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. Author manuscript; available in PMC: 2009 May 15.
Published in final edited form as: Bioorg Med Chem Lett. 2007 Nov 1;18(10):3107–3111. doi: 10.1016/j.bmcl.2007.10.104

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

Dario Meluzzi c, Wei Hao Zheng c, Mary Hensler b, Victor Nizet a,d, Pieter C Dorrestein a,b,c,*
PMCID: PMC2519147  NIHMSID: NIHMS52897  PMID: 18006314

Abstract

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 twelve diagnostic MS3 fragments of the phosphopantetheine moiety ejected from holo carrier proteins during MS2. We demonstrate our method by observing the formation of holo-AcpC, a putative acyl carrier protein from Streptococcus agalactiae.

The biosynthesis of polyketide synthase (PKS) and non-ribosomal peptide synthetase (NRPS) natural products, including the well-established therapeutic agents penicillin, vancomycin, and rapamycin, requires post-translational modification of carrier proteins by addition of a 4′-phosphopantetheine (PPant) arm. The sulfur at the free end of this moiety forms a thioester bond with substrates and intermediates at each step of the biosynthetic process. Because these substrates and intermediates increase the overall mass of the associated carrier proteins, the latter are ideal targets for “top-down” characterization of PKS and NRPS pathways by mass spectrometry (MS).1 A top-down approach was recently shown to facilitate the study of phosphopantetheinyl-tethered substrates.2 Thermal activation by infrared multiphoton dissociation (IRMPD) or collision-induced dissociation (CID) causes the holo form of acyl and peptidyl carrier proteins, which are found in PKS and NRPS pathways, respectively, to consistently “eject” their PPant arm, preserving the thioester linkage to the substrate (Figure 1a). This “PPant ejection assay” allows the mass of substrates loaded onto carrier proteins to be readily deduced from the mass of the corresponding PPant fragments.3 Specifically, when no substrate is linked to the PPant arm, the fragment ejected from the carrier protein has chemical formula C11H21N2O3S+, giving an MS2 peak at m/z 261.1267. When an acyl or peptidyl substrate is bound to the PPant arm, the PPant peak in the MS2 spectrum is shifted by an amount equal to the mass of the substrate less a water molecule.4

Figure 1.

Figure 1

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The MS3 phosphopantetheine fragmentation method. (a) MS2 experiments on holo-carrier proteins result in phosphopantetheine (PPant) ejection.2 (b) MS2 on the predicted 16+ charge state (m/z 796) of holo-AcpC produced a peak at m/z 261.2, which is consistent with the PPant fragment. (c) MS3 on this PPant fragment gave twelve peaks, which may be useful to confirm the mass of a substrate linked to the PPant arm. The masses shown in bold should be substrate-independent, because the corresponding proposed formulas do not include the sulfur atom necessary to bind the substrates.

The PPant ejection assay can greatly facilitate the characterization of NRPS and PKS systems by mass spectrometry, but demands instruments capable of both high sensitivity and high accuracy. Indeed, PPant ejection was originally observed on a custom-built Fourier transform ion cyclotron resonance (FTICR)-MS that utilized an accumulation octopole to collect ions before transmission to the FTICR mass analyzer.5 However, commercial FTICR-MS instruments may not produce peaks sufficiently intense for reliable detection of the ejected PPant species. Conversely, ion trap mass spectrometers, while typically more sensitive and capable of higher scan rates than FTICR instruments, do not always provide sufficient accuracy and signal-to-noise ratio to confirm the elemental composition of the PPant ejection peak.

These limitations became evident when we applied the PPant ejection assay on a Finnigan LTQ MS to characterize AcpC (accession no. NP 735098), a putative 12.4-kDa acyl carrier protein encoded by the fourth open reading frame in the cyl gene cluster of the bacterial pathogen Streptococcus agalactiae, or group B Streptococcus (GBS).6 The cyl gene cluster of GBS is involved in the production of the β-haemolytic/cytolytic activity that contributes to the virulence of GBS, a major cause of infant mortality in industrialized countries. Remarkably, the structure of the final molecule produced by the cyl gene cluster is still unknown.7 Because disruption of the acpC gene by transposon mutagenesis has suggested that AcpC is essetial for hemolytic phenotype of GBS,6 we decided to study this protein. To begin our investigation, we expressed an AcpC-His6 fusion construct in E. coli and enriched the protein by Ni-NTA affinity chromatography.16 The resulting sample was subjected to nano-electrospray ionization (ESI) and collision-induced dissociation (CID) of the predicted 16+ charge state (m/z 796) in the linear ion trap, yielding a clearly discernible peak at m/z 261.1 (Figure 1b).17 This peak matches the mass of the MS2 PPant fragment, suggesting that AcpC is a carrier protein and that AcpC can be phosphopantetheinylated by a PPant transferase (PPTase) naturally occurring in E. coli. However, the peak at m/z 261.1 was much weaker than the other peaks present in the MS2 spectrum and the accuracy of the ion trap mass analyzer was not sufficient for unequivocal assignment of this peak to the PPant fragment.

