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1.
Chemical cross-linking has proved successful in combination with mass spectrometry as a tool for low-resolution structure determination of proteins. The integration of chemical cross-linking with Fourier transform ion cyclotron resonance (FTICR) mass spectrometry to determine protein interfaces was tested on the calcium-dependent complex between calmodulin (CaM) and a 26-amino acid peptide derived from the skeletal muscle myosin light chain kinase (M13). Different amine-reactive, homobifunctional cross-linkers and a "zero-length" cross-linker were employed. The covalently attached complexes were separated from nonreacted proteins by one-dimensional gel electrophoresis, and the bands of interest were excised and in-gel digested with trypsin. Digestion of the cross-linked complexes resulted in complicated peptide mixtures, which were analyzed by nano-HPLC/nano-ESI-FTICR mass spectrometry. The distance constraints obtained by chemical cross-linking were in agreement with the published NMR structure of the CaM/M13 complex, pointing to residues Lys-18 and Lys-19 of M13 being cross-linked with the central alpha-helix of CaM. Thus, the integrated approach described herein has proven to be an efficient tool for mapping the topology of the CaM/M13 complex. As such it is applicable as a general strategy for the investigation of the spatial organization of protein complexes and complements existing techniques, such as X-ray crystallography and NMR spectroscopy.  相似文献   

2.
Chemical cross-linking combined with mass spectrometry is a viable approach to study the low-resolution structure of protein and protein complexes. However, unambiguous identification of the residues involved in a cross-link remains analytically challenging. To enable a more effective analysis across various MS platforms, we have developed a novel set of collision-induced dissociative cross-linking reagents and methodology for chemical cross-linking experiments using tandem mass spectrometry (CID-CXL-MS/MS). These reagents incorporate a single gas-phase cleavable bond within their linker region that can be selectively fragmented within the in-source region of the mass spectrometer, enabling independent MS/MS analysis for each peptide. Initial design concepts were characterized using a synthesized cross-linked peptide complex. Following verification and subsequent optimization of cross-linked peptide complex dissociation, our reagents were applied to homodimeric glutathione S-transferase and monomeric bovine serum albumin. Cross-linked residues identified by our CID-CXL-MS/MS method were in agreement with published crystal structures and previous cross-linking studies using conventional approaches. Common LC/MS/MS acquisition approaches such as data-dependent acquisition experiments using ion trap mass spectrometers and product ion spectral analysis using SEQUEST were shown to be compatible with our CID-CXL-MS/MS reagents, obviating the requirement for high resolution and high mass accuracy measurements to identify both intra- and interpeptide cross-links.  相似文献   

3.
Cross-linking can be used to identify spatial relationships between amino acids in proteins or protein complexes. A rapid and sensitive method for identifying the site of protein cross-linking using dithiobis(sulfosuccinimidyl propionate) (DTSSP) is presented and illustrated with experiments using murine cortactin, actin and acyl-CoA thioesterase. A characteristic 66 Da doublet, which arises from the asymmetric fragmentation of the disulfide of DTSSP-modified peptides, is observed in the mass spectra obtained under MALDI-TOF/TOF-MS conditions and allows rapid assignment of cross-links in modified proteins. This doublet is observed not only for linear cross-linked peptides but also in the mass spectra of cyclic cross-linked peptides when simultaneous fragmentation of the disulfide and the peptide backbone occurs. We suggest a likely mechanism for this fragmentation. We use guanidinylation of the cross-linked peptides with O-methyl isourea to extend the coverage of cross-linked peptides observed in this MALDI-MS technique. The methodology we report is robust and amenable to automation, and permits the analysis of native cystines along with those introduced by disulfide-containing cross-linkers.  相似文献   

