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Proteomic analysis of Vibrio metschnikovii under cold stress using a quadrupole Orbitrap mass spectrometer
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Research in Microbiology xx (2015) 1e8 www.elsevier.com/locate/resmic
Original article
Proteomic analysis of Vibrio metschnikovii under cold stress using a quadrupole Orbitrap mass spectrometer Juntao Jia a, Ying Chen b,*, Yinghui Jiang a, Zhengyi Li a, Liqing Zhao a, Jian Zhang a, Jing Tang a, Liping Feng a, Chengzhu Liang a, Biao Xu a, Peiming Gu c, Xiwen Ye a b
a Technological Center, Shandong Entry-Exit Inspection and Quarantine Bureau, 266002 Qingdao, China Research Institute for Food Safety, Chinese Academy of Inspection and Quarantine, No. A3, Road Gaobeidian, 100123 Beijing, China c Demo Center of Thermo Fisher Scientific Inc., 201206 Shanghai, China
Received 10 October 2014; accepted 17 July 2015 Available online ▪ ▪ ▪
Abstract Vibrio metschnikovii is a food-borne pathogen found in seafood worldwide. We studied the global proteome responses of V. metschnikovii under cold stress by nano-flow ultra-high-performance liquid chromatography coupled to a quadrupole Orbitrap mass spectrometer. A total of 2066 proteins were identified, among which 288 were significantly upregulated and 572 were downregulated. Functional categorization of these proteins revealed distinct differences between cold-stressed and control cells. Quantitative reverse transcription polymerase chain reaction analysis was also performed to determine the mRNA expression levels of seventeen cold stress-related genes. The results of this study should improve our understanding of the metabolic activities of cold-adapted bacteria and will facilitate a better systems-based understanding of V. metschnikovii. © 2015 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.
Keywords: Proteomics; Vibrio metschnikovii; Cold stress
1. Introduction Although Vibrio metschnikovii is present in a wide range of aquatic products and is associated with human diseases, this species has not been extensively studied and little published work is available [7,37]. Bacteria may encounter temperature downshifts (cold stress) during food processing and storage. This adverse condition may not only inhibit bacterial growth, but can also enhance virulence and resistance of the stressed bacteria [3,32,39,53], and even induce development of crossprotection against other stresses [15]. Therefore, cold stress potentially increases risks with regard to food safety. Reports on bacterial cold stress responses primarily concern Vibrio spp. [1,3,6,10,11,20,27,32,33,54,55] and species such as * Corresponding author. Tel./fax: þ86 010 85783587. E-mail address: [email protected] (Y. Chen).
Escherichia coli [25,39], Listeria monocytogenes [15,40,43] and Morganella morganii [58]. However, most published studies focus on morphological and physiological changes during cold stress, with few reports on the molecular responses of cold-stressed Vibrio spp. in general, and V. metschnikovii in particular. In addition, global comparative proteomic analysis of bacteria under cold stress remains poorly reported. Bacteria exposed to low temperatures during food processing and preservation are stressed for relatively long periods. Nearly all studies to date focused on the bacterial response to transient cold stress. However, a comprehensive understanding of the molecular response during adaptation and growth at low temperatures is vital if we are to design targeted processing and preservation strategies to effectively control pathogens [25]. In this study, we conducted a comparative proteomic analysis of V. metschnikovii exposed to cold stress for a prolonged duration. Proteomics provide microbial researchers with
http://dx.doi.org/10.1016/j.resmic.2015.07.011 0923-2508/© 2015 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved. Please cite this article in press as: Jia J, et al., Proteomic analysis of Vibrio metschnikovii under cold stress using a quadrupole Orbitrap mass spectrometer, Research in Microbiology (2015), http://dx.doi.org/10.1016/j.resmic.2015.07.011
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unparalleled insights into cellular processes and functions [17]. The techniques of proteomics, commonly two-dimensional electrophoresis and protein characterization, have been widely used for microbiological research. Despite continuing improvements in proteomics, little effort has been invested in microbial proteomics using nano-flow ultra-high performance liquid chromatography (nLC) coupled to a high resolution benchtop quadrupole Orbitrap mass spectrometer. This technique offers excellent run-to-run reproducibility, high sensitivity and the labor-saving capability of online sample processing. This study aimed to construct a novel and advantageous microbial proteomic technique and to study the proteome of V. metschnikovii exposed to prolonged cold stress. To the best of our knowledge, this work is the most comprehensive quantitative proteomics study on cold-stressed bacterial cells available to date.
CA, USA). Peptide samples of 10 ml, dissolved in 0.1% formic acid, were eluted with an acetonitrile gradient and 0.1% formic acid in 240 min at a flow rate of 250 nl/min.
2. Materials and methods
Raw files obtained from the Orbitrap Q-Exactive spectrometer were queried using Proteome Discoverer 1.3 (Thermo Fisher Scientific) against a database of V. metschnikovii from UniProt (taxon identifier 28172) containing 3079 proteins. The false discovery rate (FDR) of all peptide identifications was set at less than 1%. All proteins identified with at least two unique peptides were taken into consideration. To quantify the proteins, we used a label-free quantification approach based on the spectra counts [9]. Protein expression was considered significantly different only if protein ratios differed more than twofold [42] and the differential expressions were determined by chi-square test or Fisher's exact test when sample sizes were small.
