Chemoenzymatic approaches for streamlined detection of active site modifications on thiotemplate assembly lines using mass spectrometry

For the direct interrogation of peptides harboring covalently modified serines in nonribosomal peptide synthetases, streamlined methodologies described here employ proteolysis and reporter-coenzyme A analogues of four types. The chromophoric and fluorescent coenzyme A analogues pyrene-maleimidyl-S-CoA and BODIPY-FL-N-(2-aminoethyl)maleimidyl-S-CoA were enzymatically loaded onto the active site serines harbored in the ArCP, PCP1, and PCP2 thiolation domains of PchE and PchF, the nonribosomal peptide synthetases responsible for the biosynthesis of the siderophore pyochelin. During the chromatographic separation of cyanogen bromide digests, observation of the absorbance (at 338 and 504 nm) or fluorescence (after irradiation at 365 nm) enabled the selective detection of peptides containing each active site serine. This resulted in quick detection of each active site peptide by Fourier transform mass spectrometry in the fully reconstituted pyochelin system. The loading of short acyl chain reporters in equimolar quantities permitted further insights into digestion heterogeneity and side reactions by virtue of a mass shift signature on each active site peptide. The chromatographic shift of the reporter-loaded peptides relative to peptides carrying on pathway intermediates was 2 min at 7 kDa, providing a general strategy for efficient localization of "carrier" peptides in complex digests of thiotemplate enzymes. Also, the use of the affinity reporter, biotin-maleimidyl-S-coenzyme A, permitted the isolation of intact synthetases at high purity via removal of contaminating Escherichia coli proteins.     

Site-specific observation of acyl intermediate processing in thiotemplate biosynthesis by fourier transform mass spectrometry: the polyketide module of yersiniabactin synthetase

Complex arrays of thioester bound intermediates are present on 100-700 kDa enzymes during the biogenesis of diverse types of pharmacophores and natural product drugs. These multidomain enzymes, known as nonribosomal peptide synthetases and polyketide synthases (NRPSs and PKSs, respectively), synthesize from simple, physiologically available substrates bioactive compounds that can be further tailored by a host of modifying domains (e.g., methylation, cyclization, and epimerization) to increase the complexity of the mature final product. Interrogation of such covalent intermediates using mass spectrometry (MS) presents an underutilized method for understanding the covalent catalysis executed by NRPS and PKS enzymes. For the PKS module (205 kDa) from the yersiniabactin (Ybt) gene cluster of Yersinia pestis, limited proteolysis afforded a key 11 kDa peptide from the acyl-carrier protein (ACP) domain upon which at least five covalent intermediates could be detected (42, 70, 86, 330, and 358 Da). The isotopic resolution achieved by Fourier transform mass spectrometry (FTMS) allowed for the incorporation of substrates with stable isotopes to confirm the structural assignments of three intermediates (86, 330, and 358 Da) on the Ybt biosynthetic pathway to within 1 Da. Approximately 75% of the enzyme capacity is lost to unproductive decarboxylation of malonyl-S-ACP partly constraining the 1.4 min(-)(1) rate of Ybt production in vitro. Acyl transfer to the ACP domain (on the Ybt pathway) was promoted by a factor of approximately 10 over unproductive CO(2) loss in the presence of the cosubstrate S-adenosylmethionine (SAM), with S-adenosylhomocysteine unable to restore the condensation yield observed with SAM. The data are consistent with Claisen condensation from KS to the ACP carrier site being reversible, with the absence of downstream methylation providing more opportunity for unproductive CO(2) loss. Extension of such FTMS-based studies will allow the direct visualization of multiple intermediates in determining the catalytic order of events and kinetics of NRPS and PKS systems.     

Contemporary mass spectrometry for the direct detection of enzyme intermediates

The field of enzymology has long used small-molecule mass spectrometry. However, the direct interrogation of covalent and non-covalent intermediates by large-molecule mass spectrometry of enzymes or large peptide substrates is illuminating an increasingly diverse array of chemistries used in nature. Recent advances now allow improved detection of several modifications formed at sub-stoichiometric levels on the same polypeptide, and elucidation of intermediate dynamics with low millisecond temporal resolution. Highlighting recent applications in both ribosomal and non-ribosomal biosynthesis of natural products, along with acetyl transferases, sulfonucleotide reducatases, and PEP-utilizing enzymes, the utility of small- and large-molecule mass spectrometry to reveal enzyme intermediates and illuminate mechanism is described briefly. From ever more complex mixtures, mass spectrometry continues to evolve into a key technology for a larger number of today's enzymologists.     

