ARTICLE pubs.acs.org/ac
Identification of c-Type Heme-Containing Peptides Using Nonactivated Immobilized Metal Affinity Chromatography Resin Enrichment and Higher-Energy Collisional Dissociation Haizhen Zhang, Feng Yang, Wei-Jun Qian, Roslyn N. Brown, Yuexi Wang, Eric D. Merkley, Jea H. Park, Matthew E. Monroe, Samuel O. Purvine, Ronald J. Moore, Liang Shi, James K. Fredrickson, Ljiljana Pasa-Tolic, Richard D. Smith, and Mary S. Lipton* Biological Sciences Division and Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, Washington 99352, United States
bS Supporting Information ABSTRACT: The c-type cytochromes play essential roles in many biological activities of both prokaryotic and eukaryotic cells, including electron transfer, enzyme catalysis, and induction of apoptosis. We report a novel enrichment strategy for identifying c-type hemecontaining peptides that uses nonactivated IMAC resin. The strategy demonstrated at least 7-fold enrichment for heme-containing peptides digested from a cytochrome c protein standard, and quantitative linear performance was also assessed for heme-containing peptide enrichment. Heme-containing peptides extracted from the periplasmic fraction of Shewanella oneidensis MR-1 were further identified using higher-energy collisional dissociation tandem mass spectrometry. The results demonstrated the applicability of this enrichment strategy to identify c-type heme-containing peptides from a highly complex biological sample and, at the same time, confirmed the periplasmic localization of heme-containing proteins during suboxic respiration activities of S. oneidensis MR-1.
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he c-type cytochromes (c-Cyt) exist in nearly all living organisms. The major biological function of c-Cyt is electron transfer in reactions associated with respiration.1,2 They also are involved in enzyme catalysis3,4 and inducing apoptosis that leads to programmed cell death.5 All c-Cyt contain at least one heme group (i.e., an iron atom located at the center of a porphyrin structure) that is covalently linked via thioether bonds to the cysteine side chains of the heme-binding motif, whose common sequence is “CXXCH”.6 8 In addition to the covalent linkage to cysteine residues, the iron ion of the heme group in the center of porphyrin is coordinated by two other amino acid side chains to form a hexacoordinate. For example, in the crystal structure of cytochrome c, an iron ion of the c-type heme group coordinates to a histidine side chain on one side of porphyrin and to an oxidized methionine side chain on the other side.9 Some bacteria, especially facultative and strict anaerobic organisms such as dissimilatory metal-reducing bacteria (DMRB) Shewanella, Geobacter, and Anaeromyxobacter and sulfate respiring bacteria Desulfovibrio possess large numbers of c-Cyt, many of which contain more than one heme, including one with 40 putative heme-binding motifs.10 14 Recent results show that the numbers of c-Cyt, especially those with multihemes, found in the bacteria are positively correlated to the extent of the respiratory versatility associated with these bacteria, which emphasizes the critical role of c-Cyt in bacterial respiration.14 These c-Cyt often r 2011 American Chemical Society
work together to form pathways to transfer electrons to different terminal acceptors. For example, the extracellular electron transfer pathway used by DMRB S. oneidensis MR-1 for reducing solid metal oxides contains at least four different multiheme c-Cyt that transfer electrons from the inner membrane through the periplasmic space and then across the outer membrane to metal oxides external to the bacterial cells.15 17 Identification and functional characterization of c-Cyt stands to advance our understanding of the electron transfer mechanisms underlying the diverse bacterial respiration capabilities, which has important implications for biogeochemical cycling of carbon and metals, biotransformation of contaminants, and bioenergy production. In spite of the widespread use of liquid chromatography mass spectrometry (LC MS)-based strategies for identifying and quantifying proteins/peptides,18,19 only a few c-type hemecontaining peptides have been identified, even from bacterial samples rich in c-Cyt.20 In LC tandem MS (LC MS/MS), the low number of c-type heme-containing peptide observations are due to the inherent low abundance, low ionization efficiency, as well as reduced peptide fragmentation during collision-induced dissociation (CID). Yang et al.21 characterized CID fragmentation Received: Accepted:
January 11, 2011 July 8, 2011
Published: July 08, 2011 7260
dx.doi.org/10.1021/ac2000829 | Anal. Chem. 2011, 83, 7260–7268
Analytical Chemistry behaviors of a trypsin-digested heme-containing peptide from cytochrome c that generated a singly charged heme ion in the tandem mass spectra. In an earlier study,22 c-type heme-containing peptides derived from other purified heme proteins were identified using LC MS/MS and SEQUEST analysis that included a dynamic modification of heme group neutral mass applied to cysteine residues. However, the confidence of some of the identification was quite low and extensive manual confirmation was required. To date, identification of c-type hemecontaining peptides from complex biological samples has not been reported, most likely because they are more difficult to observe than nonheme-containing peptides during LC MS/ MS analysis. Herein, we report a novel strategy for enriching c-type hemecontaining peptides prior to LC MS/MS. The enrichment strategy uses the nonactivated immobilized metal ion affinity based chromatography (IMAC) resin coated by an iminodiacetic acid (IDA) stationary phase on the POROS 20MC (polystyrenedivinylbenzene) matrix.23,24 In common practice, the POROS 20 MC resin is first activated with metal ions such as Fe3+ and Cu2+ (through chelation between the IDA group and metal ions) to enrich phosphorylated peptides and histidine-containing peptides/proteins, respectively.25,26 Because iron ions are inherent in c-type heme groups, we hypothesized that c-type hemecontaining peptides can be potentially enriched through the metal ion affinity mechanism of the POROS 20MC resin. In this study, we evaluated enrichment performance by enriching hemecontaining horse heart cytochrome c peptides spiked into bovine serum albumin digested peptides as well as enriching c-Cyt and OmcA purified from Shewanella oneidensis MR-1. Multiple fragmentation technologies were compared to optimize fragmentation of the heme-containing peptides, and higher-energy collisional dissociation (HCD)27 resulted in the best performance of heme-containing peptide identifications. Furthermore, the IMAC enrichment method coupled with HCD fragmentation was utilized to identify heme-containing peptides and proteins from a periplasmic fraction of S. oneidensis MR-1. Our results demonstrate the effective applications of the MS-based proteomics strategy for identifying heme-containing peptides from highly complex biological samples.
