Previous Article |
Table of Contents
| Next Article 
From the Cover
Biochemistry
Global analysis of the Deinococcus radiodurans proteome by using accurate mass tags
a-Toli
*
*
*Environmental Molecular Sciences Laboratory,
Biogeochemistry, ¶Molecular Biosciences,
Pacific Northwest National Laboratory, P.O. Box 999, MSIN: K8-98,
Richland, WA 99352; Department of Biological Sciences,
Louisiana State University, Baton Rouge, LA 70803; and
Department of Pathology, Uniformed Services University
of the Health Sciences, Bethesda, MD 20814
Edited by Samuel Karlin, Stanford University, Stanford, CA, and approved June 26, 2002 (received for review March 22, 2002)
 |
Abstract |
|---|
 Top Abstract Experimental ProtocolResultsDiscussionReferences |
|---|
Abbreviations: LC, liquid chromatography; FTICR, Fourier transform ion cyclotron resonance; PMT, potential mass tag; AMT, accurate mass tag; MS/MS, tandem MS; TCA, tricarboxylic acid cycle; MMA, mass measurement accuracy; log, logarithm; SOD, superoxide dismutase
To address this need, we have developed a method for identifying peptides based on accurate mass tags (AMTs) for each protein expressed by a given organism. This approach provides greater sensitivity and dynamic range than previously achievable, more comprehensive coverage of expressed proteins, a basis for precise quantitation, and greater throughput for measurements of proteomes, because protein identification using AMTs circumvents the need for routine tandem MS (MS/MS).
Our initial study has focused on the extremely radiation-resistant bacterium Deinococcus radiodurans, but the general approach can be used for any organism whose genome has been sequenced. Two independent annotations are available for the sequenced genome of D. radiodurans (2, 3). This organism has been the target of genetic manipulation for a decade, a candidate for bioremediation of radioactive waste sites, and a subject for the study of DNA repair pathways (3, 4). D. radiodurans has an extraordinary ability to tolerate both acute and chronic exposure to high levels of ionizing radiation. Exponentially growing cultures of the Gram-positive, nonmotile, red-pigmented, nonpathogenic bacterium are able to withstand 50–100 times more ionizing radiation than Escherichia coli (5, 6). D. radiodurans can survive 5,000–15,000 Gy of acute ionizing radiation with no loss of viability (depending on the culture conditions) (7), can grow continuously under 60 Gy/hr (8), and has the ability to reduce contaminant metals and radionuclides including Cr, Tc, and U to less soluble species (9). Its resistance to radiation and to other DNA-damaging conditions (e.g., UV light, hydrogen peroxide) and desiccation (10) is likely because of its efficient DNA damage repair (6, 11). It has been suggested that the multiple copies of the D. radiodurans genome (4–10 genome equivalents) (11) may be organized to facilitate recombinational repair processes (3, 12). However, the set of predicted genes for D. radiodurans appears conventional and does not reveal the basis for its extreme radiation resistance (3). The number of annotated DNA repair enzymes (2, 3) is less than reported for E. coli.
Most likely the DNA damage-resistance phenotype is determined collectively by a complex array of interacting proteins (3) as well as by many more subtle structural peculiarities of proteins and DNA. Neither of these resistance motifs is readily inferred from the D. radiodurans sequence, underscoring the potential importance of global studies to obtain a better understanding of the interactions involved, such as determining protein expression patterns under stressed and nonstressed conditions (i.e., proteome-wide analyses).