2.1. Bacterial strain and culture V. metschnikovii F5-1 was isolated from the American lobster Homarus americanus, and identified in our laboratory [30]. Bacterial stock cultures were stored in physiological saline with 10% glycerol at 80 C, and cultured on tryptic soy agar with yeast extract (TSA-YE, Difco) slants at 37 C. 2.2. Cold treatment V. metschnikovii was subjected to cold stress by incubation at 4 C for 2 weeks. This temperature is often used to store food products [15]. Cultures incubated at 37 C for 12 h were used as control. Two biological replicates were prepared for proteomic analysis. 2.3. Sample preparation Bacterial culture samples were harvested from agar slants and weighed. About 0.3 g of bacterial culture was rinsed with 50 mmol/l TriseHCl (pH ¼ 7.4) three times and centrifuged to obtain bacterial pellets. Lysis buffer comprising 4% (w/v) sodium dodecyl sulfate (SDS), 100 mM Tris/HCl pH 7.6 and 0.1 M dithiothreitol (DTT) was added to the bacterial pellets, mixed and kept on ice. Cells were disrupted by intermittent sonic oscillation for 2 min and centrifuged at 12,000 g for 20 min at 4 C. The supernatants were subjected to insolution tryptic digestion as previously described [35]. Peptide concentrations were determined with the Bradford assay (Bio-Rad). Peptide solutions were stored frozen at 20 C. Two analytical replicates were prepared and analyzed. 2.4. Online nLC system Peptide solutions were separated by the nLC system (EasynLC 1000, Thermo Fisher Scientific, San Jose, CA, USA) equipped with a C18 column (Easy-spray column, C18, 2 mm, 100 Å, 75 mm 50 cm, Thermo Fisher Scientific, San Jose,
2.5. Mass spectrometric analysis Effluents of the online nLC were analyzed using a high resolution mass spectrometer (Orbitrap Q-Exactive, Thermo Fisher Scientific, Bremen, Germany). The Orbitrap Q-Exactive settings were as follows: spray voltage 2.4 kV, data-dependent scan mode for top-20 precursor ions with a resolution of 70,000@m/z 200 for the full MS scans from m/z 300 to 1800, and 17,500@m/z 200 for MS/MS scans generated with normalized collision energy of 27%. 2.6. Identification and quantification of proteins
2.7. Functional categorization Identified proteins were functionally categorized according to Gene Ontology (GO) annotation [2] at http://www.uniprot. org. GO annotation results were plotted by Web Gene Ontology (WEGO) annotation plotting [56]. 2.8. Quantitative analysis of mRNA levels mRNA expression levels of the selected genes were determined by quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) analysis using the 2 TDDC method as described by Livak et al. [34]. Significance of differential expression was determined by Student's t-test ( p < 0.05). Total RNAwas extracted using a High Pure RNA isolation kit (Roche, Mannheim, Germany) according to the manufacturer's instructions. Reverse transcription was performed using a Transcriptor First Strand cDNA synthesis kit (Roche, Mannheim, Germany). Reactions were incubated at 50 C for 60 min, terminated by heating at 85 C for 5 min followed by storage at 20 C. Primers for target genes in qRT-PCR were designed using Primer Premier 5.0 (Premier, Palo Alto, CA) based on the DNA sequence of V. metschnikovii CIP 69.14 in the UniProt database (http://www.uniprot.org/). Seventeen target genes were selected and amplified in triplicate using the LightCycler 480 System (Roche, Mannheim,
Please cite this article in press as: Jia J, et al., Proteomic analysis of Vibrio metschnikovii under cold stress using a quadrupole Orbitrap mass spectrometer, Research in Microbiology (2015), http://dx.doi.org/10.1016/j.resmic.2015.07.011
J. Jia et al. / Research in Microbiology xx (2015) 1e8
Germany), with 16S rRNA gene as internal control. cDNA corresponding to 0.5 mg of input RNAwas used as template. The 20 ml reaction mix contained 1 mM each of forward and reverse primers, 10 ml of SYBR Green Master (Roche, Mannheim, Germany), 2 ml of cDNA sample and 6 ml of RNase-free water. Thermocycling was programmed at 95 C for 5 min, followed by 45 cycles of denaturation at 95 C for 10 s, annealing at 60 C for 15 s and extension at 72 C for 20 s. Melting curves were also plotted to verify that a single target was amplified for each pair of primers. 3. Results and discussion
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4464) of the ‘cellular component’ domain, catalytic (GO:000 3824) and binding (GO:0005488) in ‘molecular function’ and metabolic process (GO:0008152) and cellular process (GO:0009987) in ‘biological process’. In total, 1941 and 2093 proteins were identified in the cold-stressed cells and the control, respectively. As shown in Fig. 1, both the number and percent of proteins in various categories did not differ significantly, except for a decrease in proteins in the categories pigmentation (GO:0043473), biological regulation (GO:0065007), transcription regulator activity (GO:0030528) and transporter activity (GO:0005215). We ignored categories with minor differences because of extreme caution in interpreting minor differences in functional classification [57].
3.1. Identification and quantification of proteins 3.3. Key proteins involved in response to cold stress A total of 2066 proteins were identified and quantified (Table S1). A comparison between cold-treated cells and control cells revealed an overlap of 1743 proteins. Among these mutual proteins, 288 were significantly upregulated and 572 were significantly downregulated. Compared with the control, the cold-stressed cells had increased quantities of cold shock proteins and ribosomal proteins, and decreased amounts of proteins involved in energy conversion and metabolism. Among the identified proteins, the most abundant included cold shock proteins, elongation factors, chaperone proteins, ribosomal proteins, outer membrane proteins and some proteins related to energy conversion and metabolism. 3.2. Functional categorization Using GO annotation [2,56], most proteins were classified into the categories cell (GO:0005623) and cell part (GO:004
Bacteria are frequently exposed to cold stress during food processing and preservation. To adapt to different environmental conditions, bacteria must alter their metabolism, cellular structure or both by inducing or eliminating some proteins [50]. Whereas cold shock response and adaption have been quite extensively studied in E. coli [8,13,23e25,29,36,39], the responses to cold stress by V. metschnikovii have not yet been examined. Reduced membrane fluidity and impaired protein synthesis are major problems resulting from cold shock [48]. Membrane fluidity is controlled by their fatty acid composition and maintaining protein synthesis relies on induced cold shock proteins (Csp), which initiate and facilitate ribosomal function at low temperatures. Cold shock proteins, especially those of the CspA family, are generally regarded as the proteins responding to an abrupt temperature downshift, and our results are in agreement
Fig. 1. Functional categorization of identified proteins according to the Gene Ontology Consortium classifications, visualized with WEGO. The vertical scales are logarithmic. Blue numbers on the right vertical axis refer to proteins from cold-stressed cells; black numbers refer to proteins from control cells. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) Please cite this article in press as: Jia J, et al., Proteomic analysis of Vibrio metschnikovii under cold stress using a quadrupole Orbitrap mass spectrometer, Research in Microbiology (2015), http://dx.doi.org/10.1016/j.resmic.2015.07.011
