taken for high-resolution isobarically labeled MS/MS. To demonstrate the effectiveness and importance of preprocessing, we examined how the combination of preprocessing steps improved peptide/protein sensitivity in database searching. Several combinatorial ways of data-preprocessing were applied for high-throughput data analysis including deisotoping to keep simple monoisotopic mass peaks, deconvolution of ions with multiple charge states, and preservation of top 10 peaks in every 100 Dalton mass range. After systematic analysis of high-resolution isobarically labeled spectra, we further processed the spectra and removed interferential ions that were not related to the peptide. Our results suggested that the preprocessing of isobarically labeled high-resolution tandem mass spectra significantly improved the peptide/protein identification sensitivity.     

Profiling Protein Kinases and Other ATP Binding Proteins in Arabidopsis Using Acyl-ATP Probes

Many protein activities are driven by ATP binding and hydrolysis. Here, we explore the ATP binding proteome of the model plant Arabidopsis thaliana using acyl-ATP (AcATP)¹ probes. These probes target ATP binding sites and covalently label lysine residues in the ATP binding pocket. Gel-based profiling using biotinylated AcATP showed that labeling is dependent on pH and divalent ions and can be competed by nucleotides. The vast majority of these AcATP-labeled proteins are known ATP binding proteins. Our search for labeled peptides upon in-gel digest led to the discovery that the biotin moiety of the labeled peptides is oxidized. The in-gel analysis displayed kinase domains of two receptor-like kinases (RLKs) at a lower than expected molecular weight, indicating that these RLKs lost the extracellular domain, possibly as a result of receptor shedding. Analysis of modified peptides using a gel-free platform identified 242 different labeling sites for AcATP in the Arabidopsis proteome. Examination of each individual labeling site revealed a preference of labeling in ATP binding pockets for a broad diversity of ATP binding proteins. Of these, 24 labeled peptides were from a diverse range of protein kinases, including RLKs, mitogen-activated protein kinases, and calcium-dependent kinases. A significant portion of the labeling sites could not be assigned to known nucleotide binding sites. However, the fact that labeling could be competed with ATP indicates that these labeling sites might represent previously uncharacterized nucleotide binding sites. A plot of spectral counts against expression levels illustrates the high specificity of AcATP probes for protein kinases and known ATP binding proteins. This work introduces profiling of ATP binding activities of a large diversity of proteins in plant proteomes. The data have been deposited in ProteomeXchange with the identifier PXD000188.ATP binding and hydrolysis are the driving processes in all living organisms. Hundreds of cellular proteins are able to bind and hydrolyze ATP to unfold proteins, transport molecules over membranes, or phosphorylate small molecules or proteins. Proteins with very different structures are able to bind ATP. A large and important class of ATP binding proteins is that of the kinases, which transfer the gamma phosphate from ATP to substrates. Kinases, and particularly protein kinases, play pivotal roles in signaling and protein regulation.The genome of the model plant Arabidopsis thaliana encodes for over 1099 protein kinases and hundreds of other ATP binding proteins (1, 2). Protein kinases are involved in nearly all signaling cascades and regulate processes ranging from cell cycle to flowering and from immunity to germination. Many protein kinases in plants are receptor-like kinases (RLKs), often carrying extracellular leucine-rich repeats (LRRs). The RLK class contains at least 610 members (3), including famous examples such as receptors involved in development (e.g. BRI1, ER, CLV1) and immunity (e.g. FLS2, EFR). Other important classes are mitogen-activated protein (MAP) kinases (MPKs) (20 different members), MPK kinase kinase kinases (MAP3Ks) (60 different members (4)), and calcium-dependent protein kinases (CPKs) (34 different members (5)). Because of their diverse and important roles, protein kinases have been intensively studied in plant science. The current approach is to study protein kinases individually—a daunting task, considering the remaining hundreds of uncharacterized protein kinases. New approaches are necessary in order to study protein kinases and other ATP binding proteins globally rather than individually.ATP binding activities of protein kinases and other proteins can be detected globally by acyl-ATP (AcATP) probes (6, 7) (Fig. 1A). AcATP binds to the ATP pocket of ATP binding proteins and places the acyl group in close proximity to conserved lysine residues in the ATP binding pocket. The acyl phosphonate moiety serves as an electrophilic warhead that can be nucleophilically attacked by the amino group of the lysine, resulting in a covalent attachment of the acyl reporter of the AcATP probe on the lysine and a concomitant release of ATP. The reporter tag is usually a biotin to capture and identify the labeled proteins. Labeled proteins can be displayed on protein blots using streptavidin-HRP. However, because AcATP labels many ATP binding proteins and protein kinases are of relatively low abundance, mass spectrometry is more often used to identify and quantify labeling with AcATP probes. The analysis is preferably done using Xsite, a procedure that involves trypsination of the entire labeled proteome, followed by analysis of the biotinylated peptides rather than the biotinylated proteins (8). This “KiNativ ” approach provides enough depth and resolving power to monitor ∼160 protein kinases in a crude mammalian proteome (7). Of the 518 human protein kinases (9), 394 (76%) have been detected via AcATP labeling (6).Open in a separate windowFig. 1.Structure and mechanism of labeling with BHAcATP. A, BHAcATP contains ATP, an acyl phosphate reactive group, and a biotin tag. When BHAcATP binds to the ATP binding pocket of a protein, the amino group of the nearby lysine reacts with the carbonyl carbon, which results in the covalent binding of the biotin tag to the protein while ATP is released. B, typical BHAcATP labeling profile of Arabidopsis leaf proteome. Arabidopsis leaf extracts were labeled with BHAcATP and the biotinylated proteins were detected on protein blots using streptavidin-HRP. Coomassie Brilliant Blue staining indicates equal loading. Asterisks indicate endogenously biotinylated proteins MCCA and BCCP. White, black, and gray arrowheads indicate bands containing ATBP+RBCL, PGK1, and a mix of ATP binding proteins, respectively. Abbreviations: MCCA, 3-methylcrotonyl-CoA carboxylase; BCCP, biotin carboxyl carrier protein; ATPB, chloroplastic ATPase; RBCL, ribulose-bisphosphate carboxylase; PGK1, phosphoglycerate kinase-1.KiNativ has mostly been used to validate targets of human drugs that target protein kinases using competitive labeling experiments. This approach has been used to identify selective inhibitors of, for example, Parkinson''s disease protein kinase LRRK2 (10), the BMK1 and JNK MAP kinases (11, 12), and the mTOR kinase (13). Importantly, the correlation of the biological activity of protein-kinase-inhibiting drugs with inhibitor affinity detected using KiNativ is better than that achieved when affinities are determined by assays using heterologously expressed protein kinases (7). This improved correlation illustrates that assays in the native environment provide a more realistic measure of protein kinase function.In addition to characterizing inhibitors selectively, AcATP probes can also display differential ATP binding activities of protein kinases. For example, labeling with AcATP probes during infection with dengue virus displayed a 2- to 8-fold activation of a DNA-dependent protein kinase (14) Similarly, AcATP labeling revealed an unexpected Raf kinase activation in extracts upon protein kinase inhibitor treatment (7). In conclusion, profiling with AcATP probes is a powerful approach for monitoring protein kinases and offers unprecedented opportunities to identify selective protein kinase inhibitors and discover protein kinases with differential ATP binding activities.In this work, we introduce AcATP profiling of plant proteomes. In addition to the analysis of labeled peptides, we characterized labeling using gel-based approaches and discovered that biotin is often oxidized in this procedure. We also performed an in-depth analysis of labeling sites in proteins other than protein kinases, which had not been done before. We discuss labeling outside known nucleotide binding pockets and investigate the correlation of labeling sites with protein abundance. We describe 63 labeling sites of known nucleotide binding pockets, of which 24 represent a remarkable diversity of protein kinases, including several LRR-RLKs. This work launches a new approach to study ATP binding proteins in plant science.     

