Anal. Chem. 2001, 73, 2836-2842
Proteolytic 18O Labeling for Comparative Proteomics: Model Studies with Two Serotypes of Adenovirus Xudong Yao, Amy Freas, Javier Ramirez, Plamen A. Demirev, and Catherine Fenselau*
Department of Chemistry and Biochemistry, University of Maryland, College Park, Maryland 20742
A new method for proteolytic stable isotope labeling is introduced to provide quantitative and concurrent comparisons between individual proteins from two entire proteome pools or their subfractions. Two 18O atoms are incorporated universally into the carboxyl termini of all tryptic peptides during the proteolytic cleavage of all proteins in the first pool. Proteins in the second pool are cleaved analogously with the carboxyl termini of the resulting peptides containing two 16O atoms (i.e., no labeling). The two peptide mixtures are pooled for fractionation and separation, and the masses and isotope ratios of each peptide pair (differing by 4 Da) are measured by high-resolution mass spectrometry. Short sequences and/or accurate mass measurements combined with proteomics software tools allow the peptides to be related to the precursor proteins from which they are derived. Relative signal intensities of paired peptides quantify the expression levels of their precursor proteins from proteome pools to be compared, using an equation described in the paper. Observation of individual (unpaired) peptides is mainly interpreted as differential modification or sequence variation for the protein from the respective proteome pool. The method is evaluated here in a comparison of virion proteins for two serotypes (Ad5 and Ad2) of adenovirus, taking advantage of information already available about protein sequences and concentrations. In general, proteolytic 18O labeling enables a shotgun approach for proteomic studies with quantitation capability and is proposed as a useful tool for comparative proteomic studies of very complex protein mixtures. Quantitation of relative expression of proteins in different proteomes presents a challenging task. In a traditional proteomics study, proteins are resolved by 2-D electrophoresis, and their expression levels are monitored by the intensities of stained spots on gels. Recently, a number of laboratories have proposed alternative approaches for proteomic studies.1-3 Other separation * To whom correspondence should be addressed. Phone: 1(301)4058614. Fax: 1(301)4058615. E-mail:
[email protected]. (1) Gygi, S. P.; B., R.; Gerber, S. A.; Turecek, F.; Gelb, M. H.; Aebersold, R. Nat. Biotechnol. 1999, 17, 994-999. (2) Link, A. J.; Eng, J.; Schieltz, D. M.; Carmack, E.; Mize, G. J.; Morris, D. R.; Garvik, B. M.; Yates, J. R. I. Nat. Biotechnol. 1999, 17, 676-682. (3) Pandey, A.; Mann, M. Nature 2000, 405, 837-846.
2836 Analytical Chemistry, Vol. 73, No. 13, July 1, 2001
methods, such as multidimensional liquid chromatography and capillary electrophoresis, can provide separation power superior to that of 2-D electrophoresis. Furthermore, these methods are suitable for high-throughput studies, using a “shotgun” approach.2,4 Mass spectrometry (MS) dominates the field of proteomics as an analytical tool, allowing the characterization of peptides and proteins based on their molecular masses. Therefore, stable isotope labeling of proteins is an inherent choice for protein quantitation via MS. Stable isotope labeling has been used successfully to provide absolute quantitation of polypeptide levels in tissue5,6 and to quantitate site-specific modification of proteins by poly(ethylene glycol).7 The basis of labeling approaches for proteomic studies is the generation of two proteome poolssone unlabeled, the other isotopically taggedsthat would behave indistinguishably until their MS characterization.1,8 Combination of the pools during sample preparation allows minimal differential loss of proteins and peptides and, thus, more accurate quantitation of the expression levels of proteins in counterpart proteomes. In principle, isotope tags can be incorporated into proteins during cell growth8-10 or after cell lysis.1,11 Using isotopically enriched nutrients during cell growth, proteins can be encoded with stable isotope tags at particular amino acid residues. This early introduction of isotopes allows earlier combination of the two proteome pools and, therefore, minimal differential loss of proteins during separation. This approach has been used to identify proteins9 and to quantify protein expression and sitespecific phosphorylation.8 Perhaps, a more versatile and less expensive approach (at least for mammalian cells) is the introduction of stable isotope tags after cells are harvested.1,11 As an elegant example of introduction of isotope tags after culture, isotope-coded affinity tags (ICAT) have recently been applied in comparative studies of Saccharomyces cerevisiae, grown on either ethanol or (4) Spahr, C.; Susin, S.; Bures, E.; Robinson, J.; Davis, M.; McGinley, M.; Kroemer, G.; Patterson, S. Electrophoresis 2000, 21, 1635-1650. (5) Kusmierz, J. J.; Dass, C.; Robertson, J. T.; Desiderio, D. M. Int J. Mass Spectrom. Ion Processes 1991, 111, 247-262. (6) Agbas, A.; Ahmed, M. S.; Millington, W.; Cemerikic, B.; Desiderio, D. M.; Tseng, J. L.; Dass, C. Peptides 1995, 16, 623-7. (7) Vestling, M. M.; Murphy, C. M.; Keller, D. A.; Fenselau, C.; Dedinas, J.; Ladd, D. L.; Olsen, M. A. Drug Metab. Dispos. 