4.
This study describes a novel method for improving the specific recognition, detection, and identification of proteins involved in multiprotein complexes. The method is based on a combination of coimmunoprecipitation, chemical cross-linking, and specific fluorescent tagging of protein components in close association with one another. Specific fluorescent tagging of the protein complex components was achieved using the cleavable, fluorescent cross-linker sulfosuccinimidyl 2-(7-azido-4-methylcoumarin-3-acetamido) ethyl-1,3'-dithiopropionate (SAED). Following dissociation and separation by SDS-PAGE, the fluorescently tagged proteins are then visualized by UV illumination, excised, and, following in-gel digestion, identified by mass spectrometry. In this study, a complex of the HIV-envelope protein gp120 and its cellular receptor CD4 was used as a model system. The sensitivity of detection of fluorescent SAED-labeled proteins in SDS gels, and the sensitivity of the mass spectrometric identification of fluorescent proteins after in-gel digestion, is in the range of a few hundred femtomoles of protein. This sensitivity is comparable to that achieved with silver-staining techniques, but fluorescence detection is protein independent and no background interference occurs. Furthermore, fluorescence labeling is significantly more compatible with mass spectrometric identification of proteins than is silver staining. The first application of this strategy was in the investigation of the mechanism of spermiation, the process by which mature spermatids separate from Sertoli cells. For the coimmunoprecipitation experiment, an antibody against paxillin, a protein involved in spermatid-Sertoli cell junctional complexes, was used. More components of the paxillin protein complex were visible by fluorescence detection of SAED-labeled proteins than were visible on comparable silver-stained gels. Mass spectrometric analysis of the fluorescently labeled proteins identified integrin alpha6 precursor as a protein associated in a complex with paxillin. The identification of integrin alpha6 precursor was confirmed by Western blot analysis and verifies the applicability of this novel approach for identifying proteins involved in protein complexes.  相似文献   

5.
Complex copolymers are heated to slowly increasing temperatures on a direct probe (DP) inside the plasma of the atmospheric pressure chemical ionization (APCI) source of a quadrupole ion trap. Slow heating allows for temporal separation of the thermal degradation products according to the stabilities of the bonds being cleaved. The products released from the DP are identified in situ by APCI mass spectrometry and tandem mass spectrometry. DP-APCI experiments on amphiphilic copolymers provide conclusive information about the nature of the hydrophobic and hydrophilic components present and can readily distinguish between copolymers with different comonomer compositions as well as between cross-linked copolymers and copolymer blends with similar physical properties. The dependence of DP-APCI mass spectra on temperature additionally reveals information about the thermal stability of the different domains within a copolymer.  相似文献   

6.
The ambient mass spectrometry technique, desorption electrospray ionization mass spectrometry (DESI-MS), is applied for the rapid identification and spatially resolved relative quantification of chlorophyll degradation products in complex senescent plant tissue matrixes. Polyfunctionalized nonfluorescent chlorophyll catabolites (NCCs), the "final" products of the chlorophyll degradation pathway, are detected directly from leaf tissues within seconds and structurally characterized by tandem mass spectrometry (MS/MS) and reactive-DESI experiments performed in situ. The sensitivity of DESI-MS analysis of these compounds from degreening leaves is enhanced by the introduction of an imprinting technique. Porous polytetrafluoroethylene (PTFE) is used as a substrate for imprinting the leaves, resulting in increased signal intensities compared with those obtained from direct leaf tissue analysis. This imprinting technique is used further to perform two-dimensional (2D) imaging mass spectrometry by DESI, producing well-resolved images of the spatial distribution of NCCs in senescent leaf tissues.  相似文献   

7.
The use of biomolecular interaction analysis mass spectrometry to selectively isolate, detect, and characterize epitope-tagged peptides present in total cell lysates is demonstrated. Epitope-tagged tryptic peptides were captured via affinity interactions with either chelated Ni2+ or monoclonal antibodies and detected using surface plasmon resonance biomolecular interaction analysis (SPR-BIA). After SPR-BIA the tagged peptides were either eluted from the biosensor chips for mass spectrometric analysis or analyzed directly from the biosensor chip using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF). Protein database searches were performed using the masses of the tagged tryptic peptides, resulting in identification of the protein into which the epitope tag was inserted. Detection limits for both SPR-BIA and MALDI-TOF were at the low-femtomole to subfemtomole level. The approach represents a (multiplexed) high-sensitivity chip-based technique capable of identifying epitope-tagged proteins as they are present in complex mixtures.  相似文献   