|
Experimental Protocol |
|---|
|
TopAbstractExperimental ProtocolResultsDiscussionReferences |
|---|
Cell Lysis and Tryptic Digestion. Cell lysis was achieved by bead beating using three 90-sec cycles at 4,500 rpm in a Biospec (Bartlesville, OK) Minibeadbeater, with a 5-min cool down on ice between cycles. Lysates were immediately placed on ice to inhibit proteolysis. Protein concentration was determined by the BCA assay kit (Pierce). Before liquid chromatography (LC) MS analysis, the protein samples were denatured and reduced by the addition of guanidine hydrochloride (Gdn•HCl) (6 M) and DTT (1 mM) and boiled for 5 min. On reducing the Gdn•HCl concentration to below 2 M with 100 mM NH4HCO3 and 5 mM EDTA (pH 8.4), protein samples were digested by using bovine pancreas sequencing grade modified trypsin (Promega) (trypsin/protein, 1:50, wt/wt) at 37°C for 16 hr. Protein lysates were ultracentrifuged for 30 min at 356,000 x g, and clear supernatant was dialyzed against 50 mM Tris•HCl at 4°C with 500 molecular weight cutoff cellulose ester membrane. Lysates were subsequently aliquoted and quick frozen in nitrogen and stored in –80°C freezer until analyzed.
Capillary LC Separations.
The capillary LC system consisted of a pair of syringe pumps (100-ml
ISCO model 100DM) and controller (series D ISCO) and an in-house
manufactured mixer, capillary column selector, and sample loop for
manual injections. Separations were achieved with 5,000 psi
reversed-phase packed capillaries (150 µm i.d. x 360 µm o.d.;
Polymicro Technologies, Phoenix) (14) by using two mobile-phase
solvents consisting of 0.2% acetic acid and 0.05% trifluoroacetic
acid (TFA) in water (A) and 0.1% TFA in 90% acetonitrile/10% water
(B). The mobile-phase selection valve was switched from position A to B
10 min after injection, creating an exponential gradient as mobile
phase B displaced A in the mixer. Flow through the capillary HPLC
column was 
1.8 µl/min when equilibrated to 100% mobile-phase A.
Initial Mass Tag Development.
Eluant from the HPLC was infused into a conventional ion trap MS (LCQ,
ThermoFinnigan, San Jose, CA) operating in a data-dependent
MS/MS mode over a series of segmented m/z
ranges and also with prior fractionation by using ion exchange
chromatography. For each cycle, the three most abundant ions from MS
analysis were selected for MS/MS analysis by using a collision energy
setting of 35%. Dynamic exclusion was used to discriminate against
previously analyzed ions. The collision induced dissociation
spectra from the conventional ion trap mass spectrometer were analyzed
using SEQUEST (15) and the genome sequence of
D. radiodurans. Preliminary identifications were based on a
minimum crosscorrelation score of 2, and these peptides were used as
potential mass tags (PMTs) for validation by Fourier transform ion
cyclotron resonance (FTICR) measurements. These analyses identified
large numbers of polypeptide PMTs, of which 70% were then validated
as AMTs based on the detection of a species having the predicted mass
for the PMT to <1 ppm at the corresponding elution time in the
LC-FTICR analysis. The methodology for PMT generation is described in
detail elsewhere (16).
Validation of Mass Tags.
The 11.4-tesla FTICR mass spectrometer developed at our laboratory uses
an electrospray ionization (ESI) interface to an electrodynamic ion
funnel assembly coupled to a radio frequency quadrupole for collisional
ion focusing and highly efficient ion accumulation and transport to the
cylindrical FTICR cell for analysis (17). Mass spectra were
acquired with 105 resolution. To obtain the
desired <1-ppm mass measurement accuracy (MMA), a program that uses
the multiple charge states (e.g., 2+, 3+) produced by ESI for many
protonated polypeptides (18) is applied, followed by the use of
"lock masses" (i.e., confidently known species that serve as
effective internal calibrants) for each spectrum derived from commonly
occurring polypeptides that are identified with high confidence from
one capillary LC MS/MS analysis (for each organism) obtained by using
FTICR with accurate mass measurements (16). Our calculations show
50% of the peptides predicted from in silico tryptic
digestion of D. radiodurans have unique masses at MMA 
1
ppm, and these peptides can be used to identify 99.5% of the predicted
proteins potentially expressed by the organism. The fraction of
peptides useful as AMTs is actually greater than 50% due to our use of
elution time data, which serves to distinguish previously identified
peptides having otherwise indistinguishable masses.