We therefore investigated whether subjecting the putative MS2 PPant fragment of holo-AcpC to another round of fragmentation would yield a more informative mass spectrum. Indeed, we observed a rich pattern of MS3 peaks that appeared to be uniquely associated with the ejected PPant arm (Figure 1c).18 We reasoned that this fragmentation pattern could be generally exploited as a diagnostic tool to detect the presence of carrier proteins bearing the PPant arm and to determine the mass of the PPant-linked species. Hence, we developed an alternative top-down method for the unambiguous characterization of substrates and intermediates bound to carrier proteins of PKS and NRPS biosynthetic pathways.

The likely molecular formulas for twelve of the observed MS3 PPant ions (Figure 1c) indicate that nine of the fragments, whose calculated m/z values are 132.0, 149.1, 159.1, 175.1, 205.1, 215.1, 225.1, 231.1, and 243.1, contain the PPant thiol group, which bears the substrate during the biosynthesis of natural products. Loading a substrate onto the PPant arm would then be expected to shift these thiol-containing MS3 fragments to higher masses, but should leave the mass of the other three MS3 fragments unchanged at the calculated m/z values of 142.1, 166.1, and 184.1. The latter MS3 ions provide a diagnostic pattern for detecting PPant ejection, while the nine thiol-containing MS3 ions can be used to infer the mass of the loaded substrate. The m/z value of the MS2 PPant peak resulting from fragmentation of a given carrier protein, as well as the m/z values of the MS3 peaks resulting from fragmentation of the PPant fragment itself, can all be calculated a priori from the theoretical masses listed in Figure 1c and from the mass of the given substrate.8

To evaluate our approach and to optimize the instrumental settings, CouN5, a 11.8-kDa peptidyl carrier protein from the coumermycin A1 NRPS biosynthetic pathway of Streptomyces rishiriensis DSM 40489,9 was used as a model system, because CouN5 is known to produce an abundant MS2 PPant fragment ion. Holo-CouN5 was prepared by incubation of apo-CouN519 with CoA and Sfp (accession no. P39135), a 26.1-kDa promiscuous PPTase from the surfactin biosynthetic pathway of Bacillus subtilis.10 The reaction products were ionized by nano-capillary liquid chromatography (LC)-ESI and analyzed with the linear ion trap. The predicted 15+ charge state (m/z 807) of holo-CouN5 and the PPant ion (m/z-261.1) were sequentially subjected to CID, resulting in MS3 peaks at m/z values of 132.1, 142.1, 149.0, 159.1, 166.1, 175.0, 184.1, 205.3, 215.0, 225.1, 231.2, and 243.2 (Figure 2a).20 These values agree with those observed for AcpC (Figure 1c). We verified the expected substrate-dependent mass shifts by performing MS3 experiments on CouN5 loaded with acetamide-S-CoA by Sfp, and by observing the expected +57-Da shift of the nine thiol-containing fragments (Figure 2b).21 Since a total of twelve MS3 PPant fragments must be present at m/z values predictable from the mass of the PPant-linked substrate, this mass can be determined with high confidence.

Figure 2.

Figure 2

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Confirming the diagnostic potential of the MS3 PPant fragments. (a) The MS3 spectrum holo-CouN5, with precursors at the 16+ charge state and at the PPant fragment, includes the same twelve peaks observed for holo-AcpC (Figure 1c). (b) The MS3 spectrum of acetamide-S-CouN5, with precursors at the 15+ charge state and at the PPant fragment, shows that nine of the diagnostic peaks are shifted by the mass of the linked substrate (57 Da).