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with this (Table 1). A previous time course analysis of global gene expression of Vibrio vulnificus during cold stress also indicated induction of CspA [54]. Ironically, another V. vulnificus strain was observed to express csp1 (a gene homologous to cspA) constitutively when cells were incubated at 35 C and then subjected to 4 C for one week [31]. This inconsistency is likely due to heterogeneity of these genes despite their sequence similarity, and perhaps to the fact that transcription levels do not necessarily correlate with protein levels. Other proteins involved in cold responses include RecA [51], H-NS [13], GyrA [46], NusA [16], PNP [14], Hsc66 and Hsp70 (DnaK) [48], HscB [29], OtsA and OtsB [23] and TF [24]. However, Hsc66, HscB, OtsA and OtsB were not detected in this study. Among detected proteins, only the amount of PNP increased significantly in our study, and the levels of the other proteins remained unchanged (Table 1). In addition, three ribosome-associated proteins involved in translation are considered to be required for efficient ribosomal function at low temperatures [48]: IF-2 [18], CsdA [49] and RbfA [12]. In our experiments, cold stress dramatically (169/20) induced CsdA, which encodes a presumed ATPdependent RNA helicase [49] involved in the biogenesis of the 50S ribosomal subunit [8]. IF-2 and RbfA remained unchanged. A recent study revealed upregulation of CsdA in E. coli exposed to a low temperature of 14 C [25]. We assume that the V. metschnikovii cells in our study share a similar mechanism with E. coli to counteract the effect of low temperatures. Our assumption is based on the fact that elongation factors and proteins of the CspA family were expressed at high levels, and most of the related proteins functioning in the cold shock response, such as CsdA, were also present. Nevertheless, there were many differences between the two mechanisms, such as stable expression levels of cold shock proteins in our study (Table 1). Another case in point is the difference in the role of trehalose. According to a previous study [23], genes otsA and otsB, which encode trehalose biosynthetic enzymes, are induced and trehalose synthesis is activated during cold shock, which plays an important role in resistance
of E. coli (and probably other organisms) to low temperatures. However, in our study, we did not detect OtsA and OtsB in V. metschnikovii cells during their adaption to low temperatures. We propose that all these discrepancies result mainly from cold treatment conditions and partially from bacterial diversity and methods of proteomic analysis, including protein extract preparation, the mass spectrometer used and the set threshold of protein ratios (twofold in our study). As for the first reason, the sudden cold shock in most studies is different from our prolonged cold stress treatment, in which cells reached a final adapted and stabilized phase. We assume that V. metschnikovii cells entered this stabilized phase in our study. It is widely accepted that the bacterial response to prolonged periods of cold stress may be divided into different stages, as described in previous reviews [48,52]. Among others, these stages include the initial but transient cold shock response, which lasts a few hours, and the much longer restoration period during which cells are cold-adapted. Bacteria in a stress-adapted physiological state can re-establish disrupted homeostasis by resuming cellular functions such as DNA and protein synthesis, cell growth and cellular division [15]. In terms of food safety, bacteria in a stress-adapted physiological state presumably pose the greatest threat, because they are able to multiply in the food matrix and attain numbers high enough to cause infection upon consumption by susceptible individuals [15]. We therefore focused on longer periods of cold stress, because food processing and storage last more than a few hours. As mentioned above, ribosomal proteins are among the most abundant proteins. Cold stress significantly increased their expression (Table S1), illustrating their crucial role in the cold stress response. Low temperatures alter the structural integrity of ribosomal subunits [47]. This results in translational stalling and a transient decrease in polysome numbers, accompanied by an increase of single 70S ribosomes and 50S and 30S subunits [22]. Consistent with this, quantities of ribosomal proteins increased following cold shock in the present study. Genes associated with ribosomes were among the most strongly activated gene sets in response to hyperosmotic and
Table 1 Key proteins identified in this study involved in cold stress. Protein
Description
Protein ratios
Differential expression
CspA family RecA H-NS GyrA NusA PNP Hsp70 TF
Chaperone proteins Recombination and induction of the cold shock response Nucleoid-associated DNA-binding protein a-subunit of topoisomerase DNA gyrase Termination and antitermination of transcription Exoribonuclease Chaperone protein DnaK Trigger factor catalyzing cis/trans isomerization of peptide bonds N-terminal to the praline residue Binding of the initiation tRNA to the 30S subunit A member of the DEAD-box family of helicases 30S ribosomal binding factor trigging the cold shock response Elongation factor thermo-unstable Elongation factor thermostable
178/49 61/57 12/14 109/98 63/48 267/123 461/460 154/108
up unchanged unchanged unchanged unchanged up unchanged unchanged
185/98 169/20 14/10 522/370 316/245
unchanged up unchanged unchanged unchanged
IF-2 CsdA RbfA EF-Tu EF-Ts
Note: Protein ratio is based on the quantity of a protein extracted from bacterial cells under cold stress, divided by the control. Please cite this article in press as: Jia J, et al., Proteomic analysis of Vibrio metschnikovii under cold stress using a quadrupole Orbitrap mass spectrometer, Research in Microbiology (2015), http://dx.doi.org/10.1016/j.resmic.2015.07.011
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cold stress when L. monocytogenes strains were exposed to both stresses [15]. In our experiments, we observed that nearly all of 30S and 50S ribosomal proteins were upregulated, including those encoded by rplK, rplA, rpsD, rpmB and rpmE. These five genes consistently showed strong upregulation in all three strains of L. monocytogenes following independent adaptation to hyperosmotic and cold stress [15]. This was in agreement with our results acquired at mRNA expression levels. However, when V. vulnificus was exposed to a temperature drop of 35 Ce4 C within about an hour, no significant fold increases were found for expression of genes encoding 30S and 50S ribosomal proteins [54]. The discrepancy may be caused by the differences in cold stress duration. In contrast, some proteins related to energy conversion (such as pyruvate formate-lyase (C9P3U0), alcohol dehydrogenase (C9P3R8), succinate dehydrogenase flavoprotein subunit (C9P1Z0)), carbohydrate catabolism (such as phosphoglycerate kinase (C9P2N3), NAD-dependent glyceraldehyde-3-phosphate dehydrogenase (C9P3K7), pyruvate kinase (C9P2M6), triose-phosphate isomerase (C9P1X7), enolase (C9P1J8), fructose-bisphosphate aldolase class II (C9P2N2)), amino acid metabolism (such as aspartate ammonia-lyase (C9P1V3) and carbamate kinase (C9P1U8)) were distinctly downregulated (Table S1). This is consistent with the observations of Wood et al. [54]. In another study [15], overall downregulation of gene sets associated with carbohydrate transport and utilization were observed in cold- and osmoadapted cells, and gene sets associated with phosphotransferase systems were strongly suppressed in L. monocytogenes cells adapted to cold and salt stresses. In addition, a recent study [25] in which E. coli O157:H7 was stressed by an abrupt downshift from 35 C to 14 C also revealed repression of genes and proteins involved in DNA replication, protein synthesis, and carbohydrate catabolism. It is widely accepted that protein synthesis is impaired upon cold shock [48].