8.
Separation and mass spectrometric analysis of intact noncovalent protein-protein complexes from mixtures is described. Protein complexes were separated using isoelectric focusing in a capillary under native conditions. During the mobilization, molecular masses of the intact complexes were measured on-line (as they emerged from the capillary) using Fourier transform ion cyclotron resonance (FTICR) mass spectrometry. An FTICR "in-trap" ion cleanup procedure was necessary for some complexes to reduce high levels of adduction and to obtain accurate molecular mass measurements. Optimization of the conditions for analysis of different intact complexes is discussed. We have shown that either the intact noncovalent complexes or their constituent protein subunits can be detected by variation of sheath liquid (i.e., NH4OAc vs HOAc) added at the electrospray-mass spectrometer interface. Thus, two successive experiments permit a fast and efficient characterization of intact complex stoichiometry, the individual complex subunits and the possible presence of metal or other adducted species.  相似文献   

9.
One of the challenges in protein interaction studies with chemical cross-linking stems from the complexity of intra-, inter-, and dead-end cross-linked peptide mixtures. We have developed new cross-linkers to study protein-protein interactions with mass spectrometry to improve the ability to deal with this complexity. Even the accurate mass capabilities of FTICR-MS alone cannot unambiguously identify cross-linked peptides from cell-labeling experiments due to the complexity of these mixtures resultant from the enormous number of possible cross-linked species. We have developed novel cross-linkers that have unique fragmentation features in the gas phase. The characteristics of these cross-linkers combined with the accurate mass capability of FTICR-MS can help distinguish cross-linking reaction products and assign protein identities. These cross-linkers that we call protein interaction reporters (PIRs) have been constructed with two reactive groups attached through two bonds that can be preferentially cleaved by low-energy CID of the respective protonated precursor ions. After cleavage of the labile bonds, the middle part of the linker serves as a reporter ion to aid identification of cross-linked peptides. This report highlights three new PIRs with new features that have been developed to improve the efficiency of release of reporter ions. The new cross-linkers reported here were tuned with the addition of an affinity tag, a hydrophilic group, a photocleavable group, and new low-energy MS/MS cleavable bonds. This report presents our investigation of the MSMS fragmentation behavior of selected protonated ions of the new compounds. The comprehensive fragmentation of these PIRs and PIR-labeled cross-linked peptides with low-energy collisions and an example of electron capture dissociation in FTICR-MS is presented. These new cross-linkers will contribute to current systems biology research by allowing acquisition of global or large-scale data on protein-protein interactions.  相似文献   

10.
The composition, stoichiometry, and organization of protein complexes can be determined by collision-induced dissociation (CID) coupled to tandem mass spectrometry (MS/MS). The increased use of this approach in structural biology prompts a better understanding of the dissociation mechanism(s). Here we report a detailed investigation of the CID of two dodecameric, heat-stable and toroidally shaped complexes: heat shock protein 16.9 (HSP16.9) and stable protein 1 (SP-1). While HSP16.9 dissociates by sequential loss of unfolded monomers, SP-1 ejects not only monomers, but also its building blocks (dimers), and multiples thereof (tetramers and hexamers). Unexpectedly, the dissociation of SP-1 is strongly charge-dependent: loss of the building blocks increases with higher charge states of this complex. By combining MS/MS with ion mobility (IM-MS/MS), we have monitored the unfolding and dissociation events for these complexes in the gas phase. For HSP16.9 unfolding occurs at lower energies than the ejection of subunits, whereas for SP-1 unfolding and dissociation take place simultaneously. We consider these results in the light of the structural organization of HSP16.9 and SP-1 and hypothesize that SP-1 is unable to unfold extensively due to its particular quaternary structure and unusually high charge density. This investigation increases our understanding of the factors governing the CID of protein complexes and moves us closer to the goal of obtaining structural information on subunit interactions and packing from gas-phase experiments.  相似文献   