We applied our approach to confirm the phosphopantetheinlylation of the acyl carrier protein AcpC after incubation with CoA in the presence or absence of Sfp. In both cases, analysis of the reaction products by nano-capillary LC-ESI and linear ion trap MS revealed charge state distributions consistent with the presence of both holo- and apo-AcpC. However, the presence of Sfp correlated with an increased proportion of holo-AcpC relative to apo-AcpC (Figure 3a). CID on the 16+ charge state (m/z 796) of Sfp-treated AcpC, followed by CID with precursor at m/z 261 yielded peaks at m/z 132.0, 142.1, 149.1, 159.0, 166.1, 175.1, 184.1, 205.1, 215.2, 225.1, 231.2, and 243.2 (Figure 3b), which agree with the pattern of MS3 ions observed for holo-CouN5 (Figure 1c).22 These results indicate that AcpC was phosphopantetheinylated by Sfp and, to a lesser extent, by an endogenous PPTase of E. coli. One such protein is the 14.1-kDa protein AcpS (accession no. P24224).11 To determine whether AcpS can phosphopantetheinylate AcpC, we incubated the latter with a BODIPY® FL-linked CoA derivative12 together with either Sfp or AcpS. SDS-PAGE analysis of both the Sfp and AcpS reaction products gave fluorescent bands consistent with the molecular weight of AcpC (11.4 kDa), indicating that both Sfp and AcpS can phosphopantetheinylate AcpC (Figure 4).23

Figure 3.

Figure 3

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Sfp and AcpS can phosphopantetheinylate AcpC. (a) Broadband MS spectra of AcpC, enriched after overexpression in E. coli and incubated with CoA in the absence (top) or presence (bottom) of Sfp, indicate that the amount of holo-AcpC increased relative to apo-AcpC when Sfp was present. (b) The MS3 spectra of the same samples shown in part (a), with precursors at the 16+ charge state and at the PPant fragment, show that holo-AcpC was present in both cases.

Figure 4.

Figure 4

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AcpC was fluorescently labeled by incubation with BODIPY-S-CoA (left) in the presence of Sfp (lane 1) or AcpS (lane 2). For comparison, apo-CouN5 was found to be labeled in the presence of Sfp (lane 4) but not in the presence of AcpS (lane 5).

Our investigations to date show that ion trap mass spectrometers capable of MS3 experiments can be used to recognize phosphopantetheinylated carrier proteins of PKS and NRPS pathways and to determine the masses of substrates bound to the holo form of such proteins. The results from our first application of the proposed method indicate that AcpC can be phosphopantetheinylated by two PPTases of rather different substrate specificity. Since expression of AcpC is required for production of hemolitic activity in GBS,6 our results suggest that formation of holo-AcpC may also be necessary. We propose that this post-translational modification may be accomplished by CylK (accession no. NP 735106), another product of the cyl gene cluster with very low homology to Sfp and CesP, the PPTase of Bacillus cereus.13 Interestingly, in-frame deletion of cylK abolishes most, but not all, of the hemolytic activity of GBS.14 Since we have shown that E. coli AcpS can phosphopantetheinylate AcpC, the residual hemolytic activity of the cylK deletion mutants may be due to phosphopantetheinylation of AcpC by a “borrowed” PPTase, such as the 4'-phosphopantetheinyl transferase of S. agalactiae NEM316 (accession no. NP 736164), a protein with 50% similarity to AcpS of E. coli and therefore likely involved in fatty acid biosynthesis.

In conclusion, our top-down approach based on multiple stages of tandem MS has the potential to facilitate the study of NRPS and PKS systems with low-resolution ion trap mass spectrometers. Possible applications of this method include the routine characterization of orphan gene clusters,15 the implementation of substrate screens, and the identification of phosphopantetheinylated proteins in a proteomic setting on LC time scales. This method may benefit other groups having access to affordable ion trap instruments but not to the costly FTICR-MS instruments currently used for top-down characterization of NRPS and PKS pathways.

Acknowledgments

We thank Dr. Christopher Walsh and Dr. Nathan A. Magarvey for their gift of a plasmid encoding AcpS-His6. We acknowledge support by the UCSD Skaggs School of Pharmacy and Pharmaceutical Sciences, by the Department of Chemistry and Biochemistry, by the NIH (Hemoglobin and Blood Protein training grant, no. 2T32DK07233), and by a U. S. Grant. Maintenance of our mass spectrometer was supported by the American Cancer Society (grant no. IRG-70-002-29).

