Observation ofEscherichia coliRibosomal Proteins and Their Posttranslational Modifications by Mass Spectrometry

Analytical Biochemistry 269, 105–112 (1999) Article ID abio.1998.3077, available online at http://www.idealibrary.com on

Observation of Escherichia coli Ribosomal Proteins and Their Posttranslational Modifications by Mass Spectrometry Randy J. Arnold and James P. Reilly Department of Chemistry, Indiana University, Bloomington, Indiana 47405

Received September 14, 1998

Ribosomes from the K-12 strain of Escherichia coli were analyzed with good sensitivity and high mass accuracy using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Fifty-five of the 56 subunit proteins were observable. Mass spectral peak locations were consistent with previously reported post-translational modifications involving Nterminal methionine loss, methylation, thiomethylation, and acetylation for all but one case. The speed and accuracy of mass spectrometry make it a good candidate for phylogenetic studies of ribosomes and the observation of posttranslational modifications in other organisms. © 1999 Academic Press

Although their role as the center of protein synthesis in cells has been appreciated for decades, ribosomes remain a popular target of research. The process of translating messenger RNA into proteins is complex and many of its steps have been elucidated. Nevertheless the ribosomes of prokaryotes, which are somewhat simpler than those of eukaryotes, contain three ribonucleic acids and more than 50 proteins intricately bound together into large and small subunits. A molecular level understanding of the role that each of these molecules plays in the translation process remains to be attained (1, 2). Because they are present in all organisms, the RNA and protein components of ribosomes provide the material for fingerprinting and evolutionary studies (3–5). Certain regions of the 16S rRNA are highly conserved among bacteria while others are unique to particular genera (3). Likewise, the existence of altered or missing ribosomal proteins conveys structural, genetic, and functional information (5). It has been reported that nine Escherichia coli ribosomal proteins experience posttranslational modifications involving acetylation and methylation (5); 0003-2697/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.

others are believed to be carboxylated (6) and one thiomethylated (7). In general the significance of these modifications is not understood (8). An understanding of such modifications is required to interpret the bacterial proteome, since they are not directly predictable from the genome. Despite its limited speed, two-dimensional gel electrophoresis has been the preferred method for resolving ribosomal proteins derived from a single source (1, 9, 10). Unfortunately, this does not resolve most posttranslational modifications. Mass spectrometry is an optimal method for detecting mutations and modifications in biological macromolecules. Two methods developed in the 1980s, electrospray ionization (11) and matrix-assisted laser desorption/ionization (MALDI) 1 (12) have made it possible to record mass spectra of large nucleic acids, proteins, and carbohydrates. Recent progress in timeof-flight instrumentation (13–15) has improved the quality of MALDI mass spectra to the point where the masses of molecules smaller than about 30,000 Da can typically be measured to an accuracy on the order of 1 Da or better. Several recent studies have demonstrated that MALDI mass spectra of bacteria display peaks that are characteristic of different species (16, 17) and strains (18, 19). Preliminary assignments of the peaks in our spectra suggested that a number of them could be assigned to ribosomal proteins. Since up to 45% of the mass of rapidly growing E. coli cells correspond to ribosomes, and up to 21% of the cell’s protein content is ribosomal (5, 20), the appearance of ribosomal proteins in our mass spectra did not seem surprising. It did suggest that if we extracted ribosomes from bacteria and recorded their mass spectra directly then it might be possible to detect and identify a substantial fraction 1

Abbreviations used: MALDI, matrix-assisted laser desorption/ ionization; TFA, trifluoroacetic acid; MWCO, molecular weight cutoff. 105

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of the ribosomal proteins. Previously assigned posttranslational modifications should be observable, providing further motivation for the present study. MATERIALS AND METHODS