Mutational analysis of peptidyl carrier protein and acyl carrier protein synthase unveils residues involved in protein-protein recognition

4'-Phosphopantetheinyl transferases (PPTases) are essential for the production of fatty acids by fatty acid synthases (primary metabolism) and natural products by nonribosomal peptide synthetases and polyketide synthases (secondary metabolism). These systems contain carrier proteins (CPs) for the covalent binding of reaction intermediates during synthesis. PPTases transfer the 4'-phosphopantetheine moiety from coenzyme A (CoA) onto conserved serine residues of the apo-CPs to convert them to their functionally active holo form. In bacteria, two types of PPTases exist that are evolutionary related but differ in their substrate spectrum. Acyl carrier protein synthases (AcpSs) recognize CPs from primary metabolism, whereas Sfp- (surfactin production-) type PPTases have a preference for CPs of secondary metabolism. Previous investigations showed that a peptidyl carrier protein (PCP) of secondary metabolism can be altered to serve as substrate for AcpS. We demonstrate here that a single mutation in PCP suffices for the modification of this CP by AcpS, and we have identified by mutational analysis several other PCP residues and two AcpS residues involved in substrate discrimination by this PPTase. These altered PCPs were still capable of serving their designated function in NRPS modules, and selective use of AcpS or Sfp leads to production of two different products by a trimodular NRPS.     

Facile detection of specific RNA-polypeptide interactions by MALDI-TOF mass spectrometry.

A simple method for the detection of specific RNA-polypeptide interactions using MALDI-TOF mass spectroscopy is described. Instead of direct observation of the RNA-polypeptide complex, we attempted the indirect observation of the binding event by focusing on the disappearance of the free polypeptide signal upon interaction with RNA. As a result, specific binding of the Rev-response element (RRE) RNA of the HIV with two RRE-binding peptide aptamers, DLA and RLA peptides, as well as the bacteriophage lambda boxB RNA with the lambda N peptide was observed. We also show that specific RNA-binding peptides can be identified from a mixture of peptides with varying RNA-binding affinity, showing that the method could be applied to high-throughput screening from simple peptide libraries. The method described in this study provides a quick and simple method for detecting specific RNA-polypeptide interactions that avoids difficulties associated with direct observation of RNA and RNA-polypeptide complexes, which may find various applications in the analysis of RNA-polypeptide interactions and in the identification of novel RNA-binding polypeptides. Copyright (c) 2008 European Peptide Society and John Wiley & Sons, Ltd.     

Determination of anandamide and other fatty acyl ethanolamides in human serum by electrospray tandem mass spectrometry

We developed a new selective liquid chromatography-electrospray ionization-tandem mass spectrometry method for the identification and quantification of anandamide (AEA), an endogenous cannabinoid receptor ligand, and other bioactive fatty acid ethanolamides (FAEs) in biological samples. Detection limit (0.025 pmol for AEA and 0.1 pmol for palmitoylethanolamide (PEA) and oleoylethanolamide (OEA)) and quantification limit (0.2 pmol for AEA and 0.4 pmol for OEA and PEA) were in the high fmol to low pmol range for all analytes. Linear correlations (r(2)=0.99) were observed in the calibration curves for standard AEA over the range of 0.025-25 pmol and for standard PEA and OEA over the range of 0.1-500 pmol. This method provides a time-saving and sensitive alternative to existing methods for the analysis of FAEs in biological samples.     

Structural elucidation of minor components of peptidyl antibiotic P168s (leucinostatins) by tandem mass spectrometry.

Tandem mass spectrometry with a four-sector type mass spectrometer was used to elucidate the structures of minor components of the peptidyl antibiotic P168s (leucinostatins). As N-terminal fragments, ions by B-type cleavage were dominant, while V-type cleavages were observed along with X, Y, and Z types as C-terminal ions. The V-type ions were predominant in the cleavages of the amino terminals of leucyl and hydroxyleucyl residues. The structures of several minor components could be deduced from the tandem mass spectra.     