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In our study, proteins involved in translation such as elongation factors and initiation factor IF-2, chaperone proteins participating in protein folding and proteins involved in transcription such as DNA-directed RNA polymerase were found unchanged but highly expressed during cold stress. This indicates that V. metschnikovii entered a stabilized phase, distinct from the state upon cold shock. As in the case of elongation factors, we found that synthesis of elongation factor thermo-unstable (EF-Tu) and elongation factor thermostable (EF-Ts) remained unchanged. Both were produced at a high level during cold treatment, indicating that protein synthesis was maintained to resist cold stress. However, overexpression of EF-Tu in the outer membrane and underexpression of the EF-Ts in the cytoplasm was reported for an E. coli strain following cold stress adaption [39], and EF-Tu downregulation and EF-Ts upregulation were observed in cold-induced viable but non-culturable cells of Enterococcus faecalis [19]. It is well known that EF-Ts mediates regeneration of the EF-Tu-GDP complex into the active form, EF-TuGTP, in order to allow protein synthesis to continue for a limited time [19]. These observations were different from ours, showing that the response to cold stress differs from cold shock. Finally, it is noteworthy that we observed various differential expression levels of outer membrane proteins (OMPs) in this study. For example, OmpT was sharply downregulated, OmpU was upregulated and expression of other proteins, such as OmpA (C9P350), OmpK (C9P6G4) and Omp (C9P589) remained unchanged (Table S1). This may due to the functional complexity of outer membrane proteins. 3.4. Transcriptional analysis of selected target genes We used qRT-PCR for transcriptional analysis of the following genes (with their corresponding protein names in
Fig. 2. Ratios of relative expression levels of seventeen target genes involved in the cold stress response from cold-stressed and control V. metschnikovii cells. Gene expression was estimated using qRT-PCR and the comparative critical threshold (2DDC ) method. The 16S rRNA gene was used as internal control. The results in T this figure are the means of three independent experiments. Error bars denote standard deviations. Please cite this article in press as: Jia J, et al., Proteomic analysis of Vibrio metschnikovii under cold stress using a quadrupole Orbitrap mass spectrometer, Research in Microbiology (2015), http://dx.doi.org/10.1016/j.resmic.2015.07.011
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parentheses): cspA, recA, gyrA, nusA, pnp, tig (TF), infB (IF2), csdA, rplK, rplA, rpsD, rpmB, rpmE, tuf (EF-Tu), tsf (EFTs), ompT and ompU. The proteins encoded by these genes are key proteins involved in response to cold stress. Our results (Fig. 2) indicate that the mRNA expression levels of cspA, pnp, rplK, rplA, rpsD, rpmB, rpmE and ompU (significantly upregulated under cold stress) and the expression level of recA (unchanged under cold stress) correlated well with protein expression levels; for the remaining genes, protein expression levels were inconsistent with the corresponding transcription levels. The inconsistency is most likely due to the fact that mRNA and protein profiles are often poorly correlated [44], and perhaps also because of the different rules applied to determine whether differences in levels of protein expression (two-fold rule) and transcription (Student's t-test) were considered relevant. 3.5. Approaches of proteomic analysis Many bacterial proteomic analysis approaches have been developed. In the past, bacterial lysate digests were separated by two-dimensional polyacrylamide gel electrophoresis (2DE), and peptides were identified by MS including tandem MS methods such as matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) MS [21,28,41,45] and quadruple time-of-flight (Q-TOF) MS [26]. However, 2DE has at least two major limitations. First, 2DE protein spots on the gel must be visualized and excised prior to MS, which is inconvenient and inaccurate. Second, many important regulatory proteins are expressed at low levels, and their concentrations are often too low for effective analysis [4]. Recently, shotgun proteomics, which combines high performance liquid chromatography (HPLC) with MS, has made proteomic analysis automated and convenient. Shotgun proteomics based on a combination of nLC and MS made it possible to identify thousands of proteins in a single LC-MS analysis [38]. The results of the present study support the use of nLC coupled with Orbitrap for global discovery of microbial proteomes. In our study, we identified 2066 proteins, covering about twothirds (2066/3079) of all expressed proteins of V. metschnikovii listed in UniProt (taxon identifier 28172). In other reports on proteomic responses of bacteria to different stresses, the number of proteins identified was significantly lower than in our study. For example, a total of 1048 spots obtained from E. coli strains incubated at 4 C were resolved by 2DE and subsequent MALDI-TOF MS analysis [45]. In another example, Bihan and co-workers used capillary-HPLC coupled to a hybrid LTQ-Orbitrap XL, and identified 966 proteins from Photobacterium profundum exposed to pressure [5]. However, our approach based on nLC coupled with Orbitrap is not flawless. First, Orbitrap is coupled with electrospray ionization (ESI), but compared with MALDI-MS, the effectiveness of protein analysis by ESI-MS is severely undermined by the presence of salts, detergents and contaminants in sample buffer. Second, the label-free quantitative MS we used is only a semi-quantitative method. There is still much work needed to address the drawbacks in proteomic analysis and to devise
accurate proteomic tools and methods such as ionization mechanisms, reliable reference databases, search algorithms for identification and accurate annotation and classification of proteins. In conclusion, we describe a novel and advantageous microbial proteomic technique. Our results clearly indicate that although the cold-stressed and control cells share a substantial number of proteins, each has a lot of unique proteins and quite a number of the common proteins display different expression levels. Our proteomics-based results should help to improve our understanding of the response of V. metschnikovii to prolonged exposure to low temperatures, which may contribute to a more accurate system-based understanding of this food-borne pathogen. Conflict of interest There is no conflict of interest. Acknowledgments We thank Dr. Yongwei Wang for his suggestions and help concerning proteomic analysis by Orbitrap. This work was supported by the Chinese State High-Tech Development Plan (863 program, 2012AA101605), the Science Foundation of General Administration of Quality Supervision, Inspection and Quarantine of the People's Republic of China (2009 IK176, 2009IK254, 2012IK305 and 2013IK175) and the Scientific and Technological Plan in Public Domain of Qingdao (12-1-3-80-jh). Appendix A. Supplementary data Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.resmic.2015.07.011. References [1] Asakura H, Ishiwa A, Arakawa E, Makino S-i, Okada Y, Yamamoto S, et al. Gene expression profile of Vibrio cholerae in the cold stress-induced viable but non-culturable state. Environ Microbiol 2006;9:869e79. [2] Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM, et al. Gene ontology: tool for the unification of biology. Nat Genet 2000;25:25e9. [3] Bang W, Drake MA. Resistance of cold- and starvation-stressed Vibrio vulnificus to heat and freeze-thaw exposure. J Food Prot 2002;65:975e80. [4] Belova ME, Andersona GA, Wingerda MA, Udsetha HR, Tanga K, Priora DC, et al. An automated high performance capillary liquid chromatography-Fourier transform ion cyclotron resonance mass spectrometer for high-throughput proteomics. J Am Soc Mass Spectrom 2004;15:212e32. [5] Bihan TL, Rayner J, Roy MM, Spagnolo L. Photobacterium profundum under pressure: a MS-Based label-free quantitative proteomics study. PLOS ONE 2013;8:e60897. [6] Burnham VE, Janes ME, Jakus LA, Supan J, Depaola A, Bell J. Growth and survival differences of Vibrio vulnificus and Vibrio parahaemolyticus strains during cold storage. J Food Sci 2009;74:314e8. [7] Cao J, Xu J, Zheng Q, Yan P. Rapid detection of Vibrio metschnikovii in aquatic products by real-time PCR. Folia Microbiol 2010;55:607e13.