Materials. Tryptone and yeast extract were obtained from Difco Labs (Detroit, MI). Sodium chloride and trifluoroacetic acid were purchased from EM Science (Gibbstown, NJ). Trizma–HCl, sucrose, a-cyano4-hydroxycinnamic acid, ubiquitin, cytochrome c, trypsinogen, and carbonic anhydrase were supplied by Sigma Chemical Co. (St. Louis, MO). Magnesium acetate and acetonitrile were obtained from Fisher Scientific (Fair Lawn, NJ). Ammonium chloride, ferulic acid, and sinapinic acid were purchased from Aldrich Chemical Co. (Milwaukee, WI). EDTA and 2-mercaptoethanol were supplied by Mallinckrodt Specialty Chemicals Co. (Paris, KY). Alumina was obtained from Buehler Ltd. (Evanston, IL). MWCO Nano-Spin filters (10 kDa) were purchased from Gelman Sciences (Ann Arbor, MI). MWCO membrane tubing (6 kDa) was supplied by Spectrum (Laguna Hills, CA). Ribosome isolation. E. coli strain K-12 (ATCC No. 25404) cells were grown in normal Luria broth (LB) medium and ribosomes from these cells were extracted using a method described by Spedding (1). This method involves harvesting the cells by centrifugation, grinding them with alumina, extracting the soluble components, and sucrose/salt-washing the ribosomes by ultracentrifugation through a sucrose cushion. The ribosomal pellets were suspended in low-salt buffer and dialyzed against the same buffer using membrane tubing that blocked molecules having masses nominally above 6000 Da. Aliquots of the approximately 25 mg/ml solution of ribosomes were stored at 280°C prior to mass spectral analysis. Mass Spectrometry. MALDI samples were prepared by mixing one part ribosome solution with nine parts matrix (either a-cyano-4-hydroxycinnamic acid, ferulic acid, or sinapinic acid at a concentration of 10 mg/ml in 2:1 0.1% trifluoroacetic acid (TFA)/acetonitrile) and applying 1 ml of this solution to the sample probe. For most samples, 1% TFA was added to the ribosome sample before mixing with matrix in order to precipitate the ribosomal RNA and enhance our MALDI signal. On the order of 2.5 mg of ribosomal material which corresponds to about 3 pmol of each protein are deposited in each sample spot. Since MALDI is commonly known to discriminate against the ionization of larger molecules in a mixture in favor of smaller components, some of the TFA-treated samples were passed through a 10-kDa MWCO membrane to remove smaller masses. Analysis of the retained components improved the sensitivity for masses larger than 20 kDa. Samples were allowed to air dry before

inserting them into the mass spectrometer. Positive and negative ion mass spectra were recorded on a home-built linear time-of-flight mass spectrometer similar to one previously described (21) using either 20 or 30 keV of total acceleration energy. External calibration was performed using a combination of ubiquitin, cytochrome c, trypsinogen, and/or carbonic anhydrase with the same matrix as the sample. After identifying the subunit proteins that produced strong, well-resolved peaks, their flight times and genomedetermined masses were used to internally calibrate the spectrum. RESULTS AND DISCUSSION

Peaks corresponding to ribosomal subunit proteins are observed at masses between 3 and 30 kDa, as shown in Fig. 1. Figure 1 is the composite of three separately acquired spectra, and each of the five segments was separately mass calibrated. The portion of the spectrum between 3 and 15 kDa was acquired in a single spectrum using 20 keV of acceleration energy, and each segment was internally calibrated separately. Doubly charged ions of L30, L29, S21, S16, and S19 and singly-charged ions of L33, L30, L31f, and L29 were used as internal calibrants for the 3- to 7-kDa segment, while singly charged ions of L31f, L29, L31, S21, L27, S16, S15, L25, and S14 were used as internal calibrants for the 7- to 11-kDa segment and singly charged ions of L25, S14, L18, S11, and L17 were used as internal calibrants for the 11- to 15-kDa segment. The 15- to 20-kDa segment of the spectrum was acquired using 30 keV of acceleration energy and was internally calibrated using singly charged ions of L17, L9, S5, L6, and L3. The 20- to 30-kDa segment of the spectrum was obtained by first removing low-mass subunits and impurities, such as salts and buffers, by using the 10-kDa molecular weight cutoff (MWCO) filter. The portion retained by the filter was analyzed using 30 keV of acceleration energy and calibrated using singly charged ions of S5, L10, L6, S7, L3, L1, and S3. Attempts to detect the 61-kDa S1 protein were made by using either a 50-kDa MWCO filter to remove low-mass subunits, a milder RNA removal by the addition of RNase, or magnesium chloride and acetic acid in combination with 30 keV of acceleration energy, but these attempts were unsuccessful. Every significant peak in Fig. 1 can be assigned to singly or doubly charged ribosomal protein ions. Some of the smaller features are due to matrix adducts of the most intense parent ions. The masses measured from these spectra, the masses predicted for the subunit proteins based on their amino acid sequences, and the previously established identities of posttranslational modifications are listed in Table 1. Although at first glance it may appear that there are 58 ribosomal proteins listed, L8 has been

