Improved proteomic analysis following trichloroacetic acid extraction of Bacillus anthracis spore proteins

Journal of Microbiological Methods 118 (2015) 18–24

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Journal of Microbiological Methods journal homepage: www.elsevier.com/locate/jmicmeth

Improved proteomic analysis following trichloroacetic acid extraction of Bacillus anthracis spore proteins Brooke L. Deatherage Kaiser ⁎, David S. Wunschel, Michael A. Sydor, Marvin G. Warner, Karen L. Wahl, Janine R. Hutchison Chemical and Biological Signature Science Group, National Security Directorate, Pacific Northwest National Laboratory, Richland, WA, USA

a r t i c l e

i n f o

Article history: Received 31 December 2014 Received in revised form 10 July 2015 Accepted 7 August 2015 Available online xxxx Keywords: Bacillus anthracis spores Protein extraction Proteomics Trichloroacetic acid

a b s t r a c t Proteomic analysis of bacterial samples provides valuable information about cellular responses and functions under different environmental pressures. Analysis of cellular proteins is dependent upon efficient extraction from bacterial samples, which can be challenging with increasing complexity and refractory characteristics. While no single method can recover 100% of the bacterial proteins, selected protocols can improve overall protein isolation, peptide recovery, or enrichment for certain classes of proteins. The method presented here is technically simple, does not require specialized equipment such as a mechanical disrupter, and is effective for protein extraction of the particularly challenging sample type of Bacillus anthracis Sterne spores. The ability of Trichloroacetic acid (TCA) extraction to isolate proteins from spores and enrich for spore-specific proteins was compared to the traditional mechanical disruption method of bead beating. TCA extraction improved the total average number of proteins identified within a sample as compared to bead beating (547 vs 495, respectively). Further, TCA extraction enriched for 270 spore proteins, including those typically identified by first isolating the spore coat and exosporium layers. Bead beating enriched for 156 spore proteins more typically identified from whole spore proteome analyses. The total average number of proteins identified was equal using TCA or bead beating for easily lysed samples, such as B. anthracis vegetative cells. As with all assays, supplemental methods such as implementation of an alternative preparation method may simplify sample preparation and provide additional insight to the protein biology of the organism being studied. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Proteomic analysis using liquid chromatography tandem mass spectrometry (LC–MS/MS) is a valuable tool to study the functional output of organisms. Applications of LC–MS/MS to characterize bacterial pathogens have increased our understanding of how pathogens respond to changes in environmental conditions. Bacillus anthracis, a Gram-positive, spore forming bacterium, is of particular interest due to its pathogenicity and potential use as a biological weapon (Centers for Disease Control and Prevention and National Institute for Occupational Safety and Health, 2012). Characterization of the global protein profile of B. anthracis, including the environmentally stable spore form, can provide insight into the cell life cycle as well as valuable information during a forensics investigation. B. anthracis is the etiological agent of anthrax, a zoonotic agent that can infect herbivores and occasionally humans (Dragon et al., 2005; Hugh-Jones and Blackburn, 2009; Turnbull, 2008). The life cycle of B. anthracis involves the dormant spore phase that transitions ⁎ Corresponding author at: Pacific Northwest National Laboratory, 902 Battelle Boulevard, P.O. Box 999, MSIN: P7-50, Richland, WA 99352, USA. E-mail address: [email protected] (B.L. Deatherage Kaiser).

http://dx.doi.org/10.1016/j.mimet.2015.08.008 0167-7012/© 2015 Elsevier B.V. All rights reserved.

into the metabolically active vegetative form in favorable conditions. B. anthracis spores persist in many environments, including black steppe soils rich in organic matter and calcium that are typically alkaline (Hugh-Jones and Blackburn, 2009). Once the vegetative form leaves the host, sporulation can be induced to continue the natural cycle. Proteomic analysis of B. anthracis vegetative cells and spores has provided insight in the areas of germination, sporulation, and cellular response to media conditions and environmental pressures (Huang et al., 2004; Jagtap et al., 2006; Lai et al., 2003; Redmond et al., 2004). While the vegetative form of B. anthracis is easily manipulated using established sample preparation techniques, many of these methods do not provide reliable and complete disruption of bacterial spores due to their robust physical structure. Many studies have used methods of mechanical disruption of spores, such as bead beating, followed by gelbased protein separation and purification before mass spectrometry (Bergman et al., 2006; Liu et al., 2004). More recent efforts to characterize the spore proteome have employed non-gel based preparation methods that rely on upstream chemical or mechanical disruption of the Bacillus spore. For example, Chenau et al. utilized trifluoroacetic acid (TFA) digestions of the spore to enrich for small acid soluble proteins (Chenau et al., 2011). This approach detected low numbers of spores, but also differentiated B. anthracis spores from other B. cereus