1993, 21, 911-917. (8) Oda, Y.; Huang, K.; Croos, F. R.; Cowburn, D.; Chait, B. T. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 6591-6596. (9) Veenstra, T. D.; Martinovic´, S.; Anderson, G. A.; Pasˇa-Tolic´, L.; Smith, R. D. J. Am. Soc. Mass Spectrom. 2000, 11, 78-82. (10) Chen, X.; Smith, L. M.; Bradburg, E. M. Anal. Chem. 2000, 72, 11341143. (11) Geng, M.; Ji, J.; Regnier, F. E. J. Chromatogr., A 2000, 870, 295-313. 10.1021/ac001404c CCC: $20.00
© 2001 American Chemical Society Published on Web 05/02/2001
galactose as a carbon source.1 ICATs are designed to code and select particular components in total peptide digests, such as cysteine-containing proteolytic peptides, for study.1 In addition to functional groups on the peptide side chains, two additional potential sites (N-termini11,12 and C-termini) are opened as targets for stable isotope tagging during proteolysis. Free N- and C-termini occur in nearly all proteolytic peptides and thus allow global coding of the quantitative information for individual proteins. In such a strategy, specific sequences or derivatives can be used for affinity-based separation later on. Geng et al. recently reported the labeling of tryptic peptides by acetylation of the peptide N-termini, using acetyl N-hydroxysuccinimide or its trideuterioacetylated analogue.11 Isotope ratios of peptides were determined by MALDI mass spectrometry and used to determine the concentration of a peptide relative to that of the labeled internal standard.11 Proteins can be also isotopically labeled at the C-termini of the tryptic peptides. Peptides that have been labeled with 18O facilitate protein sequencing via mass spectrometry.13-15 Schno¨lzer et al.16 systematically studied protease digestion of proteins in highly 18O-enriched water. While chymotrypsin and Asp-N incorporate only one 18O atom, trypsin, Glu-C or Lys-C can incorporate two 18O atoms into the C-termini of the resulting peptides.16 Also important, 18O labels in carboxylate group on peptides and amino acids are resistant to back exchange.16,17 Thus, under common conditions for liquid chromatography, electrospray ionization (ESI), and matrix-assisted laser desorption/ionization (MALDI), covalent bonds between oxygen atoms and carbonyl carbon in a C-terminal carboxylate group are stable. Its universal incorporation into all peptide products during proteolysis makes 18O a ready and stable isotope tag for comparative proteomics supported by mass spectrometry. In the present study, this approach is evaluated, using the proteomes of two adenovirus serotypes (type 2 and type 5) as a model system, for which protein sequences and concentrations are known.18 This model system is of additional interest itself, since adenoviruses are often used as vectors in genetic engineering and gene therapy.19 EXPERIMENTAL SECTION Culture and Purification of Adenovirus. The KB cell line and adenovirus types 2 and 5 stocks were received from the American Type Cell Collection (Manassas, VA). The KB cells were grown to confluence in viral flasks (Eagle’s minimal essential media, 10% fetal bovine serum, and 1% penicillin/streptomycin), incubated at 37 °C under 5% CO2. The cells were then incubated with 1-3 mL of the starter viral culture at 37 °C with 5% CO2 and shaken intermittently for 1 h. Then fresh medium was added. The flasks were incubated for 2-4 more days until >80% of the cells (12) Munchbach, M.; Quadroni, M.; Miotto, G.; James, P. Anal. Chem. 2000, 72, 4047-4057. (13) Takao, T. H. H.; Okamoto, K.; Harada, A.; Kamachi, M.; Shimonishi, Y. Rapid Commun. Mass Spectrom. 1991, 5, 312-315. (14) Shevchenko, A.; Chernushevich, I.; Ens, W.; Standing, K. G.; Thomson, B.; Wilm, M.; Mann, M. Rapid Commun. Mass Spectrom. 1997, 11, 10151024. (15) Kosaka, T.; Takazawa, T.; Nakamura, T. Anal. Chem. 2000, 72, 1179-1185. (16) Schnolzer, M.; Jedrzejewski, P.; Lehmann, W. D. Electrophoresis 1996, 17, 945-953. (17) Murphy, R. C. Biomed. Mass Spectrom. 1979, 6, 309-314. (18) Chroboczek, J.; Bieber, F.; Jacrot, B. Virology 1992, 186, 280-285. (19) Walther, W.; Stein, U. Drugs 2000, 60, 249-271.