OBSERVATION OF E. coli RIBOSOMAL PROTEINS

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FIG. 1. Composite positive ion MALDI mass spectrum of ribosome subunits generated using sinapinic acid matrix. Ribosomes were extracted from E. coli strain K-12 and treated with TFA as described in the text. Peaks labeled M* are matrix adduct ions.

shown to be an aggregate of L7/L12 and L10 (22, 23) and L26 is identical to S20 (22, 24) so there are in fact only 56. The most common posttranslational modification involves loss of the N-terminal methionine. This occurs

in 34 of the 56 proteins in our spectra. As previously discussed (25, 26), this modification occurs most often in cases where the amino acid in position 2, next to this methionine, has a short side chain. Sterically large side chains are believed to prevent proteins from docking in

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ARNOLD AND REILLY TABLE 1

E. coli Strain K-12 Ribosomal Subunit Proteins Observed by MALDI-MS Sequence mass (Da)

Experimental mass (Da)

61158.3 26612.6 25852.0 23338.0 17472.3 15187.1 19888.0 13995.4 14725.1 11735.6 13713.8 13605.9 12968.3 11449.3 10137.6 9190.6 9573.3 8855.3 10299.1 9553.2 8368.8 5095.8

— 26610.5 25851.9 23339.5 17514.8 15187.2 19888.7 13993.2 14723.3 11734.5 13727.7 13651.3 12968.1 11449.3 10137.6 9190.5 9573.0 8897.0 10299.6 9553.6 8368.8 5095.9

Subunit

Sequence mass (Da)

Experimental mass (Da)

L1 L2 L3 L4 L5 L6 L7 L9 L10 L11 L12 L13 L14 L15 L16 L17 L18 L19 L20 L21 L22 L23 L24 L25 L26 L27 L28 L29 L30 L31 L31 frag L32 L33 L34 L35 L36

24598.6 29729.4 22243.6 22086.6 20170.5 18772.7 12164.1 15769.1 17580.5 14744.3 12164.1 16018.6 13541.1 14980.5 15281.3 14364.7 12769.7 13002.1 13365.8 11564.4 12226.4 11199.2 11185.1 10693.5 9553.2 8993.3 8875.3 7273.5 6410.6 7871.1 6971.1 6315.2 6240.4 5380.4 7157.8 4364.4

24598.9 29732.3 22257.2 22086.2 20169.8 18772.7 12206.7 15769.7 17581.1 14870.2 12174.4 16018.0 13540.2 14980.1 15326.2 14364.7 12769.8 13001.7 13366.9 11562.7 12225.3 11198.0 11186.5 10693.4 9553.6 8993.5 8875.0 7273.4 6410.3 7871.0 6971.1 6315.1 6254.1 5380.5 7158.0 4364.2

Subunit S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11 S12 S13 S14 S15 S16 S17 S18 S19 S20 S21 S22

Part A: 30S subunit Methionine Error (Da) lost? n/a 22.1 20.1 11.5 142.5 10.1 10.7 22.2 21.8 21.1 113.9 145.4 20.2 0.0 0.0 20.1 20.3 141.7 10.5 10.4 0.0 10.1

Amino acid in position 2

n/a Yes Yes Yes Yes No Yes Yes Yes No Yes Yes Yes Yes Yes No Yes Yes Yes Yes Yes No

T A G A A R P S A Q A A A A S V T A P A P K

Error (Da)

Methionine lost?