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group members. Finally, studies focused on identifying proteins found in the exosporium and/or spore coat layers have required initial separation of the outer layer of interest from the remaining inner spore mass, and subsequent proteome analysis of the cell fractions in isolation. Using these techniques, many coat- or exosporium-localized proteins have been identified, including alanine racemase, inosine hydrolase, ExsF, CotY, ExsY, CotB and a novel protein, designated ExsK (Abhyankar et al., 2013; Lai et al., 2003; Redmond et al., 2004). However, deeper proteome coverage and inclusion of proteins from particular layers is more labor intensive and requires multiple rounds of separation/extraction and mass spectrometric analysis to capture a more complete view of the spore proteome. While bead beating and/or sonication have been commonly used for spore protein extraction, there are downsides to these techniques. In addition to concerns of possible sample loss, sample abundance requirements, potential for aerosolization, and the relative labor-intensive nature of these mechanical disruption techniques, the spore structure is often not fully disrupted (especially without use of buffers containing detergent and/or denaturant), and abundant proteins from the outer layers of the spore may not be easily extracted (Thompson et al., 2011). We sought to evaluate two spore protein extraction methods, with the goal of increasing overall protein yield and/or spore protein extraction to facilitate increased protein identification via LC–MS/MS analysis. In this study, we applied either a mechanical or chemical disruption method to both B. anthracis vegetative cells and spores. The chosen mechanical disruption method was bead beating, as it is prevalent in the literature for disrupting biomass (Mehmeti et al., 2013; Thompson et al., 2011). For the chemical approach, we used Trichloroacetic acid (TCA) treatment, which is typically used to purify teichoic acids, precipitate proteins, and extract LPS from bacterial cells (Caldwell and Lattemann, 2004; Nandakumar et al., 2003; Norris and Ribbons, 1971; Shannon et al., 2005). To assess the performance of these two methods, the total number of proteins and number of spore-specific proteins identified by LC–MS/MS was compared following bead beating or TCA extraction. For vegetative cell mass, TCA extraction and bead beating yielded similar results. However, TCA extraction yielded more protein identifications from spore samples than bead beating, and facilitated the extraction of more spore coat and exosporium-localized proteins without upstream spore fractionation procedures, making it an attractive complementary procedure for broad spore proteome analyses. 2. Materials and methods

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to experiments by serial dilution in 0.1% peptone with plating on TSA at 37 °C. Vegetative cultures were made fresh for each experiment to avoid sporulation. For proteomic analysis, three independent spore and vegetative cultures were used for n = 3 biological reps.

2.2. Peptide sample preparation All chemicals were purchased from Sigma-Aldrich unless indicated otherwise. Vegetative cell and spore samples (B. anthracis Sterne) were prepared by both bead beating and TCA extraction (Fig. S1). A total of 1 x 10 8 CFU B. anthracis spores or vegetative cells were used for each method. For bead beating, the biomass was resuspended in 100 μL lysis buffer (6 M urea, 14.3 mM 2-Mercaptoethanol in 50 mM Tris–HCl), and an equivalent volume of 0.1 mm zirconia beads were added to an equal volume as the cell suspension. Tubes were secured into the MP FastPrep®-24 and homogenized for six 20 second pulses with a 15 second recovery period. Samples were centrifuged for 5 min at maximum speed to pellet the beads. The lysate was removed, transferred to a fresh tube, and incubated at 60 °C for one hour with shaking (400 rpm) as directed by manufacturer's recommendation for protein denaturation prior to enzyme digestion (see below; Promega Gold Trypsin Protocol). After incubation, 900 μL of 50 mM ammonium bicarbonate was added to the sample to dilute urea to b 1 M, and Promega Gold Trypsin (V5280) was added to a final ratio of between 1:100–1:20 as directed by the manufacturer protocol. Samples were incubated overnight (17 h) at 37 °C with shaking (150 rpm). Following trypsin digestion, samples were cleaned using C18 solid phase extraction (SPE) columns (Phenomenex) according to the manufacturer protocol. Cleaned peptide samples were brought to near dryness (Eppendorf Vacufuge plus), resuspended in 0.1% formic acid, and analyzed by the bicinchoninic acid (BCA; Pierce #23225) assay to determine peptide concentration. All samples were adjusted to 1 μg/μL prior to LC–MS/MS analysis. For TCA extraction, cell mass was pelleted and resuspended in 0.5 mL of 20% TCA in water, and incubated for 24 h at −20 °C. Samples were thawed, pelleted (16,000 ×g for 5 min), and washed twice in icecold acetone. Following drying in an Eppendorf Vacufuge plus to drive off excess acetone, pellets were resuspended in 100 μL lysis buffer (see above) and incubated at 60 °C for one hour with shaking (400 rpm). Samples were diluted with 50 mM ammonium bicarbonate, trypsin digested, and cleaned using SPE columns as described above. Each sample was adjusted to 1 μg/μL for LC–MS/MS analysis.