Serial protein labeling with infrared maleimide dyes to identify cysteine modifications

Cysteine thiol modifications are increasingly recognized to occur under both physiological and pathophysiological conditions, making their accurate detection, identification and quantification of growing importance. However, saturation labeling of thiols with fluorescent dyes results in poor protein recuperation and therefore requires the use of large quantities of starting material. This is especially important in sequential dye-labeling steps when applied for an identification of cysteine modifications. First, we studied the effects of different detergents during labeling procedure, i.e. Tween 20, Triton X-100 and CHAPS, on protein yield and composition. Tween 20 and Triton X-100 resulted in yields of around 50% labeled proteins compared to only 10% with PBS alone and a most diversified 2-DE protein pattern. Secondly, Tween 20 was used for serial protein labeling with maleimid fluorophores, first to conjugate to accessible thiols and after a reduction to label with another fluorophore previously masked di-sulphide and/or oxidized proteins in frontal cortex autopsy tissue of a subject with mild Alzheimer's disease. Two-DE DIGE revealed a complex protein pattern of readily labeled thiols and di-sulphide and/or oxidized proteins. Seventeen proteins were identified by MALDI-TOF and by peptide fingerprints. Several proteins were oxidized and involved in Alzheimer's disease. However methionine oxidation was prevalent. Infrared DIGE may provide an additional tool for an identification of oxidation susceptible proteins.     