Footnotes

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References and notes

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  • 17.A 4-µL sample of enriched AcpC was desalted with C4 ZipTip® pipette tips (Millipore) following the manufacturer’s protocol, diluted to 50 µL in 50:49:1 MeOH:H2O:AcOH, infused by nano-electrospray ionization with a Biversa Nanomate (Advion Biosystems, Ithaca, NY), and analyzed by MS2 (normalized collision energy: 13; width: 4; data type: profile; precursor: m/z 796) on a Finnigan LTQ-MS (Thermo-Electron Corporation, San Jose, CA) running Tune Plus software version 1.0 (Thermo). The final spectrum (Figure 1b) was obtained by averaging 298 MS2 scans with QualBrowser software version 1.4 SR1 (Thermo). The chemical formulas of the MS3 fragment ions listed in Figure 1c were confirmed by locating the corresponding ions in MS2 spectra of CoA, acquired with the Fourier transform ion cyclotron resonance (FTICR) mass analyzer portion of a Finnigan LTQ-FT MS.
  • 18.The MS3 scans of AcpC were collected and averaged as described for the MS2 scans of AcpC (precursor m/z: 261; normalized energy: 14; width: 2; microscans: 10; data type: profile; average: 114 scans).17
  • 19.The expression and purification of CouN5 have been described.25 To obtain holo-CouN5, 20 µL of 60 µM purified and desalted CouN5 were incubated for six hours at room temperature with 1 µL of 10 mM CoA (Aldrich), 1 µL of 150 µM Sfp (or 1 µL of 100 mM Tris-HCl pH 7.5, for the control reaction), 5 µL of 10 mM tris(2-carboxyethyl)-phosphine (TCEP) (Fluka BioChemika), and 1 µL of 100 mM MgCl2 (Teknova). Sfp was expressed and purified as described.10
  • 20.Nano-capillary columns were prepared by drawing a 360-µm O.D., 100-µm I.D. deactivated, fused silica tubing (Agilent) with a Model P-2000 laser puller (Sutter Instruments Co.) (Heat: 330, 325, 320; Vel: 45; Del: 125) and were packed at ~600 psi to a length of ~10 cm with C18 reverse-phase resin suspended in methanol. The column was equilibrated with 90% of solvent A (water, 0.1% AcOH) and loaded with 10 µL of a 1:20 dilution in water of the reaction mixture by flowing 90% of solvent A and 10% of solvent B (ACN, 0.1% AcOH) at 20 µL/min for 5 min, 15 µL/min for 3 min, and 10 µL/min for 12 min. Proteins were eluted at ~500 nL/min with a time-varying solvent mixture [break points (min.,% of solvent A): (20, 90), (23, 75), (43,10)] and directly electro-sprayed into the LTQ MS inlet (source voltage: 1.8 kV; capillary temperature: 180 °C). MSn scans of holo-CouN5 (Figure 2a) were acquired (normalized collision energy: 15, 16 for n = 2, 3; width: 10, 6 for n = 2, 3; data type: centroid; precursors: m/z 807.00, 261.17 for n = 2, 3) under the control of Xcalibur software version 1.4 SR1 (Thermo) and averaged (RT 32.22–32.39 min) using QualBrowser.
  • 21.Acetamide-S-CouN5 was prepared by incubating 20 µL of 60 µM CouN5 for six hours at room temperature with 2 µL of 5 mM acetamide-S-CoA, 1 µL of 150 µM Sfp, 5 µL of 10 mM TCEP, and 1 µL of 100 mM MgCl2. Acetamide-S-CoA was prepared by reacting 1 µL of 100 mM acetamide (Aldrich) in DMSO with 2 µL of 10 mM CoA in water for ~10 min, and then neutralizing exess acetamide with 1 µL of 100 mM β-mercaptoethanol in DMSO. MSn scans of Acetamide-S-CouN5 (Figure 2b) were acquired (MSn precursors: m/z 811.00, 318.15 for n = 2, 3) and averaged (RT 31.96–32.15 min) as described for holo-CouN5.20
  • 22.Holo-AcpC was prepared by incubating 200 µL of 30 µM enriched AcpC with 10 µL of 10 mM CoA, 4 µL of 10 mM TCEP, 4 µL of 100 mM MgCl2, and 5 µL of 150 µM Sfp for ~16 hours at room temperature. Broadband MS and MS3 scans of a 1:7 dilution of Sfp-treated AcpC in water (bottom of Figure 3) were acquired (MSn precursors: m/z 795.6, 261.13 for n = 2, 3) and averaged (RT 33.6–41.00 min) as described for holo-CouN5.20 Scans of untreated AcpC (top of Figure 3) were similarly acquired and averaged (RT 32.10–42.00 min).
  • 23.Preparation of BODIPY-S-CoA from CoA and BODIPY® FL N-(2-aminoethyl)maleimide (Invitrogen) was described.12 CouN5 and AcpC were separately incubated for one hour at room temperature with catalytic amounts of Sfp, AcpS, or Tris-HCl pH 7.6 buffer, in the presence of BODIPY-S-CoA and MgCl2. The products were analyzed by 16% SDS-PAGE (PAGEgel, Inc.) and the resulting bands were visualized by fluorescence imaging with an AlphaImager® HP (Alpha Innotech).
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