Please cite this article in press as: Jia J, et al., Proteomic analysis of Vibrio metschnikovii under cold stress using a quadrupole Orbitrap mass spectrometer, Research in Microbiology (2015), http://dx.doi.org/10.1016/j.resmic.2015.07.011
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MODEL
Research in Microbiology xx (2015) 1e8 www.elsevier.com/locate/resmic
Original article
Proteomic analysis of Vibrio metschnikovii under cold stress using a quadrupole Orbitrap mass spectrometer Juntao Jia a, Ying Chen b,*, Yinghui Jiang a, Zhengyi Li a, Liqing Zhao a, Jian Zhang a, Jing Tang a, Liping Feng a, Chengzhu Liang a, Biao Xu a, Peiming Gu c, Xiwen Ye a b
a Technological Center, Shandong Entry-Exit Inspection and Quarantine Bureau, 266002 Qingdao, China Research Institute for Food Safety, Chinese Academy of Inspection and Quarantine, No. A3, Road Gaobeidian, 100123 Beijing, China c Demo Center of Thermo Fisher Scientific Inc., 201206 Shanghai, China
Received 10 October 2014; accepted 17 July 2015 Available online ▪ ▪ ▪
Abstract Vibrio metschnikovii is a food-borne pathogen found in seafood worldwide. We studied the global proteome responses of V. metschnikovii under cold stress by nano-flow ultra-high-performance liquid chromatography coupled to a quadrupole Orbitrap mass spectrometer. A total of 2066 proteins were identified, among which 288 were significantly upregulated and 572 were downregulated. Functional categorization of these proteins revealed distinct differences between cold-stressed and control cells. Quantitative reverse transcription polymerase chain reaction analysis was also performed to determine the mRNA expression levels of seventeen cold stress-related genes. The results of this study should improve our understanding of the metabolic activities of cold-adapted bacteria and will facilitate a better systems-based understanding of V. metschnikovii. © 2015 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.
Keywords: Proteomics; Vibrio metschnikovii; Cold stress
1. Introduction Although Vibrio metschnikovii is present in a wide range of aquatic products and is associated with human diseases, this species has not been extensively studied and little published work is available [7,37]. Bacteria may encounter temperature downshifts (cold stress) during food processing and storage. This adverse condition may not only inhibit bacterial growth, but can also enhance virulence and resistance of the stressed bacteria [3,32,39,53], and even induce development of crossprotection against other stresses [15]. Therefore, cold stress potentially increases risks with regard to food safety. Reports on bacterial cold stress responses primarily concern Vibrio spp. [1,3,6,10,11,20,27,32,33,54,55] and species such as * Corresponding author. Tel./fax: þ86 010 85783587. E-mail address: [email protected] (Y. Chen).
Escherichia coli [25,39], Listeria monocytogenes [15,40,43] and Morganella morganii [58]. However, most published studies focus on morphological and physiological changes during cold stress, with few reports on the molecular responses of cold-stressed Vibrio spp. in general, and V. metschnikovii in particular. In addition, global comparative proteomic analysis of bacteria under cold stress remains poorly reported. Bacteria exposed to low temperatures during food processing and preservation are stressed for relatively long periods. Nearly all studies to date focused on the bacterial response to transient cold stress. However, a comprehensive understanding of the molecular response during adaptation and growth at low temperatures is vital if we are to design targeted processing and preservation strategies to effectively control pathogens [25]. In this study, we conducted a comparative proteomic analysis of V. metschnikovii exposed to cold stress for a prolonged duration. Proteomics provide microbial researchers with
http://dx.doi.org/10.1016/j.resmic.2015.07.011 0923-2508/© 2015 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved. Please cite this article in press as: Jia J, et al., Proteomic analysis of Vibrio metschnikovii under cold stress using a quadrupole Orbitrap mass spectrometer, Research in Microbiology (2015), http://dx.doi.org/10.1016/j.resmic.2015.07.011
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unparalleled insights into cellular processes and functions [17]. The techniques of proteomics, commonly two-dimensional electrophoresis and protein characterization, have been widely used for microbiological research. Despite continuing improvements in proteomics, little effort has been invested in microbial proteomics using nano-flow ultra-high performance liquid chromatography (nLC) coupled to a high resolution benchtop quadrupole Orbitrap mass spectrometer. This technique offers excellent run-to-run reproducibility, high sensitivity and the labor-saving capability of online sample processing. This study aimed to construct a novel and advantageous microbial proteomic technique and to study the proteome of V. metschnikovii exposed to prolonged cold stress. To the best of our knowledge, this work is the most comprehensive quantitative proteomics study on cold-stressed bacterial cells available to date.
CA, USA). Peptide samples of 10 ml, dissolved in 0.1% formic acid, were eluted with an acetonitrile gradient and 0.1% formic acid in 240 min at a flow rate of 250 nl/min.
2. Materials and methods
Raw files obtained from the Orbitrap Q-Exactive spectrometer were queried using Proteome Discoverer 1.3 (Thermo Fisher Scientific) against a database of V. metschnikovii from UniProt (taxon identifier 28172) containing 3079 proteins. The false discovery rate (FDR) of all peptide identifications was set at less than 1%. All proteins identified with at least two unique peptides were taken into consideration. To quantify the proteins, we used a label-free quantification approach based on the spectra counts [9]. Protein expression was considered significantly different only if protein ratios differed more than twofold [42] and the differential expressions were determined by chi-square test or Fisher's exact test when sample sizes were small.