2.1. Bacterial strains, growth conditions, and spore preparation 2.3. Liquid chromatography mass spectrometry For spore preparations, a B. anthracis Sterne starter culture was grown for 16 h in tryptic soy broth without dextrose (TSB; BD 2862) at 37 °C with agitation at 200 rpm. The starter culture was diluted 1:10 in 0.1% peptone (BD; 211677) and 100 μL was plated onto bovine blood agar plates (Hardy Diagnostics A10). The culture was incubated for 7 days at 37 °C and spores were harvested by scraping from agar plates with sterile water. The spore suspension was transferred to a conical tube and centrifuged for 10 min at 5000 ×g. The supernatant was discarded and spores were resuspended in 30 mL sterile water prior to incubation at 4 °C for 7 days to promote vegetative cell lysis. Suspensions were then washed three additional times in sterile water. Spores were stored at 4 °C in water as per ASTM Designation E28900-11 (E54 Committee, 2011). Spores were visualized with phase contrast microscopy and were N 95% phase-bright prior to use. For vegetative stock cultures, a fresh colony was inoculated into 10 mL TSB in a 50 mL glass Erlenmeyer flask. The culture was incubated for 8 h at 37 °C with shaking at 200 rpm. Two mL aliquots were pelleted by centrifugation for 10 min at 16,000 ×g prior to washing three times in 0.1% peptone. Spore and vegetative cultures were enumerated prior

An Agilent 1200 HPLC with a 40 cm long 0.15 mm ID fused silica packed with Jupiter 5 μm C-18 resin was used to separate 1 μg of each peptide sample (each at 1 μg/μL concentration). The relative concentrations of two solutions, 5% acetonitrile, 0.1% formic acid in nano-pure H2O (solution A) and 95% acetonitrile, 0.1% formic acid in nano-pure H2O (solution B), were adjusted to establish the gradient. The flow rate was 2 μL per minute with the reversed phase gradient transitioning from 0% solution B to 45% solution B over the course of 60 min. A wash and a regeneration step were performed following this step. Eluate from the HPLC was directly ionized and transferred into the gas phase with an electrospray emitter (operated at 3.5 kV relative to the mass spectrometer interface). The ion transfer tube on the Orbitrap XL system (Thermo Electron, Thousand Oaks, CA) was maintained at 200 °C and 200 V, with an ion injection time set for automatic gain control with a maximum injection time of 200 ms for 5x107 charges in the linear ion trap. Dynamic parent ion selection was used, in which the top five most abundant ions were selected for MS/MS using a 3 m/z window. Each sample was analyzed in technical triplicate for these studies.