were rounded, a sign of cytopathic effect. The infected KB cells were then harvested by using trypsin/EDTA to detach them. The cell suspensions were spun at 1000g for 15 min at 4 °C, and the pellet was washed with a half volume of phosphate-buffered saline solution three times and then resuspended in 10 mM Tris‚HCl. After three cycles of freeze/thawing (-80 °C for 30 min and then a 37 °C water bath until thawed), the cell suspension was sonicated in an ice bath (with a hand-held sonicator for three cycles of 20 s each). Spun at 8000g for 20 min, the supernatant was collected and loaded (up to 10 mL) on a discontinuous CsCl gradient (6 mL of 1.2 g/cm3 CsCl over 8 mL of 1.4 g/cm3 CsCl). The virus formed a band when spun in a Beckman SW 28 rotor (Palo Alta, CA) for 90 min at 4 °C, which was collected and diluted to a volume of 14 mL with 10 mM Tris-HCl buffer. Cesium chloride (7.2 g) was dissolved, and the solution was centrifuged in a Beckman Ti50 rotor at 35 000 rpm for 16 h at 4 °C to collect a band of the mature virus. The virus in the CsCl gradient solution was then interfacially precipitated using acetonitrile. When anhydrous acetonitrile was added, a white thin pellet of the virus appeared at the interface of the organic and aqueous phases after centrifugation. Characterization of Adenovirus Virion Proteins. The virion protein pellets were mixed with sinapinic acid in acetonitrile/water (70:30, with 0.1 TFA%) sinapinic acid/TFA and analyzed by a Kompact MALDI 4 time-of-flight mass spectrometer (Kratos Analytical Instruments). The instrument was externally calibrated. Proteolytic Digestion of Adenovirus Virions. The virion protein pellets, recovered from 150 µL of CsCl stock (type 2) or 50 µL of CsCl stock (type 5), were washed twice with 50 µL of Millipore deionized water and dried in a SpeedVac to remove residue H216O. The two dried pellets were then incubated with 2 µL of endopeptidase Lys-C (1 µg/µL, Boehringer Mannheim, Germany) in the presence of 6.2 M urea and 310 mM Tris (pH 8.0) for 5 h in 16O (type 5) or 18O (type 2) water (Isotec, Inc., Miamisburg, OH; normalized, 97.7 atom % 18O) of a total volume of 25 µL. Each reaction mixture was then incubated with 2 µL of modified trypsin (1 µg/µL, sequencing grade, Promega, Madison, WI) for 36 h (0.8 M urea, 100 mM Tris, pH 8.0, a total volume of 200 µL). All proteolysis reactions were carried out at 37 °C. Fourier Transform Ion Cyclotron Resonance Characterization of Adenovirus Peptides. To identify and quantify the virion proteins, high-resolution mass spectra of the peptide fractions were obtained on a 4.7-T actively shield Fourier transform ion cyclotron resonance spectrometer (IonSpec, Irvine, CA). The combined type 2 (18O) and type 5 (16O) digests, 30 µL each, were separated on a C18 reversed-phase HPLC column (5 µm, 300 Å, 250 mm × 4.6 mm; Jupiter, Phenomenex, Torrance, CA). Solvent A was 0.1% TFA, and solvent B was 0.08% TFA in acetonitrile. The elution profile was 0% B 1 min, 0-60% B for 30 min, 60-90% B for 5 min, and 90-0% B for 5 min. Peptides were monitored by their absorbance at 216 nm. A total of 36 fractions (∼400 µL each) were collected in 100 µL of 100 mM amonium bicarbonate (pH 7.0) separately and dried by SpeedVac. The solidified fractions were redissolved with Millipore deionized water and desalted by Poros R2 50 (10 µL; Applied Biosystems, Framingham, MA) packing in a gel-loading pipet tip. Some of these peptide fractions were further separated by step elution of the peptides from the tip with 20 or 40% acetonitrile in 0.1% TFA water. The peptide solution (0.6 µL) and dihydroxybenzoic acid (DHB) solution (500 Analytical Chemistry, Vol. 73, No. 13, July 1, 2001
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Figure 1. MALDI-TOF mass spectrum of virion proteins from adenovirus type 5.