11.
A new method is presented to screen proteolytic mass maps of cross-linked protein complexes for the presence of cross-linked peptides and for the verification of proposed structures. On the basis of the incorporation of 18O from isotopically enriched water into the C-termini of proteolytic peptides, cross-linked peptides are readily distinguished in mass spectra by a characteristic 8 amu shift. This is due to the incorporation of two 18O atoms in each C-terminus, so that normal and surface-labeled peptides shift 4 amu and cross-linked peptides containing two C-termini will shift 8 amu compared with their unlabeled counterparts. The method is fast, sensitive, and reliable and can be combined with any available cross-linking reagent and a wide range of proteolytic agents. As proof of principle, we successfully applied the method to a complex of two DNA repair proteins (Rad18-Rad6) and identified the interaction domain.  相似文献   

12.
The interaction of proteins with polysaccharides represents a major and challenging topic in glycobiology, since such complexes mediate fundamental biological mechanisms. A new strategy based on the hyphenation of frontal analysis capillary electrophoresis (FACE) with electrospray ionization mass spectrometry (ESIMS) is reported for the characterization of protein/carbohydrate complexes. While most of the previously reported CE-MS experiments were performed using capillary electrophoresis in zone format, we report for the first time CE-MS experiments in which CE was performed in frontal analysis (FACE-MS). We showed that the frontal mode offered a better sensitivity than zone mode and was well suited for the CE-MS coupling. This FACE-MS coupling was applied to the analysis of the complex between antithrombin and the sulfated pentasaccharide reproducing the antithrombin-binding sequence in heparin. The mixture of coincubated antithrombin and heparin pentasaccharide was continuously injected into the capillary, and the electrophoretic separation of the free and bound forms of the protein was achieved. The intact noncovalent complex antithrombin/heparin pentasaccharide was detected on-line by ESIMS in positive ionization mode and in nondenaturing sheath liquid conditions. The complex stoichiometry was determined from the mass measurement of the complex. In addition, the characterization of the sulfated pentasaccharide ligand dissociated from the complex was performed in negative ionization mode using a denaturing sheath liquid, allowing the determination of its molecular mass and sulfation features. This FACE-ESIMS strategy opens the way to ligand fishing experiments performed on heterogeneous carbohydrate mixtures and subsequent characterization of specifically bound carbohydrates.  相似文献   

13.
In protein interaction analysis, one promising method to identify the involved proteins and to characterize interacting sites at the same time is the mass spectrometric analysis of enzymatic hydrolysates of covalently cross-linked complexes. While protein identification can be accomplished by the methodology developed for proteome analysis, the unequivocal detection and characterization of cross-linked sites remained involved without selection criteria for linked peptides in addition to mass. To provide such criteria, we incorporated cross-links with a distinct isotope pattern into the microtubule-destabilizing protein Op18/stathmin (Op18) and into complexes formed by Op18 with tubulin. The deuterium-labeled cross-linking reagents bis(sulfosuccinimidyl)-glutarate-d4, -pimelate-d4, and -sebacate-d4 were prepared together with their undeuterated counterparts and applied as a 1:1 mixture of the respective d0 and d4 isotopomers. The resulting d0/d4 isotope tags allowed a straightforward mass spectrometric detection of peptides carrying the linker even in complex enzymatic protein hydrolysates. In the structure elucidation of the linked peptides by MS/MS, the assignment of the linked amino acids was again greatly facilitated by the d0/d4 tag. By applying two cross-linkers with similar reactivity but different spacer length in parallel, even doublets with very low intensity could be assigned with high confidence in MS and MS/MS spectra. Since in the Op18-tubulin complexes only a limited number of peptides carried the linker, the identification of the involved proteins per se was not impeded, thus accomplishing both protein identification and characterization of interacting sites in the same experiment. This novel methodology allowed us to significantly refine the current view of the complex between Op18 and tubulin corroborating the tubulin "capping" activity of the N-terminal domain of Op18.  相似文献   