Glycoproteomics based on tandem mass spectrometry of glycopeptides

Next to the identification of proteins and the determination of their expression levels, the analysis of post-translational modifications (PTM) is becoming an increasingly important aspect in proteomics. Here, we review mass spectrometric (MS) techniques for the study of protein glycosylation at the glycopeptide level. Enrichment and separation techniques for glycoproteins and glycopeptides from complex (glyco-)protein mixtures and digests are summarized. Various tandem MS (MS/MS) techniques for the analysis of glycopeptides are described and compared with respect to the information they provide on peptide sequence, glycan attachment site and glycan structure. Approaches using electrospray ionization and matrix-assisted laser desorption/ionization (MALDI) of glycopeptides are presented and the following fragmentation techniques in glycopeptide analysis are compared: collision-induced fragmentation on different types of instruments, metastable fragmentation after MALDI ionization, infrared multi-photon dissociation, electron-capture dissociation and electron-transfer dissociation. This review discusses the potential and limitations of tandem mass spectrometry of glycopeptides as a tool in structural glycoproteomics.     

Probing neuropeptide signaling at the organ and cellular domains via imaging mass spectrometry

Imaging mass spectrometry (IMS) has evolved to be a promising technology due to its ability to detect a broad mass range of molecular species and create density maps for selected compounds. It is currently one of the most useful techniques to determine the spatial distribution of neuropeptides in cells and tissues. Although IMS is conceptually simple, sample preparation steps, mass analyzers, and software suites are just a few of the factors that contribute to the successful design of a neuropeptide IMS experiment. This review provides a brief overview of IMS sampling protocols, instrumentation, data analysis tools, technological advancements and applications to neuropeptide localization in neurons and endocrine tissues. Future perspectives in this field are also provided, concluding that neuropeptide IMS would greatly facilitate studies of neuronal network and biomarker discovery.     

Top-down mass spectrometry on low-resolution instruments: characterization of phosphopantetheinylated carrier domains in polyketide and non-ribosomal biosynthetic pathways

Mass spectrometry (MS) is an important tool for studying non-ribosomal peptide, polyketide, and fatty acid biosynthesis. Here we describe a new approach using multi-stage tandem MS on a common ion trap instrument to obtain high-resolution measurements of the masses of substrates and intermediates bound to phosphopantetheinylated (holo) carrier proteins. In particular, we report the chemical formulas of 12 diagnostic MS(3) fragments of the phosphopantetheine moiety ejected from holo carrier proteins during MS(2). We demonstrate our method by observing the formation of holo-AcpC, a putative acyl carrier protein from Streptococcus agalactiae.     

High-throughput quantification of soy isoflavones in human and rodent blood using liquid chromatography with electrospray mass spectrometry and tandem mass spectrometry detection

Soy-containing foods and dietary supplements are widely consumed for putative health benefits (e.g., cancer chemoprevention, beneficial effects on serum lipids associated with cardiovascular health, reduction of osteoporosis, relief of menopausal symptoms). However, studies of soy isoflavones in experimental animals suggest possible adverse effects as well (e.g., enhancement of reproductive organ cancer, modulation of endocrine function, anti-thyroid effects). This paper describes the development and validation of a sensitive high throughput method for quantifying isoflavones in blood from experimental animal and human studies. Serum samples containing genistein, daidzein, and equol were processed using reverse phase solid-phase extraction in the 96-well format for subsequent LC-ES/MS/MS or LC-ES/MS analysis using isotope dilution in conjunction with labeled internal standards. The method was validated by repetitive analysis of spiked blank serum and the intra-day and inter-day accuracy (88-99%) and precision (relative standard deviations from 3 to 13%) of measurement determined. The lower limit of quantification for all isoflavones was approximately 0.005 micro M using MS/MS detection, and 0.03 micro M using MS for genistein and daidzein. The degree of method performance, with respect to throughput, sensitivity and selectivity, makes this approach practical for analysis of large sample sets generated from mechanistic animal studies and human clinical trials of soy isoflavones.     

Analysis of mammalian fatty acyl-coenzyme A species by mass spectrometry and tandem mass spectrometry

Acyl-CoAs are intermediates of numerous metabolic processes in eukaryotic cells, including beta-oxidation within mitochondria and peroxisomes, and the biosynthesis/remodeling of lipids (e.g. mono-, di-, and triglycerides, phospholipids and sphingolipids). Investigations of lipid metabolism have been advanced by the ability to quantitate acyl-CoA intermediates via liquid chromatography coupled to electrospray ionization-tandem mass spectrometric detection (LC-ESI-MS/MS), which is presently one of the most sensitive and specific analytical methods for both lipids and acyl-CoAs. This review of acyl-CoA analysis by mass spectrometry focuses on mammalian samples and long-chain analytes (i.e. palmitoyl-CoA), particularly reports of streamlined methodology, improved recovery, or expansion of the number of acyl chain-lengths amenable to quantitation.     