mM, 0.5 µL) were mixed on the probe. A N2 laser (337 nm) was used for desorption. Standard scripts were used to trap and detect the ions in the broad-band mode. For protein identification, measured molecular masses were searched against those of the expected peptides, generated through the program PeptideMass.20 The baseline noises were from 0.5 to 1%. Quadrupole Time-of-Flight Tandem Mass Spectrometric Characterization of Differentially Labeled Peptide Mixtures. Product ion scans were conducted on a quadrupole time-of-flight mass spectrometer (Applied Biosystems, Foster City, CA). The combined type 2 (18O) and type 5 (16O) digests, fractionated as described above, were loaded into a nanospray tip and analyzed with aid of a nanospray source (MDS Protana, Odense, Denmark). Instrument settings were as follows: ion spray voltage, 950 V, curtain gas, 25; collision gas, 6; focusing potential 2, 20.0 V; declustering potential 1, 40 V; declustering potential 2, 10.0 V; and ion energy 1, 1.6 V. Precursor ions were selected for m/z 496.8 with a low-resolution selection window, and TOF mass range was set m/z from 50 to 1000. During an acquisition period of 2 min, collision energy for precursor ions was manually changed from 15 to 35 V to provide optimized MS/MS spectra. Determination of Concentrations of Tryptic Peptides. UV absorbances of diluted tryptic peptide solutions from the type 2 and type 5 virion proteins were recorded at 216 nm and room temperature. In 1000 µL of double distilled water, three aliquots of 0.5 µL of peptide digest were sequentially added and the absorbances were recorded, using water as reference. RESULTS MADLI-TOF Characterization of Virion Proteins. In the spectrum of the proteins from adenovirus type 5 (Figure 1), 8 or 9 (protein II, protein III/protein IIIa, protein IV, protein V, protein VI, protein VII, protein VIII, protein IX) out of 11 structural virion proteins18,21,22 were identified. The peak around m/z 64 000 was not resolved, with protein III (63 292 Da) and IIIa (65 252 Da)22 plausibly overlapping. The terminal protein was not expected to appear in the MALDI-TOF spectra, since it is covalently attached to the virus DNA.21 A small processing product (2000-2500 Da) has been observed in previous studies of the type 2 virus.18,21,22 The sequence is not available for type 5 and we did not look for this peptide in the present study. Postdigestion Fractionation of Proteolytic Peptides from Adenovirus. After the lysate from each population of cells was (20) PeptideMass, http://expasy.cbr.nrc.ca/tools/peptide-mass.html. (21) Shenk, T. Fundam. Virol. 1996, 979-1016. (22) Lehmberg, E.; Traina, J. A.; Chakel, J. A.; Chang, R. J.; Parkman, M.; McCaman, M. T.; Murakami, P. K.; Lahidji, V.; Nelson, J. W.; Hancock, W. S.; Nestaas, E.; Pungor, E. J. J. Chromatogr., B 1999, 732, 411-423.
2838 Analytical Chemistry, Vol. 73, No. 13, July 1, 2001
Figure 2. HPLC profile for C18 separation of the mixture of labeled and unlabeled tryptic peptides pooled from adenovirus type 2 and type 5.