14.
MALDI (matrix-assisted laser desorption/ionization) imaging mass spectrometry (IMS) is a new technology that generates molecular profiles and two-dimensional ion density maps of peptide and protein signals directly from the surface of thin tissue sections. This allows specific information to be obtained on the relative abundance and spatial distribution of proteins. One important aspect of this is the opportunity to correlate these specific ion images with histological features observed by optical microscopy. To facilitate this, we have developed protocols that allow MALDI mass spectrometry imaging and optical microscopy to be performed on the same section. Key components of these protocols involve the use of conductive glass slides as sample support for the tissue sections and MS-friendly tissue staining protocols. We show the effectiveness of these with protein standards and with several types of tissue sections. Although stain-specific intensity variations occur, the overall protein pattern and spectrum quality remain consistent between stained and control tissue samples. Furthermore, imaging mass spectrometry experiments performed on stained sections showed good image quality with minimal delocalization of proteins resulting from the staining protocols.  相似文献   

15.
The development of rapid, global methods for monitoring states of protein phosphorylation would provide greater insight for understanding many fundamental biological processes. Current best practices use mass spectrometry (MS) to profile digests of purified proteins for evidence of phosphorylation. However, this approach is beset by inherent difficulties in both identifying phosphopeptides from within a complex mixture containing many other unmodified peptides and ionizing phosphopeptides in positive-ion MS. We have modified an approach that uses barium hydroxide to rapidly eliminate the phosphoryl group of serine and threonine modified amino acids, creating dehydroamino acids that are susceptible to nucleophilic derivatization. By derivatizing a protein digest with a mixture of two different alkanethiols, phosphopeptide-specific derivatives were readily distinguished by MS due to their characteristic ion-pair signature. The resulting tagged ion pairs accommodate simple and rapid screening for phosphopeptides in a protein digest, obviating the use of isotopically labeled samples for qualitative phosphopeptide detection. MALDI-MS is used in a first pass manner to detect derivatized phosphopeptides, while the remaining sample is available for tandem MS to reveal the site of derivatization and, thus, phosphorylation. We demonstrated the technique by identifying phosphopeptides from beta-casein and ovalbumin. The approach was further used to examine in vitro phosphorylation of recombinant human HSP22 by protein kinase C, revealing phosphorylation of Thr-63.  相似文献   

16.
Hemoglobin-based oxygen therapeutics are prepared by reaction of hemoglobin with cross-linking molecules and are utilized as blood substitutes. They can be used as doping agents to increase the oxygen-carrying capacity of hemoglobin. We have compared a glutaraldehyde-polymerized bovine hemoglobin (Oxyglobin, Biopure Corp.) with natural bovine hemoglobin by mass spectrometry in order to detect specific fragment ions of the cross-linked protein for further potential applications in doping control of human blood samples. HCl acid (6 N) hydrolysis was performed in parallel on both proteins. Hydrolysates were then analyzed by direct infusion electrospray mass spectrometry (ESIMS) using a triple quadrupole mass spectrometer. Confirmation and precision were obtained by LC-ESIMS(n) experiments performed on an ion trap mass spectrometer. Chromatographic and mass spectrometry data allowed detection of two potential Oxyglobin-specific ions--m/z 299 and 399--that were shown to lose a 159 u neutral fragment under collision-induced dissociation conditions. Thus, monitoring of constant neutral loss of 159 u on acid hydrolysates of human serum samples spiked with different amounts of Oxyglobin has proved to be an efficient screening method to specifically detect and identify Oxyglobin. LC-MS of the spiked serum sample hydrolysates enabled detection of Oxyglobin at a detection limit of 4 g x L(-1).  相似文献   