Characterization of synthetic peptide byproducts from cyclization reactions using on-line HPLC-ion spray and tandem mass spectrometry

Summary The cyclization of a linear dynorphin A (Dyn A) analogue to give the lactam derivative cyclo[d-Asp², Dap⁵]Dyn A(1–13)NH₂ (where Dap=,-diaminopropionic acid) was studied to evaluate the usefulness of different coupling reagents for side chain to side chain lactam formation. This cyclization proved to be difficult and yielded substantial byproducts that varied depending upon the activating reagent used. On-line HPLC-ion spray mass spectrometry was more practical and useful than conventional HPLC alone for characterizing the products of these cyclization reactions. Peptide byproducts could be identified from the series of multiply charged ions observed, even when some of these peptides eluted from the HPLC with similar retention times. In addition to the desired cyclic peptide, the peptide byproducts observed following the cyclization using BOP (benzotriazol-1-yl-oxy-tris(dimethylamino)phosphonium hexafluorophosphate) were the linear peptide, the cyclic dimeric peptide and the linear peptide resulting from aspartimide rearrangement. The peptide byproducts obtained following cyclization using HATU (O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate) and HAPyU (O-(7-azabenzotriazol-1-yl)-1,1,3,3-bis(tetramethylene)uronium hexafluorophosphate) were predominantly linear tetramethylguanidinium (Tmg) and dipyrrolidinylguanidinium (Dpg) derivatives resulting from alkylation of the side chain of Dap by HATU and HAPyU, respectively; in addition to monomeric guanidinium derivatives, dimeric and aspartimide-containing peptides were also produced. Peptide sequencing by ion spray tandem mass spectrometry was performed to confirm the structure of both pure peptides and peptide byproducts in the crude samples. A unique fragmentation for the ,-bond of the Dap side chain was demonstrated and could be used to identify linear peptide byproducts. The distinctive fragment ions from this cleavage were also observed for the peptides containing the Tmg and Dpg functionalities on the Dap side chain.     

Analysis of polyunsaturated aminophospholipid molecular species using isotope-tagged derivatives and tandem mass spectrometry/mass spectrometry/mass spectrometry

When aminophospholipids with only saturated and monounsaturated fatty acids esterified to the glycerol backbone were labeled with isotopically enriched N-methylpiperazine acetic acid N-hydroxysuccinimide ester reagents, it was found that they could be readily detected as N-methylpiperazine-amide-tagged aminophospholipids using a precursor scan of the stable isotope reporter ion (m/z 114-117) formed by tandem mass spectrometry/mass spectrometry. However, it was found in the current study that these precursor ion scans are not useful in determining the changes of aminophospholipids with polyunsaturated fatty acids (PUFAs) esterified to the glycerol backbone due to the presence of interfering ions in the reporter ion region. Therefore, a method was developed using tandem mass spectrometry/mass spectrometry/mass spectrometry (MS(3)) to obtain reporter ion ratios that were not distorted by interfering ions present in the collision-induced dissociation spectra of nontagged aminophospholipids with PUFAs. This new MS(3) method for N-methylpiperazine- amide-tagged aminophospholipids was used to examine the fate of diacyl, ether, or plasmalogen glycerophosphoethanolamine (GPEtn) species after exposure of human polymorphonuclear leukocytes to A23187 and granulocyte macrophage-colony-stimulating factor/formyl-methionyl-leucyl-phenylalanine stimuli, which can induce eicosanoid biosynthesis, to follow those GPEtn molecular species which were the source of arachidonic acid released. Upon stimulation of the human polymorphonuclear leukocyte, it was found that the abundant arachidonoyl GPEtn plasmalogen molecular species were uniquely reduced in relative content compared to ether or diacyl species and this subclass of GPEtn may be a source of the arachidonic acid converted to leukotrienes by the 5-lipoxygenase pathway activated in this cell.     