2.1. Bacterial strain and culture V. metschnikovii F5-1 was isolated from the American lobster Homarus americanus, and identified in our laboratory [30]. Bacterial stock cultures were stored in physiological saline with 10% glycerol at 80 C, and cultured on tryptic soy agar with yeast extract (TSA-YE, Difco) slants at 37 C. 2.2. Cold treatment V. metschnikovii was subjected to cold stress by incubation at 4 C for 2 weeks. This temperature is often used to store food products [15]. Cultures incubated at 37 C for 12 h were used as control. Two biological replicates were prepared for proteomic analysis. 2.3. Sample preparation Bacterial culture samples were harvested from agar slants and weighed. About 0.3 g of bacterial culture was rinsed with 50 mmol/l TriseHCl (pH ¼ 7.4) three times and centrifuged to obtain bacterial pellets. Lysis buffer comprising 4% (w/v) sodium dodecyl sulfate (SDS), 100 mM Tris/HCl pH 7.6 and 0.1 M dithiothreitol (DTT) was added to the bacterial pellets, mixed and kept on ice. Cells were disrupted by intermittent sonic oscillation for 2 min and centrifuged at 12,000 g for 20 min at 4 C. The supernatants were subjected to insolution tryptic digestion as previously described [35]. Peptide concentrations were determined with the Bradford assay (Bio-Rad). Peptide solutions were stored frozen at 20 C. Two analytical replicates were prepared and analyzed. 2.4. Online nLC system Peptide solutions were separated by the nLC system (EasynLC 1000, Thermo Fisher Scientific, San Jose, CA, USA) equipped with a C18 column (Easy-spray column, C18, 2 mm, 100 Å, 75 mm 50 cm, Thermo Fisher Scientific, San Jose,
2.5. Mass spectrometric analysis Effluents of the online nLC were analyzed using a high resolution mass spectrometer (Orbitrap Q-Exactive, Thermo Fisher Scientific, Bremen, Germany). The Orbitrap Q-Exactive settings were as follows: spray voltage 2.4 kV, data-dependent scan mode for top-20 precursor ions with a resolution of 70,000@m/z 200 for the full MS scans from m/z 300 to 1800, and 17,500@m/z 200 for MS/MS scans generated with normalized collision energy of 27%. 2.6. Identification and quantification of proteins
2.7. Functional categorization Identified proteins were functionally categorized according to Gene Ontology (GO) annotation [2] at http://www.uniprot. org. GO annotation results were plotted by Web Gene Ontology (WEGO) annotation plotting [56]. 2.8. Quantitative analysis of mRNA levels mRNA expression levels of the selected genes were determined by quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) analysis using the 2 TDDC method as described by Livak et al. [34]. Significance of differential expression was determined by Student's t-test ( p < 0.05). Total RNAwas extracted using a High Pure RNA isolation kit (Roche, Mannheim, Germany) according to the manufacturer's instructions. Reverse transcription was performed using a Transcriptor First Strand cDNA synthesis kit (Roche, Mannheim, Germany). Reactions were incubated at 50 C for 60 min, terminated by heating at 85 C for 5 min followed by storage at 20 C. Primers for target genes in qRT-PCR were designed using Primer Premier 5.0 (Premier, Palo Alto, CA) based on the DNA sequence of V. metschnikovii CIP 69.14 in the UniProt database (http://www.uniprot.org/). Seventeen target genes were selected and amplified in triplicate using the LightCycler 480 System (Roche, Mannheim,
Please cite this article in press as: Jia J, et al., Proteomic analysis of Vibrio metschnikovii under cold stress using a quadrupole Orbitrap mass spectrometer, Research in Microbiology (2015), http://dx.doi.org/10.1016/j.resmic.2015.07.011
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Germany), with 16S rRNA gene as internal control. cDNA corresponding to 0.5 mg of input RNAwas used as template. The 20 ml reaction mix contained 1 mM each of forward and reverse primers, 10 ml of SYBR Green Master (Roche, Mannheim, Germany), 2 ml of cDNA sample and 6 ml of RNase-free water. Thermocycling was programmed at 95 C for 5 min, followed by 45 cycles of denaturation at 95 C for 10 s, annealing at 60 C for 15 s and extension at 72 C for 20 s. Melting curves were also plotted to verify that a single target was amplified for each pair of primers. 3. Results and discussion
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4464) of the ‘cellular component’ domain, catalytic (GO:000 3824) and binding (GO:0005488) in ‘molecular function’ and metabolic process (GO:0008152) and cellular process (GO:0009987) in ‘biological process’. In total, 1941 and 2093 proteins were identified in the cold-stressed cells and the control, respectively. As shown in Fig. 1, both the number and percent of proteins in various categories did not differ significantly, except for a decrease in proteins in the categories pigmentation (GO:0043473), biological regulation (GO:0065007), transcription regulator activity (GO:0030528) and transporter activity (GO:0005215). We ignored categories with minor differences because of extreme caution in interpreting minor differences in functional classification [57].
3.1. Identification and quantification of proteins 3.3. Key proteins involved in response to cold stress A total of 2066 proteins were identified and quantified (Table S1). A comparison between cold-treated cells and control cells revealed an overlap of 1743 proteins. Among these mutual proteins, 288 were significantly upregulated and 572 were significantly downregulated. Compared with the control, the cold-stressed cells had increased quantities of cold shock proteins and ribosomal proteins, and decreased amounts of proteins involved in energy conversion and metabolism. Among the identified proteins, the most abundant included cold shock proteins, elongation factors, chaperone proteins, ribosomal proteins, outer membrane proteins and some proteins related to energy conversion and metabolism. 3.2. Functional categorization Using GO annotation [2,56], most proteins were classified into the categories cell (GO:0005623) and cell part (GO:004
Bacteria are frequently exposed to cold stress during food processing and preservation. To adapt to different environmental conditions, bacteria must alter their metabolism, cellular structure or both by inducing or eliminating some proteins [50]. Whereas cold shock response and adaption have been quite extensively studied in E. coli [8,13,23e25,29,36,39], the responses to cold stress by V. metschnikovii have not yet been examined. Reduced membrane fluidity and impaired protein synthesis are major problems resulting from cold shock [48]. Membrane fluidity is controlled by their fatty acid composition and maintaining protein synthesis relies on induced cold shock proteins (Csp), which initiate and facilitate ribosomal function at low temperatures. Cold shock proteins, especially those of the CspA family, are generally regarded as the proteins responding to an abrupt temperature downshift, and our results are in agreement
Fig. 1. Functional categorization of identified proteins according to the Gene Ontology Consortium classifications, visualized with WEGO. The vertical scales are logarithmic. Blue numbers on the right vertical axis refer to proteins from cold-stressed cells; black numbers refer to proteins from control cells. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) Please cite this article in press as: Jia J, et al., Proteomic analysis of Vibrio metschnikovii under cold stress using a quadrupole Orbitrap mass spectrometer, Research in Microbiology (2015), http://dx.doi.org/10.1016/j.resmic.2015.07.011