’ EXPERIMENTAL SECTION Chemicals and Materials. POROS 20MC was obtained from PerSeptive Biosystems (Framingham, MA). All other chemicals were purchased from Sigma (St. Louis, MO) unless otherwise noted. The purification of OmcA from S. oneidensis MR-1 is described elsewhere.28 Bacterial Culture and Periplasmic Fractionation. S. oneidensis MR-1 (ATCC 700550) was maintained in Luria Bertani (LB) broth or on LB plates containing 1.5% (wt/vol) agar. A volume of 5 mL of overnight starter cultures was diluted 1:100 into 30 mL of fresh LB broth and shaken overnight at 30 °C until cells were in the early stationary growth phase (OD600 1.0 1.2). At this stage, cells developed the reddish hue indicative of heme protein expression. In addition, periplasmic fractions from MR-1 cells grown under similar conditions showed multiple bands when stained for heme proteins using TMBZ-peroxide.29 The periplasmic fraction was prepared according to the method of Ross et al.30 with all centrifugation steps performed at 12 000g. The dilute periplasmic extract was concentrated using Ultrafree 0.5 Centrifugal Filter
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Units with 5 kDa molecular weight cutoff (Millipore Corp., Billerica, MA). Protein digest. Horse heart cytochrome c, bovine serum albumin (BSA), OmcA, and S. oneidensis MR-1 periplasmic proteins were denatured in 6 M urea and 5 mM dithiothreitol (DTT) at room temperature for 45 min and then diluted 10-fold using 100 mM ammonium bicarbonate solution. Sequencing grade modified trypsin (Promega, Madison, WI) was added at a ratio of 1:20 to digest proteins overnight at 37 °C. The digested samples were loaded onto a 1 mL SPE C18 column (Supelco, Bellefonte, PA) and washed with 4 mL of 0.1% trifluoroacetic acid (TFA)/5% acetonitrile (ACN). Peptides were eluted from the SPE column with 1 mL of 0.1% TFA/80% ACN and then lyophilized. The peptide concentration was measured using a BCA protein assay (Pierce, Rockford, IL). Optimization of IMAC Enrichment. After SPE cleanup and lyophilization, peptides were suspended in deionized water with a final pH range of 3.0 4.0. Horse heart cytochrome c and BSA digested peptides were mixed at a weight ratio of 1:40, and the peptide mixture was loaded onto 5 mg of nonactivated IMAC resin and incubated at room temperature for 1 h. The IMAC resin was then washed three times with 200 μL of aqueous buffer three times at a constant pH of 3.0 4.0 but with increasing percentages of ACN, i.e., 0%, 10%, and 20% to get rid of nonspecific binding peptides. Next, IMAC enrichment was performed using the same peptide mixture but with three different washing buffers that contained 10% ACN and with increasing pH ranges, i.e., 1.0 2.0, 3.0 4.0, and 6.0 7.0. After washing, the enriched heme-containing peptides were eluted by incubating the IMAC resin with the elution buffer consisting of 80% ACN and 0.1% trifluoroacetic acid (TFA) for 10 min. The eluted peptides were subjected to direct infusion analysis on a 12 T Fourier transform ion cyclotron resonance (FTICR) mass spectrometer (Bruker Daltonics, Billerica, MA). All other IMAC enrichments were performed using optimized washing conditions that utilized 10% acetonitrile aqueous buffer at a pH of 6.0 7.0. Quantitative Evaluation of c-Type Heme-Containing Peptide Enrichment. Six different amounts of cytochrome c tryptic digested peptides (0.1, 0.5, 1.0, 2.0, 5.0, 10.0 μg) were spiked into 100 μg of BSA tryptic peptides. The total peptide amount of the mixture was measured by BCA protein assay (Pierce, Rockford, IL), and the amount of heme-containing peptides was determined by UV absorbance intensity at 390 nm, the characteristic absorbance wavelength of the heme group, using a UV visible spectrophotometer (Agilent 8453, Santa Clara, CA). The ratios of heme-containing peptides to total peptides with and without IMAC enrichment were plotted against the spiked amount of cytochrome c peptides to demonstrate the quantitative linearity of IMAC enrichment. In addition to UV absorbance analysis, the peptide mixture after IMAC enrichment was subject to LC MS/MS analysis (three technical replicates) for hemecontaining peptide quantification. The peak area of identified heme-containing peptides were plotted against the original spiked amount of cytochrome c peptides to demonstrate the quantitative linearity of IMAC enrichment. The peak area of heme-containing peptides via LC MS/MS analysis were quantified using the MASIC program.31 Direct Infusion Analysis. Cytochrome c and BSA peptide mixtures were dissolved in electrospray solvent (50% ACN and 0.1% TFA) and infused into a modified Bruker 12 T FTICR mass spectrometer,32 equipped with a Triversa NanoMate 7261