Structure of PA1221, a nonribosomal peptide synthetase containing adenylation and peptidyl carrier protein domains

Many bacteria use large modular enzymes for the synthesis of polyketide and peptide natural products. These multidomain enzymes contain integrated carrier domains that deliver bound substrates to multiple catalytic domains, requiring coordination of these chemical steps. Nonribosomal peptide synthetases (NRPSs) load amino acids onto carrier domains through the activity of an upstream adenylation domain. Our lab recently determined the structure of an engineered two-domain NRPS containing fused adenylation and carrier domains. This structure adopted a domain-swapped dimer that illustrated the interface between these two domains. To continue our investigation, we now examine PA1221, a natural two-domain protein from Pseudomonas aeruginosa. We have determined the amino acid specificity of this new enzyme and used domain specific mutations to demonstrate that loading the downstream carrier domain within a single protein molecule occurs more quickly than loading of a nonfused carrier domain intermolecularly. Finally, we have determined crystal structures of both apo- and holo-PA1221 proteins, the latter using a valine-adenosine vinylsulfonamide inhibitor that traps the adenylation domain-carrier domain interaction. The protein adopts an interface similar to that seen with the prior adenylation domain-carrier protein construct. A comparison of these structures with previous structures of multidomain NRPSs suggests that a large conformational change within the NRPS adenylation domains guides the carrier domain into the active site for thioester formation.     

Recognition of hybrid peptidyl carrier proteins/acyl carrier proteins in nonribosomal peptide synthetase modules by the 4'-phosphopantetheinyl transferases AcpS and Sfp

The acyl carrier proteins (ACPs) of fatty acid synthase and polyketide synthase as well as peptidyl carrier proteins (PCPs) of nonribosomal peptide synthetases are modified by 4'-phosphopantetheinyl transferases from inactive apo-enzymes to their active holo forms by transferring the 4'-phosphopantetheinyl moiety of coenzyme A to a conserved serine residue of the carrier protein. 4'-Phosphopantetheinyl transferases have been classified into two types; the AcpS type accepts ACPs of fatty acid synthase and some ACPs of type II polyketide synthase as substrates, whereas the Sfp type exhibits an extraordinarily broad substrate specificity. Based on the previously published co-crystal structure of Bacillus subtilis AcpS and ACP that provided detailed information about the interacting residues of the two proteins, we designed a novel hybrid PCP by replacing the Bacillus brevis TycC3-PCP helix 2 with the corresponding helix of B. subtilis ACP that contains the interacting residues. This was performed for the PCP domain as a single protein as well as for the TycA-PCP domain within the nonribosomal peptide synthetase module TycA from B. brevis. Both resulting proteins, designated hybrid PCP (hPCP) and hybrid TycA (hTycA), were modified in vivo during heterologous expression in Escherichia coli (hPCP, 51%; hTycA, 75%) and in vitro with AcpS as well as Sfp to 100%. The designated hTycA module contains two other domains: an adenylation domain (activating phenylalanine to Phe-AMP and afterward transferring the Phe to the PCP domain) and an epimerization domain (converting the PCP-bound l-Phe to d-Phe). We show here that the modified PCP domain of hTycA communicates with the adenylation domain and that the co-factor of holo-hPCP is loaded with Phe. However, communication between the hybrid PCP and the epimerization domain seems to be disabled. Nevertheless, hTycA is recognized by the next proline-activating elongation module TycB1 in vitro, and the dipeptide is formed and released as diketopiperazine.     

Noncovalent Shiga-like toxin assemblies: characterization by means of mass spectrometry and tandem mass spectrometry

Shiga-like toxin 1 (SLTx), produced by enterohemorrhagic strains of Escherichia coli (EHEC), belongs to a family of structurally and functionally related AB(5) protein toxins that are associated with human disease. EHEC infection often gives rise to hemolytic colitis, while toxin-induced kidney damage is one of the major causes of hemolytic uremic syndrome (HUS) and acute renal failure in children. As such, an understanding and analysis of the noncovalent interactions that maintain the quaternary structure of this toxin are fundamentally important since such interactions have significant biochemical and medical implications. This paper reports on the analysis of the noncovalent homopentameric complex of Shiga-like toxin B chain (SLTx-B(5)) using electrospray ionization (ESI) triple-quadrupole (QqQ) mass spectrometry (MS) and tandem mass spectrometry (MS/MS) and the analysis of the noncovalent hexameric holotoxin (SLTx-AB(5)) using ESI time-of-flight (TOF) MS. The triple-quadrupole analysis revealed highly charged monomer ions dissociate from the multiprotein complex to form dimer, trimer, and tetramer product ions, which were also seen to further dissociate. The ESI-TOFMS analysis of SLTx-AB(5) revealed the complex remained intact and was observed in the gas phase over a range of pHs. Theses findings demonstrate that the gas-phase structure observed for both the holotoxin and the isoloated B chains correlates well with the structures reported to exist in the solution phase for these proteins. Such analysis provides a rapid screening technique for assessing the noncovalent structure of this family of proteins and other structurally related toxins.