Amino acid in position 2

10.3 12.9 113.6 20.4 20.7 0.0 142.6 10.6 10.6 1125.9 110.3 20.6 20.9 20.4 144.9 0.0 10.1 20.4 11.2 21.7 21.1 21.2 11.4 20.1 10.4 10.2 20.3 20.1 20.3 20.1 0.0 20.1 113.7 10.1 10.2 20.2

Yes Yes No No Yes Yes Yes No Yes Yes Yes No No No No No No No Yes No No No Yes No Yes Yes Yes No Yes No No Yes Yes No Yes No

A A I E A S S Q A A S K I R L R D S A Y E I A F A A S K A K K A A K P K

Comment Not observed

Acetylated (A1)

Methylated (A1) b-Methylthiolated (D88)

Acetylated (A1) Same as L26

Part B: 50S subunit Comment

Weak Methylated (Q150)

Acetylated (S1)

Methylated (9 times) Methylated (K81)

unknown mod. (R81)

Weak

Shoulder on L24

Same as S20

-RFNIPGSK (C-term) Methylated (A1)

Note. L1, L3, L6, L9, L10, L17, L18, L25, L27, L29, L30, L31, L31 frag., and L33 were used as internal calibrants. a Sequence mass is calculated by accounting for N-terminal methionine loss, but does not include any other modifications. S5, S11, S14, S15, S16, S19, S21, and S22 were used as internal calibrants.

OBSERVATION OF E. coli RIBOSOMAL PROTEINS

the methionine aminopeptidase active site. We find that when alanine is in position 2 (21 cases), the methionine is always lost. Methionine is also always cleaved when leucine, proline, and glycine are in position 2. However, when lysine, isoleucine, serine, glutamine, arginine, aspartic acid, tyrosine, glutamic acid, phenylalanine, and valine are in position 2 (20 cases) the methionine is always retained. When serine is in position 2, we observe that four times out of five, the methionine is cleaved. These results, included in Table 1, are in accord with those of Sherman et al., except for the particular case of S16 that has valine in position 2 (25). (They observed methionine loss for all cases in which the second residue was valine.) Several other posttranslational modifications, including methylation, acetylation, fragmentation, and amino acid side-chain derivatization, are listed in Table 1. In the original sequencing experiments, subunits S11, L3, L12, and L33 were found to be singly methylated, L11 methylated at nine sites, and subunits S5, S18, and L7 were found to be acetylated (24). The masses that we measure are consistent with these interpretations. We observe two peaks for subunit L31. The one at 7871.1 Da corresponds to the full protein sequence predicted from the genome (27), while the feature at 6971.1 Da corresponds to a fragment resulting from the loss of the eight-amino-acid sequence, RFNIPGSK, from the C-terminus of the predicted protein sequence, in agreement with the original amino acid sequence (28). Two other subunits contain derivatized amino acids that shift the masses of these proteins from those expected from the genome. S12 has an expected mass of 13605.9 Da. Similar to the case of L16, we do not observe a peak at this mass, but instead we see one at 13651.3 Da, 45.4 Da higher. In their original sequencing of this subunit WittmannLiebold and co-workers (29) proposed an unknown derivative of aspartic acid at position 88. In a recent very elegant study Kowalek and Walsh identified this modified residue as b-methyl thioaspartic acid (7). This modification adds 46.1 Da to the original sequence mass, close to the 45.4 Da shift that we observe. L16 should have a mass of 15281.3 Da based on the genomic sequence. We do not observe a peak at this mass, but instead see a strong peak at 15326.2 Da, 44.9 Da higher. The N-terminal methionine has been reported to be methylated (30). The original sequencing of this subunit also indicated that the arginine residue at position 81 was modified in some way; subsequent work by J. Brosius (personal communication) suggested that it was hydroxylated. These two modifications still yield a protein that is 14.9 Da lighter than the mass we observe, suggesting that another methylation or hydroxylation occurs. Further mass spectrometric fragmentation studies should enable us to es-