|
Results |
|---|
|
TopAbstractExperimental ProtocolResultsDiscussionReferences |
|---|
The D. radiodurans strain R1 genome consists of two chromosomes, one megaplasmid and one plasmid (3). Our analysis used the 3,116 protein-encoding ORFs predicted by the TIGR annotation (ftp://ftp.tigr.org/pub/data/d_radiodurans/GDR.pep) (2) (we exclude from this analysis 71 ORFs predicted to contain frame shifts). The proteomic measurements provide a physical validation that predicted ORFs actually encode a protein. A two-dimensional visualization of a portion of one analysis (Fig. 1) illustrates the ability to identify proteins from the peptides detected for D. radiodurans [grown in a defined minimal medium (13) and harvested at mid-log phase].
|
1,500 AMTs are typically detected
corresponding to 700 ORFs (depending on the culture condition) and
15–20% of the D. radiodurans proteome.
|
|
20 zmol (12,000 molecules) of protein can be detected
during an FTICR analysis (25). In this work, capillary LC-FTICR
measurements provided an overall dynamic range of
104-105 (16). The further
expansion of the dynamic range of measurements by using a new DREAMS
technology (26) offers the potential to detect proteins at less than
one copy per cell given practical cell populations
(108 cells per analysis). For more abundant
proteins, broad coverage of peptide fragments was achieved; ribosomal
proteins were identified with an average of 9 AMTs. Similarly, DNA
polymerase I, considered to be the most abundant polymerase in
prokaryotic cells (27), is predicted to be present in about 400
copies per cell in E. coli and was identified with six AMTs
providing 20% coverage of the protein. Another method for estimating protein abundance in a cell population is the use of codon adaptation index (CAI) (28) or predicted highly expressed (PHX) (29) proteins, two separate algorithms devised to estimate protein abundance on the basis of codon usage. Although many of the proteins fall into a range corresponding to proteins expressed at high abundance, about 50% of the proteins we detected have both CAI and PHX scores predicting low abundance. If codon usage were the only indicator of protein abundance, the increase in AMT coverage per ORF with higher CAI and PHX values (Table 3) would suggest a slight and expected bias toward detection of high abundance proteins. However, the significant number of ORFs identified with low PHX and CAI values also suggests that this approach effectively identifies lower abundance proteins.
|
|
Stress Response, DNA Damage Repair, and Recognition Proteins and RecA.
It is predicted that D. radiodurans encodes a spectrum of
148 stress response proteins (four are predicted to contain authentic
frame shifts) (3). We identified 74 proteins predicted from the
annotated genome with an average of six AMTs per protein corresponding
to 24% overall sequence coverage. Two classes of annotated proteins
shown to play roles in the detoxification processes are catalase (DR
1998 and DRA0259) and superoxide dismutase (SOD) (DR1279, DR1546, and
DRA0202). We confirmed the presence of both predicted catalases with 48
(DR1998) and 37 (DRA0259) AMTs covering 80% and 50% of their
sequences, respectively. Of the SOD proteins, we identified DR 1279
with 14 AMTs, corresponding to 87% coverage, whereas DR1546 and
DRA0202 were identified with two and three AMTs, respectively. Of the
predicted 75 proteins with potential DNA repair activities (23), we
identified 39 with an average of three AMTs per protein corresponding
to 12% coverage of the amino acid sequence. RecA is central to
homologous recombinational repair of irradiation-induced double-strand
breaks in D. radiodurans chromosomal and plasmid DNA (6, 7,
11, 31). Five different RecA AMTs (covering 34% of the sequence) were
identified primarily in cells recovering from exposure to ionizing
radiation.