Production in two-liter beverage bottles of proteins for NMR structure determination labeled with either 15N- or 13C-15N

The use of 2-L polyethylene terephthalate beverage bottles as a bacterial culture vessel has been recently introduced as an enabling technology for high-throughput structural biology [Sanville Millard, C. et al., 2003. Protein Express. Purif. 29, 311-320]. In the article following this one [Stols et al., this issue, pp. 95-102], this approach was elaborated for selenomethionine labeling used for multiwavelength anomalous dispersion phasing in the X-ray crystallographic determinations of protein structure. Herein, we report an effective and reproducible schedule for uniform 15N- and 13C-labeling of recombinant proteins in 2-L beverage bottles for structural determination by NMR spectroscopy. As an example, three target proteins selected from Arabidopsis thaliana were expressed in Escherichia coli Rosetta (DE3)/pLysS from a T7-based expression vector, purified, and characterized by electrospray ionization mass spectrometry and NMR analysis by 1H-15N heteronuclear single quantum correlation spectroscopy. The results show that expressions in the unlabeled medium provide a suitable control for estimation of the level of production of the labeled protein. Mass spectral characterizations show that the purified proteins contained a level of isotopic incorporation equivalent to the isotopically labeled materials initially present in the growth medium, while NMR analysis of the [U-15N]-labeled proteins provided a convenient method to assess the solution state properties of the target protein prior to production of a more costly double-labeled sample.     

Role of RNA and protein synthesis and turnover in the heat-induced increase in nuclear protein

The proteins which become associated with nuclei during hyperthermic exposure were characterized by labeled amino acid incorporation. Actinomycin-D (Act-D) or cycloheximide (CHM) pretreatment was used to determine whether concurrent RNA or protein synthesis is required for hyperthermia to induce the increase in nuclear protein content. Prior to heat exposure exponentially growing HeLa cells were (i) pulse labeled for 1 h, (ii) labeled for 36 h, or (iii) labeled for 24 h followed by 17 h chase. The nuclear specific activity (CPM/microgram protein) of [3H]lysine-labeled proteins did not change under any of the labeling conditions, whereas that of [3H]leucine-containing proteins increased significantly with (i) but not with (ii) or (iii), while that of [3H]tryptophan-labeled protein increased significantly with (i) and (ii) but not with (iii). Act-D treatment 1 h prior to and during heating did not affect nuclear protein increase, while CHM-treated cells showed generally less nuclear protein content (70% of control at 60 min) but nevertheless significant nuclear protein increase upon heating (60% increase at 60 min from 0 min). These results suggest that those proteins associated with nuclei following heat exposure are nonhistones with a high turnover rate, and the process dose not require the synthesis of RNA or proteins.     

Sequential protein expression and selective labeling for in-cell NMR in human cells

BackgroundIn-cell NMR is a powerful technique to investigate proteins in living human cells at atomic resolution. Ideally, when studying functional processes involving protein–protein interactions by NMR, only one partner should be isotopically labeled. Here we show that constitutive and transient protein expression can be combined with protein silencing to obtain selective protein labeling in human cells.MethodsWe established a human cell line stably overexpressing the copper binding protein HAH1. A second protein (human superoxide dismutase 1, SOD1) was overexpressed by transient transfection and isotopically labeled. A silencing vector containing shRNA sequences against the HAH1 gene was used to decrease the rate of HAH1 synthesis during the expression of SOD1. The levels of HAH1 mRNA and protein were measured as a function of time following transfection by RT-PCR and Western Blot, and the final cell samples were analyzed by in-cell NMR.ResultsSOD1 was ectopically expressed and labeled in a time window during which HAH1 biosynthesis was strongly decreased by shRNA, thus preventing its labeling. In-cell NMR spectra confirmed that, while both proteins were present, only SOD1 was selectively labeled and could be detected by ¹H–¹⁵N heteronuclear NMR.Conclusions and general significanceWe showed that controlling protein expression by specifically silencing a stably expressed protein is a useful strategy to obtain selective isotope labeling of only one protein. This approach relies on established techniques thus permitting the investigation of protein–protein interactions by NMR in human cells.