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tablish the identities of specific modified residues of protein L16. Isolation of the L16 subunit by highperformance liquid chromatography and subsequent digestion with trypsin should enable the localization of the modified residues. MS/MS experiments will provide information about the location and identity of the modifications. Since all ribosomal proteins are present in equal abundance, one copy per ribosome, except for L7/L12, which is present as a dimer of dimers, the variation in peak intensities that is apparent in Fig. 1 seems rather striking. Some of the strongest features are more than an order of magnitude larger than certain other ribosomal protein peaks. This observation has no simple explanation. Certain of the ribosomal proteins (e.g., S4, S8, S15, S17 and S20) are known to be directly attached to the rRNA (22), and one could imagine that if these were not released upon the addition of 1% TFA in the course of the sample preparation then they might precipitate out with the rRNA. However, these particular ribosomal proteins are by no means the weakest peaks in the mass spectra. The MALDI process is known to be somewhat discriminating because proteins vary either in their ability to incorporate into the matrix or in their propensity to become charged by proton transfer (31, 32). Indeed, many of the ribosomal proteins are relatively basic (22) and this should facilitate protonation. However, we have not been able to discern any correlation between the isoelectric points and the mass spectral ion yields for the various ribosomal proteins. Subunit S1, which may not be a bonafide ribosomal protein (2), was not detected in our experiments. Several factors may explain this observation. First, MALDI is commonly known to discriminate against the larger molecular components in a sample mixture. Concerned about our reduced sensitivity for such large mass proteins, we added a-amylase (54.7 kDa) and bovine serum albumin (66 kDa) to separate ribosome samples at molar concentrations similar to those of ribosomal proteins. In each case, we were able to detect both singly and doubly charged ions of the added protein, albeit with a signal-to-noise ratio approximately an order of magnitude less than that obtained for MALDI samples of the pure proteins at the same concentration. Second, S1 is a strong RNA binding protein and has an acidic isoelectric point (pI ' 5) (33) that may cause it to precipitate with the rRNA upon the addition of acid. SDS–PAGE analysis of our intact buffered ribosomes yielded a band at about 60 kDa that was absent in the TFA-treated ribosomes (data not shown), suggesting that S1 was removed from our sample upon the addition of acid. Third, the presence of 103 acidic compared to only 81 basic residues in S1 (33) suggests that it would have a lower propensity to form positive ions in the MALDI process than other ribo-

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FIG. 2. MALDI mass spectra of ribosomal subunit proteins. (A) Positive ions obtained from sinapinic acid matrix. (B) Negative ions obtained with ferulic acid matrix. (C) Positive ions obtained with a-cyano-4-hydroxycinnamic acid matrix. The 10-kDa MWCO filters were not used for the samples analyzed in this figure.

somal proteins whose pI’s are greater than 9. Further evidence for this was provided by the following experiment. RNase (1 ml of 1 mg/ml) was added to a 100-ml sample of buffered ribosomes and reacted overnight, yielding a protein precipitate that contained S1 according to SDS–PAGE analysis (data not shown). A separate 100 ml of buffered ribosomes was evaporated to dryness, diluted w/ 5 ml dd H 2O and its rRNA precipitated by the addition of 0.5 ml of 1 M magnesium chloride followed by 10 ml glacial acetic acid (34). This procedure also yielded a mixture that contained S1 according to SDS–PAGE. Nevertheless, MALDI analysis of these ribosome extracts yielded no observable S1 signal. Based on these arguments, we believe that the failure to detect S1 was more related to the protein than the technique. Since the ribosome sample could not be easily weighed during the extraction procedure, the amount of ribosomal material deposited in each spot is a rough estimate based on the average signal intensity of the

ribosome sample compared to signal intensities of standard proteins. This estimate is only approximate and may be in error by as much as an order of magnitude. Nevertheless, the material deposited in each spot can be used to obtain dozens of spectra, each an average of 500 individual shots. Our detection limits, single picomoles per spot and tens to hundreds of femtomoles per spectrum, do not approach the attomole level attained through picoliter sample preparations (35), but are competitive with Coomassie staining (about 10 pmol) or silver staining (about 100 fmol) for proteins of this size (36). Variations in the mass spectrometer operating conditions lead to striking effects. Figure 2A shows the low-mass region of the spectrum obtained when analyzing positive ions desorbed from a sinapinic acid matrix. Many doubly charged ions appear. Figure 2B displays a negative ion spectrum in the same mass region recorded using a ferulic acid matrix. Only singly charged ions are observed in this spectrum. Also, peak