Stable isotope labeling allows the quantitation of changes in protein expression levels. The protein expression pattern of D. radiodurans recovering from a dose of 17.5 kGy of ionizing radiation (growing on 14N media) was compared with the protein expression pattern from control cells (growing on 15N media) (Fig. 3). Initial studies indicate that the expression of RecA as well as DNA-directed RNA polymerase I is significantly induced during recovery from 17.5 kGy irradiation, consistent with previous observations (32).
|
|
Discussion |
|---|
|
TopAbstractExperimental ProtocolResultsDiscussionReferences |
|---|
When D. radiodurans is exposed to ionizing radiation, a dose-dependent delay of cellular replication suggests the existence of a DNA damage checkpoint (33). During this delay, several phases of cellular detoxification are hypothesized (34), but details remain unclear. One detoxification process in D. radiodurans involves removal of the activated oxygen species by catalase and SOD. Although the biochemical function of these two enzymes is known (35), their role in radiation resistance is unclear. Basal levels of catalase in exponentially growing cultures of D. radiodurans have been shown to be over 100-fold greater than for E. coli (10, 35). Genetic inactivation of members of either protein group render D. radiodurans cells more sensitive to ionizing radiation at doses that exceed 16,000 Gy (36), whereas mutations in DR1279 result in a greater sensitivity to ionizing radiation compared with mutations in DR1998. We confirmed the presence of both catalases under most culture conditions, suggesting constitutive expression. Of the SOD proteins, we identified DR1279 under many culture conditions but detected DR1546 and DRA0202 proteins in only a few culture conditions and in relatively low abundance. Thus, DR1279 apparently dominates SOD function and also appears to be constitutively expressed.
Additional detoxification processes in D. radiodurans export substantial portions of its DNA as large or small oligonucleotides from the cell after irradiation (37), presumably to prevent reincorporation into the genome. Homologs of UvrA (including UvrA1) are believed to play a role in these processes because they have been linked to ABC transporter proteins (2), and UvrA serves as the site of attachment for nucleotide excision repair proteins to the cell membrane in E. coli (2, 38). The dramatic DNA damage resistance of D. radiodurans has been attributed to exceedingly efficient DNA repair mechanisms, although such mechanisms remain poorly understood (3, 34). We observed most of the predicted proteins associated with nucleotide excision repair (including the UvrA1, UvrB, UvrC, Mfd, and PolA) in both stressed and unstressed cultures, which may indicate that these processes are continuously removing damaged nucleotides from the cell. UV DNA damage endonuclease and DNA helicase II (UvrD) were not detected, suggesting that these proteins may be expressed only at low basal levels. The detoxification process originally ascribed to the activity of the MutT Nudix protein family has been refuted by Xu et al., who found low levels of MutT activity for proteins putatively identified with MutT domains (39), consistent with the present observation of the MutT Nudix protein family predominantly in unstressed cells.
Although D. radiodurans can express a suite of stress response proteins, expression patterns have initially revealed few correlations between expression and stress response. One exception is for the hypothetical protein DR0070, which is observed only after alkaline treatment (Fig. 2). Closer examination of this ORF reveals a limited similarity to alkaline protease of Bacillus subtilis (39), surrounding the catalytic serine residue, and illustrates the use of such data to verify or suggest protein function.
One protein that has been closely linked with the DNA repair ability of D. radiodurans is RecA. RecA is detected predominantly in cells recovering from ionizing radiation and also at low levels in nonstressed cells, suggesting a low level of constitutive expression, contrary to previously published work. Previous studies using conventional PAGE with a RecA immunoprobe were not sufficiently sensitive to detect these low levels of RecA in D. radiodurans grown under nonstress conditions (32). Besides RecA, few proteins typically associated with presynaptic or postsynaptic recombination events were detected.