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B.L. Deatherage Kaiser et al. / Journal of Microbiological Methods 118 (2015) 18–24

2.4. Data processing and analysis Spectra were searched against the B. anthracis Sterne protein database using the open source search tool MS-GF+ and the downloaded genome from PATRIC (Kim et al., 2008; Wattam et al., 2014). Output from MS-GF + was filtered in Microsoft Access to identify confident peptide-spectrum matches using the Qvalue (≤ 0.001) to achieve a false discovery rate of ≤0.1%. Spectral counts were calculated for each protein and used as a measure of relative protein abundance. Additional filtering included requirements that: (1) the spectral count for any given observation was greater than one and (2) each protein was observed in at least 5 of 9 datasets in at least one condition of the four sample type/preparation method combinations. For example, if a protein was present in 5 of 9 bead-beat spore datasets but absent from all other datasets, it would pass filtering. However, if a protein was observed in 3 datasets of bead beat spore samples and 2 datasets of TCA extracted spore samples, it would not pass the filtering requirement. Statistical analysis (ANOVA) and visualization of data were performed in the freely downloadable program Inferno (https://code.google.com/ p/inferno4proteomics/), formerly known as DAnTE (Polpitiya et al., 2008). Differences in protein abundance were considered significant based on the following criteria: (1) p ≤ 0.01 and fold change of ≥ 1.5fold, or (2) present in ≥5 datasets (of 9) of one method and present in ≤1 dataset (of 9) of the other method (to account for presence/absence scenarios).

3.1. Preparation of B. anthracis spore and vegetative cell samples for proteomic analysis

Fig. 1. TCA extraction of spores increases the number of proteins identified by LC–MS/MS. Total numbers of proteins identified in each of 9 datasets per condition are plotted here. Statistical analysis revealed that while the preparation method did not affect the number of proteins identified from vegetative cell samples (p = 0.178), preparation method did significantly influence spore protein identification (p = 0.001). Each black circle represents an individual dataset; red bars represent the average as noted in Table 1. VEG = vegetative cell sample, SPORE = spore sample, BEAT = bead beating method, and TCA = TCA extraction method. A two-tailed t-test was used to calculate p-values. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

In order to determine the influence of protein extraction methods in the downstream analysis of B. anthracis spores and vegetative cells, equal cell numbers of cells of each sample were subjected to one of two processing methods: (1) physical disruption via bead beating or (2) chemical disruption via Trichloroacetic acid (TCA). As described in Fig. S1 and in Materials and methods, equal numbers (CFU) of spores and vegetative cells from each biological replicate (n = 3) were aliquoted in duplicate. One vegetative cell sample and one spore sample underwent the physical disruption method, beginning with bead beating performed in the context of a denaturing and reducing lysis buffer (6 M urea and 14.3 mM 2-Mercaptoethanol in 50 mM Tris–HCl). Bead beating alone does not efficiently extract protein content from Bacillus spores (Thompson et al., 2011), but treating spores with a denaturing solution (8 M urea + 1–10% w/v 2-Mercaptoethanol) increases extraction efficiency and renders cells more sensitive to lysozyme (Gould and Hitchins, 1963; Somerville et al., 1970). Although SDS can increase protein extraction in combination with urea and 2Mercaptoethanol, detergents such as SDS are incompatible with downstream mass spectrometric analyses, and therefore detergents

were excluded from experiments described here. Following bead beating, the sample lysate was incubated for one hour at 60 °C, subjected to trypsin digestion and peptide clean-up, and analyzed by LC–MS/MS. The second aliquot of spores and vegetative cells underwent chemical extraction with TCA. TCA has been used to effectively extract teichoic acids in the Gram-positive cell surface and to precipitate proteins in solution (Caldwell and Lattemann, 2004; Nandakumar et al., 2003), and we hypothesized that it may promote recovery of proteins from the spore surface and underlying layers. Samples were first treated with 20% TCA at −20 °C for 24 h, followed with an acetone wash of the sample pellet. The acetone-washed sample then underwent the same downstream processes as described for the bead-beat sample (Fig. S1). Overall peptide yields from each sample are noted in Table 1. TCA extraction yielded the greatest total amount of peptide from all samples prior to LC–MS/MS analysis (column three; average μg peptide yield (SD)). For all LC–MS/MS analyses, normalization to the same amount of peptide was performed at the sample injection stage.

3. Results and discussion

Table 1 Summary of protein yields and identifications following each preparation method. Sample

Preparation method

Average μg peptide yield (SD)

Average number of proteins identified (SD; range)1

B. anthracis Sterne vegetative cells

Bead beat

77.5 (15.6) 154.4 (15.3) 24.3 (5.7) 37.8 (5.8)

497 (21: 445–515) 507 (6.7; 497–518) 495 (32; 435–530) 547 (21; 515–581)

TCA B. anthracis Sterne spores

Bead beat TCA

1

Number of proteins uniquely identified in sample/preparation 1 5 25 72

Averages, standard deviations (SD), and ranges were calculated from protein identifications in each of 9 datasets per sample/preparation method. See Fig. 1 for visualization of range.