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with this (Table 1). A previous time course analysis of global gene expression of Vibrio vulnificus during cold stress also indicated induction of CspA [54]. Ironically, another V. vulnificus strain was observed to express csp1 (a gene homologous to cspA) constitutively when cells were incubated at 35 C and then subjected to 4 C for one week [31]. This inconsistency is likely due to heterogeneity of these genes despite their sequence similarity, and perhaps to the fact that transcription levels do not necessarily correlate with protein levels. Other proteins involved in cold responses include RecA [51], H-NS [13], GyrA [46], NusA [16], PNP [14], Hsc66 and Hsp70 (DnaK) [48], HscB [29], OtsA and OtsB [23] and TF [24]. However, Hsc66, HscB, OtsA and OtsB were not detected in this study. Among detected proteins, only the amount of PNP increased significantly in our study, and the levels of the other proteins remained unchanged (Table 1). In addition, three ribosome-associated proteins involved in translation are considered to be required for efficient ribosomal function at low temperatures [48]: IF-2 [18], CsdA [49] and RbfA [12]. In our experiments, cold stress dramatically (169/20) induced CsdA, which encodes a presumed ATPdependent RNA helicase [49] involved in the biogenesis of the 50S ribosomal subunit [8]. IF-2 and RbfA remained unchanged. A recent study revealed upregulation of CsdA in E. coli exposed to a low temperature of 14 C [25]. We assume that the V. metschnikovii cells in our study share a similar mechanism with E. coli to counteract the effect of low temperatures. Our assumption is based on the fact that elongation factors and proteins of the CspA family were expressed at high levels, and most of the related proteins functioning in the cold shock response, such as CsdA, were also present. Nevertheless, there were many differences between the two mechanisms, such as stable expression levels of cold shock proteins in our study (Table 1). Another case in point is the difference in the role of trehalose. According to a previous study [23], genes otsA and otsB, which encode trehalose biosynthetic enzymes, are induced and trehalose synthesis is activated during cold shock, which plays an important role in resistance
of E. coli (and probably other organisms) to low temperatures. However, in our study, we did not detect OtsA and OtsB in V. metschnikovii cells during their adaption to low temperatures. We propose that all these discrepancies result mainly from cold treatment conditions and partially from bacterial diversity and methods of proteomic analysis, including protein extract preparation, the mass spectrometer used and the set threshold of protein ratios (twofold in our study). As for the first reason, the sudden cold shock in most studies is different from our prolonged cold stress treatment, in which cells reached a final adapted and stabilized phase. We assume that V. metschnikovii cells entered this stabilized phase in our study. It is widely accepted that the bacterial response to prolonged periods of cold stress may be divided into different stages, as described in previous reviews [48,52]. Among others, these stages include the initial but transient cold shock response, which lasts a few hours, and the much longer restoration period during which cells are cold-adapted. Bacteria in a stress-adapted physiological state can re-establish disrupted homeostasis by resuming cellular functions such as DNA and protein synthesis, cell growth and cellular division [15]. In terms of food safety, bacteria in a stress-adapted physiological state presumably pose the greatest threat, because they are able to multiply in the food matrix and attain numbers high enough to cause infection upon consumption by susceptible individuals [15]. We therefore focused on longer periods of cold stress, because food processing and storage last more than a few hours. As mentioned above, ribosomal proteins are among the most abundant proteins. Cold stress significantly increased their expression (Table S1), illustrating their crucial role in the cold stress response. Low temperatures alter the structural integrity of ribosomal subunits [47]. This results in translational stalling and a transient decrease in polysome numbers, accompanied by an increase of single 70S ribosomes and 50S and 30S subunits [22]. Consistent with this, quantities of ribosomal proteins increased following cold shock in the present study. Genes associated with ribosomes were among the most strongly activated gene sets in response to hyperosmotic and
Table 1 Key proteins identified in this study involved in cold stress. Protein
Description
Protein ratios
Differential expression
CspA family RecA H-NS GyrA NusA PNP Hsp70 TF
Chaperone proteins Recombination and induction of the cold shock response Nucleoid-associated DNA-binding protein a-subunit of topoisomerase DNA gyrase Termination and antitermination of transcription Exoribonuclease Chaperone protein DnaK Trigger factor catalyzing cis/trans isomerization of peptide bonds N-terminal to the praline residue Binding of the initiation tRNA to the 30S subunit A member of the DEAD-box family of helicases 30S ribosomal binding factor trigging the cold shock response Elongation factor thermo-unstable Elongation factor thermostable
178/49 61/57 12/14 109/98 63/48 267/123 461/460 154/108
up unchanged unchanged unchanged unchanged up unchanged unchanged
185/98 169/20 14/10 522/370 316/245
unchanged up unchanged unchanged unchanged
IF-2 CsdA RbfA EF-Tu EF-Ts
Note: Protein ratio is based on the quantity of a protein extracted from bacterial cells under cold stress, divided by the control. Please cite this article in press as: Jia J, et al., Proteomic analysis of Vibrio metschnikovii under cold stress using a quadrupole Orbitrap mass spectrometer, Research in Microbiology (2015), http://dx.doi.org/10.1016/j.resmic.2015.07.011
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cold stress when L. monocytogenes strains were exposed to both stresses [15]. In our experiments, we observed that nearly all of 30S and 50S ribosomal proteins were upregulated, including those encoded by rplK, rplA, rpsD, rpmB and rpmE. These five genes consistently showed strong upregulation in all three strains of L. monocytogenes following independent adaptation to hyperosmotic and cold stress [15]. This was in agreement with our results acquired at mRNA expression levels. However, when V. vulnificus was exposed to a temperature drop of 35 Ce4 C within about an hour, no significant fold increases were found for expression of genes encoding 30S and 50S ribosomal proteins [54]. The discrepancy may be caused by the differences in cold stress duration. In contrast, some proteins related to energy conversion (such as pyruvate formate-lyase (C9P3U0), alcohol dehydrogenase (C9P3R8), succinate dehydrogenase flavoprotein subunit (C9P1Z0)), carbohydrate catabolism (such as phosphoglycerate kinase (C9P2N3), NAD-dependent glyceraldehyde-3-phosphate dehydrogenase (C9P3K7), pyruvate kinase (C9P2M6), triose-phosphate isomerase (C9P1X7), enolase (C9P1J8), fructose-bisphosphate aldolase class II (C9P2N2)), amino acid metabolism (such as aspartate ammonia-lyase (C9P1V3) and carbamate kinase (C9P1U8)) were distinctly downregulated (Table S1). This is consistent with the observations of Wood et al. [54]. In another study [15], overall downregulation of gene sets associated with carbohydrate transport and utilization were observed in cold- and osmoadapted cells, and gene sets associated with phosphotransferase systems were strongly suppressed in L. monocytogenes cells adapted to cold and salt stresses. In addition, a recent study [25] in which E. coli O157:H7 was stressed by an abrupt downshift from 35 C to 14 C also revealed repression of genes and proteins involved in DNA replication, protein synthesis, and carbohydrate catabolism. It is widely accepted that protein synthesis is impaired upon cold shock [48].