Although this is the first study, to our knowledge, to demonstrate such comprehensive (>61%) proteome coverage, an additional significance of our approach lies in the breadth of proteomic studies now enabled. The present results provide a large set of AMT "protein biomarkers" for quantitative expression studies that can be conducted with greater sensitivity and higher throughput due to elimination of the need for MS/MS measurements. Such studies use cells grown on stable-isotope-labeled media whose proteins (and their peptide AMTs) serve as internal standards to establish relative expression levels (40, 41). Several studies have now demonstrated that precision of better than 10–20% is achievable (41–43), a significant improvement over conventional proteomics approaches or gene expression arrays. The observation that both RNA polymerase I and RecA were induced after exposure to ionizing radiation (Fig. 3), although not surprising, illustrates that the use of AMTs for quantitation also provides the rapid throughput that will be necessary to extract biological insights from global proteomic studies.
|
Acknowledgements |
|---|
|
Footnotes |
|---|
This paper was submitted directly (Track II) to the PNAS office.
See commentary on page 10943.
|
References |
|---|
|
TopAbstractExperimental ProtocolResultsDiscussionReferences |
|---|
, N., Zhao, R., Paa-Toli, L., Li, L., Berger, S. F., Harkewicz, R., Anderson, G. A., Belov, M. E. & Smith, R. D. (2001) Anal. Chem. 73, 3011-3021.[Medline]
a-Toli, L., Shen, Y., Conrads, T. P., Veenstra, T. D. & Udseth, H. R. (2002) Proteomics 2, 513-523.[CrossRef][ISI][Medline]
a-Toli, L., Masselon, C., Prior, D., Udseth, H. & Smith, R. (2001) J. Am. Soc. Mass Spectrom. 13, 144-154.
a-Toli, L. & Smith, R. D. (2000) J. Am. Soc. Mass Spectrom. 11, 416-421.[Medline]
a-Toli, L., Veenstra, T. D., Lipton, M. S., Udseth, H. R. & Smith, R. D. (2001) Anal. Chem. 73, 1766-1775.[Medline]
a-Toli, L., Lee, S.-W., Shen, Y., Zhao, R., Lipton, M. S., Conrads, T. P., Toli, N. & Smith, R. D. (2001) Anal. Chem. 73, 3312-3322.[Medline]
a-Toli, L. & Smith, R. D. (2000) Anal. Chem. 72, 3349-3354.[Medline]
, N., Udseth, H. R. & Smith, R. D. (2001) Anal. Chem. 73, 5052-5060.[Medline]
a-Toli, L., Jensen, P. K., Anderson, G. A., Lipton, M. S., Peden, K. K., Martinovic, S., Toli, N., Bruce, J. E. & Smith, R. D. (1999) J. Am. Chem. Soc. 121, 7949-7950.[CrossRef]
a-Toli, L., Anderson, G. A., Horner, J. A., Lipton, M. S., Bruce, J. E. & Smith, R. D. (1999) Anal. Chem. 71, 2076-2084.[Medline]
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg What's this?
Related Commentary in PNAS:
This article has been cited by other articles in HighWire Press-hosted journals:
|
|
 |
|
  M. F. Romine, T. S. Carlson, A. D. Norbeck, L. A. McCue, and M. S. Lipton Identification of Mobile Elements and Pseudogenes in the Shewanella oneidensis MR-1 Genome Appl. Envir. Microbiol., May 15, 2008; 74(10): 3257 - 3265. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Ansong, S. O. Purvine, J. N. Adkins, M. S. Lipton, and R. D. Smith Proteogenomics: needs and roles to be filled by proteomics in genome annotation Brief Funct Genomic Proteomic, March 10, 2008; (2008) eln010v1. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. K. Schmid, D. J. Reiss, A. Kaur, M. Pan, N. King, P. T. Van, L. Hohmann, D. B. Martin, and N. S. Baliga The anatomy of microbial cell state transitions in response to oxygen Genome Res., October 1, 2007; 17(10): 1399 - 1413. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. C. G. Rison, J. Mattow, P. R. Jungblut, and N. G. Stoker Experimental determination of translational starts using peptide mass mapping and tandem mass spectrometry within the proteome of Mycobacterium tuberculosis Microbiology, February 1, 2007; 153(2): 521 - 528. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Satoh, H. Ohba, H. Sghaier, and I. Narumi Down-regulation of radioresistance by LexA2 in Deinococcus radiodurans. Microbiology, November 1, 2006; 152(Pt 11): 3217 - 3226. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. D. Jaffe, D. R. Mani, K. C. Leptos, G. M. Church, M. A. Gillette, and S. A. Carr PEPPeR, a Platform for Experimental Proteomic Pattern Recognition Mol. Cell. Proteomics, October 1, 2006; 5(10): 1927 - 1941. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Brim, J. P. Osborne, H. M. Kostandarithes, J. K. Fredrickson, L. P. Wackett, and M. J. Daly Deinococcus radiodurans engineered for complete toluene degradation facilitates Cr(VI) reduction. Microbiology, August 1, 2006; 152(Pt 8): 2469 - 2477. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. N. Adkins, H. M. Mottaz, A. D. Norbeck, J. K. Gustin, J. Rue, T. R. W. Clauss, S. O. Purvine, K. D. Rodland, F. Heffron, and R. D. Smith Analysis of the Salmonella typhimurium Proteome through Environmental Response toward Infectious Conditions Mol. Cell. Proteomics, August 1, 2006; 5(8): 1450 - 1461. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Wolff, A. Otto, D. Albrecht, J. S. Zeng, K. Buttner, M. Gluckmann, M. Hecker, and D. Becher Gel-free and Gel-based Proteomics in Bacillus subtilis: A Comparative Study Mol. Cell. Proteomics, July 1, 2006; 5(7): 1183 - 1192. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. Haas, B. K. Faherty, S. A. Gerber, J. E. Elias, S. A. Beausoleil, C. E. Bakalarski, X. Li, J. Villen, and S. P. Gygi Optimization and Use of Peptide Mass Measurement Accuracy in Shotgun Proteomics Mol. Cell. Proteomics, July 1, 2006; 5(7): 1326 - 1337. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. de Groot, V. Chapon, P. Servant, R. Christen, M. F.-L. Saux, S. Sommer, and T. Heulin Deinococcus deserti sp. nov., a gamma-radiation-tolerant bacterium isolated from the Sahara Desert Int J Syst Evol Microbiol, November 1, 2005; 55(6): 2441 - 2446. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. J. Ram, N. C. VerBerkmoes, M. P. Thelen, G. W. Tyson, B. J. Baker, R. C. Blake II, M. Shah, R. L. Hettich, and J. F. Banfield Community Proteomics of a Natural Microbial Biofilm Science, June 24, 2005; 308(5730): 1915 - 1920. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W.-J. Qian, M. E. Monroe, T. Liu, J. M. Jacobs, G. A. Anderson, Y. Shen, R. J. Moore, D. J. Anderson, R. Zhang, S. E. Calvano, et al. Quantitative Proteome Analysis of Human Plasma following in Vivo Lipopolysaccharide Administration Using 16O/18O Labeling and the Accurate Mass and Time Tag Approach Mol. Cell. Proteomics, May 1, 2005; 4(5): 700 - 709. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Kolker, A. F. Picone, M. Y. Galperin, M. F. Romine, R. Higdon, K. S. Makarova, N. Kolker, G. A. Anderson, X. Qiu, K. J. Auberry, et al. Global profiling of Shewanella oneidensis MR-1: Expression of hypothetical genes and improved functional annotations PNAS, February 8, 2005; 102(6): 2099 - 2104. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Wang and D. A. Julin DNA Helicase Activity of the RecD Protein from Deinococcus radiodurans J. Biol. Chem., December 10, 2004; 279(50): 52024 - 52032. [Abstract] [Full Text] [PDF]
|
|
|
|
) RecA and (
)
DNA-directed RNA polymerase in cells after exposure to 17.5 kGy of
ionizing radiation obtained be analysis of a mixture of cells grown on
unlabeled media and the 15N-labeled reference proteome
(control, nonirradiated cells). Cells were prepared at 0, 3, 7, 9, and
12 hr after exposure.