17.
Protein-protein interactions are key to function and regulation of many biological pathways. To facilitate characterization of protein-protein interactions using mass spectrometry, a new data acquisition/analysis pipeline was designed. The goal for this pipeline was to provide a generic strategy for identifying cross-linked peptides from single LC/MS/MS data sets, without using specialized cross-linkers or custom-written software. To achieve this, each peptide in the pair of cross-linked peptides was considered to be "post-translationally" modified with an unknown mass at an unknown amino acid. This allowed use of an open-modification search engine, Popitam, to interpret the tandem mass spectra of cross-linked peptides. False positives were reduced and database selectivity increased by acquiring precursors and fragments at high mass accuracy. Additionally, a high-charge-state-driven data acquisition scheme was utilized to enrich data sets for cross-linked peptides. This open-modification search based pipeline was shown to be useful for characterizing both chemical as well as native cross-links in proteins. The pipeline was validated by characterizing the known interactions in the chemically cross-linked CYP2E1-b5 complex. Utility of this method in identifying native cross-links was demonstrated by mapping disulfide bridges in RcsF, an outer membrane lipoprotein involved in Rcs phosphorelay.  相似文献   

18.
19.
A new methodology for distinguishing between specific and nonspecific protein-ligand complexes in nanoelectrospray ionization mass spectrometry (nanoES-MS) is described. The method involves the addition of an appropriate reference protein (P(ref)), which does not bind specifically to any of the solution components, to the nanoES solution containing the protein(s) and ligand(s) of interest. The occurrence of nonspecific protein-ligand binding is monitored by the appearance of nonspecific (P(ref) + ligand) complexes in the nanoES mass spectrum. Furthermore, the fraction of P(ref) undergoing nonspecific ligand binding provides a quantitative measure of the contribution of nonspecific binding to the measured intensities of protein and specific protein-ligand complexes. As a result, errors introduced into protein-ligand association constants, K(assoc), as determined with nanoES-MS, by nonspecific ligand binding can be corrected. The principal assumptions on which this methodology is based, namely, that the fraction of proteins and protein complexes that engage in nonspecific ligand binding during the nanoES process is determined by the number of free ligand molecules in the offspring droplets leading to gaseous ions and is independent of the size and structure of the protein or protein complex, are shown to be generally valid. The application of the method for the determination of K(assoc) for two protein-carbohydrate complexes, under conditions where nonspecific ligand binding is prevalent, is demonstrated.  相似文献   

20.
Cisplatin is a potent anticancer drug, which functions by cross-linking adjacent DNA guanine residues. However within 1 day of injection, 65-98% of the platinum in the blood plasma is protein-bound. It is generally accepted that cisplatin binds to methionine and histidine residues, but what is often underappreciated is that platinum from cisplatin has a 2+ charge and can form up to four bonds. Thus, it has the potential to function as a cross-linker. In this report, the cross-linking ability of cisplatin is demonstrated by Fourier transform ion cyclotron resonance (FTICR) mass spectrometry (MS) with the use of standard peptides, the 16.8 kDa protein calmodulin (CaM), but was unsuccessful for the 64 kDa protein hemoglobin. The high resolution and mass accuracy of FTICR MS along with the high degree of fragmentation of large peptides afforded by collisionally activated dissociation (CAD) and electron capture dissociation (ECD) are shown to be a valuable means of characterizing cross-linking sites. Cisplatin is different from current cross-linking reagents by targeting new functional groups, thioethers, and imidazoles groups, which provides complementarity with existing cross-linkers. In addition, platinum(II) inherently has two positive charges which enhance the detection of cross-linked products. Higher charge states not only promote the detection of cross-linking products with less purification but result in more comprehensive MS/MS fragmentation and can assist in the assignment of modification sites. Moreover, the unique isotopic pattern of platinum flags cross-linking products and modification sites by mass spectrometry.  相似文献   

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