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In our study, proteins involved in translation such as elongation factors and initiation factor IF-2, chaperone proteins participating in protein folding and proteins involved in transcription such as DNA-directed RNA polymerase were found unchanged but highly expressed during cold stress. This indicates that V. metschnikovii entered a stabilized phase, distinct from the state upon cold shock. As in the case of elongation factors, we found that synthesis of elongation factor thermo-unstable (EF-Tu) and elongation factor thermostable (EF-Ts) remained unchanged. Both were produced at a high level during cold treatment, indicating that protein synthesis was maintained to resist cold stress. However, overexpression of EF-Tu in the outer membrane and underexpression of the EF-Ts in the cytoplasm was reported for an E. coli strain following cold stress adaption [39], and EF-Tu downregulation and EF-Ts upregulation were observed in cold-induced viable but non-culturable cells of Enterococcus faecalis [19]. It is well known that EF-Ts mediates regeneration of the EF-Tu-GDP complex into the active form, EF-TuGTP, in order to allow protein synthesis to continue for a limited time [19]. These observations were different from ours, showing that the response to cold stress differs from cold shock. Finally, it is noteworthy that we observed various differential expression levels of outer membrane proteins (OMPs) in this study. For example, OmpT was sharply downregulated, OmpU was upregulated and expression of other proteins, such as OmpA (C9P350), OmpK (C9P6G4) and Omp (C9P589) remained unchanged (Table S1). This may due to the functional complexity of outer membrane proteins. 3.4. Transcriptional analysis of selected target genes We used qRT-PCR for transcriptional analysis of the following genes (with their corresponding protein names in
Fig. 2. Ratios of relative expression levels of seventeen target genes involved in the cold stress response from cold-stressed and control V. metschnikovii cells. Gene expression was estimated using qRT-PCR and the comparative critical threshold (2DDC ) method. The 16S rRNA gene was used as internal control. The results in T this figure are the means of three independent experiments. Error bars denote standard deviations. Please cite this article in press as: Jia J, et al., Proteomic analysis of Vibrio metschnikovii under cold stress using a quadrupole Orbitrap mass spectrometer, Research in Microbiology (2015), http://dx.doi.org/10.1016/j.resmic.2015.07.011
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parentheses): cspA, recA, gyrA, nusA, pnp, tig (TF), infB (IF2), csdA, rplK, rplA, rpsD, rpmB, rpmE, tuf (EF-Tu), tsf (EFTs), ompT and ompU. The proteins encoded by these genes are key proteins involved in response to cold stress. Our results (Fig. 2) indicate that the mRNA expression levels of cspA, pnp, rplK, rplA, rpsD, rpmB, rpmE and ompU (significantly upregulated under cold stress) and the expression level of recA (unchanged under cold stress) correlated well with protein expression levels; for the remaining genes, protein expression levels were inconsistent with the corresponding transcription levels. The inconsistency is most likely due to the fact that mRNA and protein profiles are often poorly correlated [44], and perhaps also because of the different rules applied to determine whether differences in levels of protein expression (two-fold rule) and transcription (Student's t-test) were considered relevant. 3.5. Approaches of proteomic analysis Many bacterial proteomic analysis approaches have been developed. In the past, bacterial lysate digests were separated by two-dimensional polyacrylamide gel electrophoresis (2DE), and peptides were identified by MS including tandem MS methods such as matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) MS [21,28,41,45] and quadruple time-of-flight (Q-TOF) MS [26]. However, 2DE has at least two major limitations. First, 2DE protein spots on the gel must be visualized and excised prior to MS, which is inconvenient and inaccurate. Second, many important regulatory proteins are expressed at low levels, and their concentrations are often too low for effective analysis [4]. Recently, shotgun proteomics, which combines high performance liquid chromatography (HPLC) with MS, has made proteomic analysis automated and convenient. Shotgun proteomics based on a combination of nLC and MS made it possible to identify thousands of proteins in a single LC-MS analysis [38]. The results of the present study support the use of nLC coupled with Orbitrap for global discovery of microbial proteomes. In our study, we identified 2066 proteins, covering about twothirds (2066/3079) of all expressed proteins of V. metschnikovii listed in UniProt (taxon identifier 28172). In other reports on proteomic responses of bacteria to different stresses, the number of proteins identified was significantly lower than in our study. For example, a total of 1048 spots obtained from E. coli strains incubated at 4 C were resolved by 2DE and subsequent MALDI-TOF MS analysis [45]. In another example, Bihan and co-workers used capillary-HPLC coupled to a hybrid LTQ-Orbitrap XL, and identified 966 proteins from Photobacterium profundum exposed to pressure [5]. However, our approach based on nLC coupled with Orbitrap is not flawless. First, Orbitrap is coupled with electrospray ionization (ESI), but compared with MALDI-MS, the effectiveness of protein analysis by ESI-MS is severely undermined by the presence of salts, detergents and contaminants in sample buffer. Second, the label-free quantitative MS we used is only a semi-quantitative method. There is still much work needed to address the drawbacks in proteomic analysis and to devise
accurate proteomic tools and methods such as ionization mechanisms, reliable reference databases, search algorithms for identification and accurate annotation and classification of proteins. In conclusion, we describe a novel and advantageous microbial proteomic technique. Our results clearly indicate that although the cold-stressed and control cells share a substantial number of proteins, each has a lot of unique proteins and quite a number of the common proteins display different expression levels. Our proteomics-based results should help to improve our understanding of the response of V. metschnikovii to prolonged exposure to low temperatures, which may contribute to a more accurate system-based understanding of this food-borne pathogen. Conflict of interest There is no conflict of interest. Acknowledgments We thank Dr. Yongwei Wang for his suggestions and help concerning proteomic analysis by Orbitrap. This work was supported by the Chinese State High-Tech Development Plan (863 program, 2012AA101605), the Science Foundation of General Administration of Quality Supervision, Inspection and Quarantine of the People's Republic of China (2009 IK176, 2009IK254, 2012IK305 and 2013IK175) and the Scientific and Technological Plan in Public Domain of Qingdao (12-1-3-80-jh). Appendix A. Supplementary data Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.resmic.2015.07.011. References [1] Asakura H, Ishiwa A, Arakawa E, Makino S-i, Okada Y, Yamamoto S, et al. Gene expression profile of Vibrio cholerae in the cold stress-induced viable but non-culturable state. Environ Microbiol 2006;9:869e79. [2] Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM, et al. Gene ontology: tool for the unification of biology. Nat Genet 2000;25:25e9. [3] Bang W, Drake MA. Resistance of cold- and starvation-stressed Vibrio vulnificus to heat and freeze-thaw exposure. J Food Prot 2002;65:975e80. [4] Belova ME, Andersona GA, Wingerda MA, Udsetha HR, Tanga K, Priora DC, et al. An automated high performance capillary liquid chromatography-Fourier transform ion cyclotron resonance mass spectrometer for high-throughput proteomics. J Am Soc Mass Spectrom 2004;15:212e32. [5] Bihan TL, Rayner J, Roy MM, Spagnolo L. Photobacterium profundum under pressure: a MS-Based label-free quantitative proteomics study. PLOS ONE 2013;8:e60897. [6] Burnham VE, Janes ME, Jakus LA, Supan J, Depaola A, Bell J. Growth and survival differences of Vibrio vulnificus and Vibrio parahaemolyticus strains during cold storage. J Food Sci 2009;74:314e8. [7] Cao J, Xu J, Zheng Q, Yan P. Rapid detection of Vibrio metschnikovii in aquatic products by real-time PCR. Folia Microbiol 2010;55:607e13.
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Please cite this article in press as: Jia J, et al., Proteomic analysis of Vibrio metschnikovii under cold stress using a quadrupole Orbitrap mass spectrometer, Research in Microbiology (2015), http://dx.doi.org/10.1016/j.resmic.2015.07.011