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3.2. Comparison of overall spore- and vegetative cell-derived protein identifications following each sample preparation

3.3. Distinct spore protein profiles are extracted by TCA treatment and bead beating

Peptide samples were subjected to LC–MS/MS analysis and resulting raw data were filtered as described in Methods to remove lowconfidence identifications. All filter-passing protein identifications are provided in Table S1A. The overall number of filter-passing protein identifications for vegetative cell samples were comparable between preparation methods, with an average of 497 and 507 proteins observed in each bead beat and TCA-prepared dataset, respectively (Tables 1, S1). While preparation method did not seem to largely impact the overall number of protein identifications in vegetative cell samples, there was an effect of preparation method on spore samples. Spore samples prepared by bead beating yielded an average of 495 filter-passing protein identifications, while 547 proteins were observed, on average, following TCA extraction. Average values were calculated from nine individual datasets of each sample type/preparation method combination; the range of individual dataset values is also included in Table 1 and visually represented in Fig. 1. A two-tailed t-test determined that the number of protein identifications following the bead beating and TCA extraction of spores, but not vegetative cell samples, was statistically significantly different by this measure (p = 0.001). In addition, both of these methods were more effective than published TFA extraction (Chenau et al., 2011), as only 19 filter-passing proteins, on average, were identified following TFA extraction of an equal starting amount of spores in this study (data not shown; range 15–29). For spore samples, the TCA treatment method resulted in an increased number of protein identifications by LC–MS/MS analysis.

In addition to an increased number of protein identifications, we hypothesized that different extraction methods may result in unique protein profiles. To test this hypothesis, we calculated Pearson's correlation coefficients from all protein abundance data (Fig. 2). This correlation calculation confirmed that the vegetative cell proteome is largely unaffected by the preparation method (bottom right of the figure; significantly red with high correlation coefficients between bead beat and TCA-prepared vegetative cells), yet the spore proteome is more significantly impacted by preparation method (darker red color and lower correlation coefficients between the two spore preparations). Interestingly, the lowest correlation coefficients were calculated between vegetative cell samples and the TCA extracted spore samples, indicating that the TCA method likely extracts more spore-specific proteins not present in vegetative cell samples. Because the proteome recovery from spore samples was benefitted by using the TCA method as compared to vegetative cell samples, and spores are more refractory to protein extraction methods, we chose to focus on the protein enrichment in spore samples using these different methods. Fig. 3 displays the relative abundance of all spore protein observations. In this format, similarly expressed proteins cluster together on the vertical axis and datasets with similar protein expression patterns cluster together on the horizontal axis. The clustering pattern revealed the differing protein observation patterns in samples prepared by bead beating (red bar across the top of the figure) and TCA prepared samples (blue bar across the top of the figure). While some proteins

Fig. 2. Correlation coefficients demonstrate that preparation method does not greatly impact B. anthracis vegetative cell proteome, but B. anthracis spore samples are more significantly affected. Heat map representation of Pearson's correlation coefficients, with bright red being most highly similar, black being somewhat similar, and green being most highly dissimilar (no green appears on this heat map). The protein profiles of vegetative cell samples prepared by TCA (Veg_TCA) and bead beating (Veg_BEAT) are highly similar. Protein profiles of spore samples prepared by TCA (Spores_TCA) are less similar to spore samples prepared by bead beating (Spores_BEAT), as shown by the darker red to black. Each block includes 3 biological replicates and 3 technical replicates of each sample type. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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Fig. 3. Protein profiles of B. anthracis Sterne spore samples prepared by bead beating and TCA extraction demonstrate significant differences. Relative protein abundances following bead beating (highlighted in red along the top of the figure) and TCA extraction (highlighted in blue along the top of the figure) are shown here, with red being highly observed and green being least observed in the heat map. Gray represents a protein not observed in the sample. Proteins are represented along the vertical axis, and samples represented along the horizontal axis. Note that samples prepared with the same method cluster together (top of the figure). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