OBSERVATION OF E. coli RIBOSOMAL PROTEINS

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FIG. 3. The effect of TFA on positive ion MALDI mass spectra of ribosomal subunit proteins using sinapinic acid matrix. (A) No TFA added. (B) 1% TFA added to the ribosome sample. The 10-kDa MWCO filters were not used for the samples analyzed in this figure.

intensities in these two spectra are vastly different. This difference in peak intensities might suggest that some of the proteins, such as L30, favor the formation of positive ions over negative ions. However, a positive ion spectrum generated with an a-cyano-4-hydroxycinnamic acid matrix (Fig. 2C), is comparable to the negative ion spectrum of Fig. 2B. Matrix-dependent variations of mass spectra are typical of the MALDI process, which is still not fully understood. Ribosomal RNA, a major component of our ribosome sample, can inhibit protein ion yield, so in order to record optimal spectra it was essential to remove it. This effect is displayed in Fig. 3. Part A of this figure was obtained by mixing the buffered ribosomes that contained the RNA with sinapinic acid matrix and analyzing positive ions. Very little signal was observed at masses above 8000 Da. The addition of 1% TFA to the buffered ribosome solution caused the RNA, but not the protein subunits with the exception of S1, to precipitate out of solution. A MALDI sample was prepared from this supernatant in the same way as for the buffered ribosome. The positive ion spectrum obtained is displayed in Fig. 3B. All ribosomal subunits having masses in this range appear in this spectrum, so the

data displayed in Fig. 1 were recorded using this procedure. Following completion of this work, we learned of a very recent electrospray mass spectrometry study of ribosomal proteins (37). Although in that work only nine proteins were observed, it may be difficult to detect many more using that approach because of the broad charge distribution produced by electrospray. S1, a protein that we did not detect, was observed along with the L7/L10/L12 complex that had at one time been called L8 (22, 23). Although we have observed a number of posttranslational modifications in E. coli proteins, many others may have gone undetected. In general, more posttranslational modifications are present in proteins than are observed. Krishna and Wold (38) point out that chemical instability of modified residues can easily impede their detection. For example, both b-carboxyaspartic acid (39) and g-carboxyglutamic acid (6) have been detected in E. coli ribosomal proteins, so the observation of mass shifts corresponding to these residues would not be surprising. However, acid hydrolysis of the additional carboxy group during sample preparation may be limiting the number of these modifications

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that we observe. The original sequencing of most subunits (see list in Ref. 24) may also have been affected by this problem since these sample preparations also involved acid hydrolysis. Milder sample handling conditions may facilitate the observation of other posttranslational modifications by mass spectrometry. In conclusion, we have demonstrated that ribosomal subunit proteins can be rapidly analyzed by MALDI mass spectrometry. We observed 98% (55 of 56) of the unique subunit masses from ribosomes extracted from strain K-12 of E. coli. A substantial number of mass shifts associated with posttranslational modifications were also observed. In one case, additional mass spectrometric work is required to elucidate the nature of the modification. Both positive and negative ion signals were observed by MALDI and several different matrices produced quality spectra. With its speed and highmass accuracy, this technique offers great promise for use in phylogenetic comparisons of ribosomes, for the rapid observation of posttranslational modifications in different organisms, and in the analysis of other similarly complex biological systems. ACKNOWLEDGMENTS The authors acknowledge John Richardson and Jonathan Karty for their help in preparing the ribosome samples, Kristen Mayer for her help with the SDS–PAGE analysis, and Juergen Brosius for insightful comments. This work has been supported by the National Science Foundation.

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