dx.doi.org/10.1021/ac2000829 |Anal. Chem. 2011, 83, 7260–7268
Analytical Chemistry 100 nanoelectrospray ionization source (Advion BioSciences, Inc., Ithaca, NY). Mass spectra were acquired using the APEX Control program (Bruker Daltonics, Billerica, MA) with a 0.2 s external accumulation time in the hexapole to average 5 scan spectra. LC MS/MS analysis. Following IMAC enrichment, tryptic peptides from the cytochrome c BSA mixture, OmcA and S. oneidensis MR-1 periplasmic proteins were analyzed using LC MS/MS. The HPLC system consisted of a custom configuration of 65 mL Isco model 65D syringe pumps (Isco, Inc., Lincoln, NE), two-position Valco valves (Valco Instruments Co., Houston, TX), and a PAL autosampler (Leap Technologies, Carrboro, NC) that allowed for fully automated sample analysis across four separate HPLC columns.33 Reversed-phase capillary HPLC columns were manufactured in-house by slurry packing 3 μm Jupiter C18 stationary phase (Phenomenex, Torrence, CA) into a 60 cm length of 360 μm o.d. 75 μm i.d. fused silica capillary tubing (Polymicro Technologies Inc., Phoenix, AZ) that incorporated a 0.5 μm retaining screen in a 1/16 in. custom laserbored 75 μm i.d. union (screen and union, Valco Instruments Co., Houston, TX; laser bore, Lenox Laser, Glen Arm, MD). Mobile phases consisted of 0.1% formic acid in water (A) and 0.1% formic acid acetonitrile (B). The mobile phases were degassed using an in-line Degassex model DG4400 vacuum degasser (Phenomenex, Torrence, CA). The HPLC system was equilibrated at 10 kpsi with 100% mobile phase A, and then a mobile phase selection valve was switched 50 min after injection, which created a near-exponential gradient as mobile phase B displaced A in a 2.5 mL active mixer. A 40 cm length of 360 μm o.d. 15 μm i.d. fused silica tubing was used to split ∼17 μL/min of flow before it reached the injection valve (5 μL sample loop). The split flow controlled the gradient speed under conditions of constant pressure operation (10 kpsi). Flow through the capillary HPLC column when equilibrated to 100% mobile phase A was ∼500 nL/min. MS analysis was performed using a ThermoFinnigan LTQ-Velos Orbitrap mass spectrometer (Thermo Scientific, San Jose, CA) coupled with an electrospray ionization interface. Data were acquired for 100 min, beginning 65 min after sample injection (15 min into gradient). Orbitrap spectra (automated gain control (AGC) 1 106) were collected from 400 to 2000 m/z at a resolution of 100k followed by data dependent ion trap MS/MS spectra (AGC 1 104) of the 10 most abundant ions. Two different MS/MS modes were utilized for OmcA trptic digests analysis: (1) CID/HCD and (2) electron transfer dissociation (ETD)/HCD alternating activation on the same precursors. CID and HCD used normalized collision energy settings of 35 and 40, respectively. The activation time for ETD was 100 ms, and a dynamic exclusion time of 60 s was employed to discriminate against previously analyzed ions. Data Analysis. Heme-containing peptides from both OmcA and S. oneidensis MR-1 periplasmic proteins were identified using the SEQUEST algorithm (Thermo Scientific, San Jose, CA) to search the whole genome database of S. oneidensis MR-1, which consists of 4198 open reading frames.34 The parameter file of SEQUEST included dynamic modifications of the heme group neutral mass (615.169 Da) on cysteine and the oxidation mass shift (15.995 Da) on methionine. The heme-containing peptides were further filtered using the following stringent criteria: mass measurement accuracy 0.05, and Xcorr >1.5. The false discovery rate of peptide identifications determined by searching against a decoy database that contained both forward
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and reversed peptide sequences was estimated to be