were observed in both conditions (often with varying abundance), other proteins were observed exclusively following one preparation condition. 3.4. Enrichment of B. anthracis spore-specific proteins following TCA extraction No single sample preparation method can isolate and identify the entire complement of proteins within a complex sample like a microorganism. In addition to the complexity of the protein composition, the physical characteristics of a sample can also influence the ease or difficulty of protein extraction. Bacterial spores are particularly refractory

sample types. Successful enrichment of proteins from the outer layers (exosporium and spore coat) has been achieved by physical removal of these layers with a French pressure cell and/or sonication, and subsequent analyses of each pre-separated fraction (Abhyankar et al., 2013; Lai et al., 2003; Redmond et al., 2004). While effective, these techniques are time consuming, labor intensive, and do not provide information about protein content of more internal layers of the spore structure in the same assay. If proteins from all spore layers could be efficiently extracted using the same process, implementing this method would easily add value to current proteomic analyses. Due to the increased protein identifications following TCA extraction of spore samples, we hypothesized that spore-specific proteins may be enriched in these samples. We approached testing this hypothesis in two ways. First, a statistical analysis was performed to identify proteins statistically enriched following each extraction method. Secondly, in order to determine whether there was any enrichment in spore-specific proteins, the lists of enriched proteins in this study were compared to published proteomes of whole spores, spore coat fractions, exosporium fractions, or combined spore coat and exosporium fractions. Importantly, as aliquots from the exact same samples were removed and processed by each selected method in this study, differences in protein abundance can be attributed to the influence of downstream sample preparation and not upstream biological influences such as different growth medium or temperature. To determine if the protein extraction method influenced the protein profile of spore samples, an ANOVA was performed that compared the nine bead-beat samples to the nine TCA-extracted samples. Across all 18 spore sample datasets, a total of 763 filter-passing proteins were identified. Of that total, 337 proteins were observed at equal relative abundances following either preparation method, and 426 proteins were determined to be significantly different based on the criteria of either (1) a p-value ≤ 0.01 and a fold-change difference of ≥ 1.5, or (2) present in ≥ 5 datasets of one condition and present in ≤1 dataset of the other method (Table S1B). These criteria allowed for consideration of proteins that were frequently observed in one condition and absent or nearly absent in the other condition, as they are otherwise excluded from ANOVA testing. In total, bead-beat and TCA-extracted spore samples were enriched for 156 and 270 proteins, respectively. With the knowledge that certain proteins were more easily harvested by one preparation method over the other, we asked whether there was any preferential enrichment of proteins previously found to be in Bacillus spores by comparing the enriched protein lists to published spore proteomes. Six published investigations of spore protein content were considered, which included three analyses of the whole spore proteome of either B. anthracis (Huang et al., 2004; Jagtap et al., 2006) or B. subtilis (which lacks the exosporium layer) (Mao et al., 2011), one analysis of the spore coat proteome of B. subtilis (Lai et al., 2003), one analysis of the exosporium layer of B. anthracis (Redmond et al.,

Table 2 Proteins previously identified as coat and/or exosporium-specific proteins are enriched in TCA-extracted spore samples. Reference (PMID)

Species

Cell fraction

Total # proteins identified in paper

# Proteins identified in this study from each paper

% Spore-specific1 proteins observed in this study

% Spore-specific1 proteins equally represented following either extraction method

% Spore-specific1 proteins enriched in bead beat samples

% Spore-specific1 proteins enriched in TCA extracted samples

16927434 15352240 21667307 12562816 14766913 23998435

Bacillus anthracis Bacillus anthracis Bacillus subtilis Bacillus subtilis Bacillus anthracis Bacillus cereus

WS WS WS SC EX SC & EX

189 36 71 53 7 100

141 26 37 22 6 47

75% 72% 52% 42% 86% 47%

64/141 = 45% 11/26 = 42% 13/37 = 35% 5/22 = 23% 0/6 = 0% 9/47 = 19%

59/141 = 42% 13/26 = 50% 11/37 = 30% 7/22 = 32% 0/6 = 0% 13/47 = 28%

18/141 = 13% 2/26 = 8% 13/37 = 35% 10/22 = 46% 6/6 = 100% 25/47 = 53%

1 Six published spore proteome studies were compared to data generated in the current study to examine whether either protein preparation method preferentially enriched for a subset of known spore proteins. Between 42–86% of published spore proteins were identified in this study (columns 4–6). Proteins identified by studies of whole spores were largely either equally represented in our datasets or found enriched in bead-beat samples. Conversely, proteins of the spore coat and exosporium layers were found to be enriched in TCA-extracted samples. Gray shading represents the sample extraction method that most mimics data for each publication (see text for details). WS = whole spore; SC = spore coat; EX = exosporium.

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2004), and one analysis of coat and exosporium layers of B. cereus (Abhyankar et al., 2013). The three publications that examined coat and/or exosporium utilized techniques that removed/extracted the layer of interest prior to analysis (separate from the remaining spore mass). In total, our datasets identified between 42–86% of proteins in these publications (Table 2, column 6; Table S1B). To determine whether these spore-specific proteins were enriched by bead beating or TCA extraction, we determined how many spore-specific proteins from each publication were found to be: (1) equally represented by either method, (2) enriched in bead beat samples, or (3) enriched in TCA extracted samples in our datasets (Table 2, columns 7–9). For any given publication, between 0–45% of proteins were equally observed following either preparation method (Table 2, column 7). However, there were some interesting trends identified among those proteins enriched by each of the methods presented herein. Sporespecific proteins identified in publications that examined whole Bacillus spore samples (Table 2, rows 1–3) were more likely to be either equally identified by either method (Table 2 column 7) or more enriched in bead beat samples (Table 2, column 8) (Huang et al., 2004; Jagtap et al., 2006; Mao et al., 2011). The exception is the examination of whole B. subtilis spores (Table 2, row 3). B. subtilis spore-specific proteins were more evenly distributed into bead beat-enriched, TCAenriched, and equally represented categories, an effect that may be due to the absence of an exosporium layer in this organism. Interestingly, spore-specific proteins that had previously been identified in isolated spore coat or exosporium fractions were enriched in TCA-extracted samples (Abhyankar et al., 2013; Lai et al., 2003; Redmond et al., 2004), regardless of the Bacillus species analyzed (Table 2, rows 4–6). In many cases, these coat/exosporium proteins were not observed at all in bead beat samples. These data suggest that an added benefit of TCA extraction is the drawing out of proteins from multiple physical layers of the spore structure (including spore coat, exosporium, and inner layers), thus alleviating the need for additional fractionation steps to provide a more complete spore proteome profile. We propose this approach as a complementary, straightforward method to enrich proteins from normally difficult samples such as spores.

4. Concluding remarks Proteomic analysis provides a valuable tool to better understand functional aspects of microbial life. A key influence on the quality of data and completeness of a bacterial proteome is the method used for cell lysis and protein extraction. In general, the approaches can be divided into two groups: mechanical disruption such as bead beating, or chemical treatment such as TCA extraction. In this study, we compared the overall peptide yields, number of proteins identified, and the number of spore-specific proteins identified from B. anthracis Sterne spores using bead beating or TCA extraction. While TCA extraction and bead beating were comparable for vegetative B. anthracis samples, TCA extraction resulted in the isolation of more peptide mass and identification of more spore-specific proteins as compared to bead beating. Interestingly, spore samples that underwent TCA extraction also demonstrated enrichment in spore coat and exosporium proteins. This is not to say that coat and exosporium proteins were the only subset of proteins observed following TCA extraction. On the contrary, the total number of proteins identified following this method was comparable with traditional bead beating, and there was significant overlap in the proteins observed following each method, which included proteins identified from the internal spore structure. While no single sample preparation method will recover 100% of the proteins in a bacterial cell sample, TCA extraction was able to extract a high number of proteins from multiple spore layers that previously were only enriched when spores were physically fractionated prior to analysis. Expanding the breadth of proteome coverage through the use of the experimentally simple TCA extraction method

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represents an attractive alternative or complementary strategy for sample processing of refractory spore samples. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.mimet.2015.08.008.

Acknowledgments The authors would like to thank Dr. Alvin Fox for helpful discussions. We would also like to thank Charlie Doll for his instrumentation expertise. Funding for this work was provided the Laboratory Directed Research and Development Program at Pacific Northwest National Laboratory, a multiprogram national laboratory operated by Battelle for the U.S. Department of Energy. Battelle Memorial Institute operates Pacific Northwest National Laboratory for the U.S. DOE under Contract DE-AC06-76RLO.

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