Figure 3. Isotope pair of peaks measured using MALDI on a Fourier transform mass spectrometer. The inset is the theoretical distribution of the peptide AWNSSTGQMLR with a ratio of 1:3.3 for 16O2 and 18O species. 2
digested with trypsin, the peptide mixtures were combined for all subsequent manipulations. Thirty-six aliquots were collected by HPLC fractionation (Figure 2). Each fraction was desalted by passing through a Poros R2 minicolumn and examined by matrixassisted laser desorption on a Fourier transform mass spectrometer. Many peaks were observed to occur as isotopic pairs. One example is shown in Figure 3. Accurate mass measurement of the monoisotopic peak (MH+obs 1250.62, MH+calc 1250.60) allowed this peptide to be assigned as AWNSSTGQMLR derived by trypsin cleavage of protein VI carrying two 16O atoms in the carboxylate group at the C-teminus for type 2 and two 18O atoms in the carboxylate group at the C-terminus for type 5. The inset is the theoretical pattern, with a ratio of 1:3.3 for the 16O2 peptide to the 18O peptide. About 5% of the type 2 sample has incorporated only 2 a single atom of 18O. Quantitation of Virion Peptides from Adenovirus Type 2 and Type 5. The volumes of suspended virion proteins used for precipitation and digestion were set at an arbitrary ratio of 3 to 1 (v/v) for type 2 to type 5. The relative concentration of total tryptic peptides from type 2 and type 5 virion proteins was initially determined by UV spectrometry at 216 nm, where the absorbance is mainly attributed to amide bonds. The UV absorbances for the dilute peptide solutions of the type 2 and type 5 were linearly fitted against the volume of the peptide solution added, giving the absorbance for the type 2 peptide solution as 0.13 ( 0.02/µL, and for the type 5 as 0.042 ( 0.001/µL. This gave a ratio of 3.1 ( 0.5 for the UV absorbances of the peptides from type 2 to those from type 5.
Figure 4. Partial high-resolution MALDI mass spectrum of labeled and unlabeled tryptic peptides from adenovirus type 2 and type 5. Table 1. Identification and Quantitation of Selected Adenovirus Peptides
a
MH+obs
MH+calc
peptide sequence
protein name
ratio 1a ( error
ratio 2b ( error
764.41 992.55 1049.64 1203.71 1750.93 1986.11 1703.90 1238.78 1356.72 1628.96 1250.62 893.48 1093.70 1603.87 2226.24 1513.85 913.49 1414.89 1526.90 1910.06 1117.58
764.38 992.52 1049.60 1203.66 1750.84 1986.01 1703.84 1238.75 1356.68 1628.96 1256.60 893.46 1093.67 1603.78 2226.18 1513.78 913.47 1414.83 1526.85 1910.00 1117.64
NWAAFR GNVYWVR GVIETVYLR LLTPNEFEIK VAITFDSSVSWPGNDR NLLLLPGSYTYEWNFR SFYNDQAVYSQLIR LSAILEAVVPAR MTVEPGLEPEVR VLRPGTTVVFTPGER AWNSSTGQMLR GNVYWVR LAAGIVTVPPR TTVDDAIDAVVEEAR NYTPTPPPVSTVDAAIQTVVR QAILTLQTSSSEPR-NH3 MPPWAGVR LTALLAQLDSLTR ELNVVSQQLLDLR GIVTDFAFLSPLASSAAS LTGPVVFTQR
II II II II II II III IIIa V V VI VII VII VII VII VIII IX IX IX IX terminal
2.5 ( 0.1 3.5 ( 0.9 3.0 ( 0.3 3.2 ( 0.6 2.8 ( 0.4 2.7 ( 0.3 4.7 ( 1.8 3.2 ( 0.7 4.0 ( 0.8 3.3 ( 1.4 3.7 ( 0.3 3.4 ( 0.1 3.9 ( 0.4 3.6 ( 0.2 4.2 ( 0.4 3.3 ( 0.5 2.8 ( 0.3 3.3 ( 0.4 3.7 ( 1.4 2.6 ( 0.4 2.6 ( 0.3
2.5 ( 0.1 3.4 ( 0.7 3.0 ( 0.3 3.1 ( 0.5 2.9 ( 0.4 2.8 ( 0.2 4.6 ( 1.6 3.1 ( 0.6 3.9 ( 0.7 3.2 ( 1.2 3.4 ( 0.2 3.3 ( 0.1 3.7 ( 0.3 3.5 ( 0.2 4.3 ( 0.4 3.3 ( 0.5 2.7 ( 0.3 3.2 ( 0.3 3.6 ( 1.2 2.5 ( 0.3 2.5 ( 0.3
Calculated using eq 1. b Calculated using peak heights.
In Fourier transform ion cyclotron resonance mass spectra of fractions from the pooled peptide fractions, paired sets of isotope clusters are observed 4 Da apart (Figure 4). The set of peaks with the smaller masses (such as 1597.78, 1603.83, and 1628.96 Da) corresponds to peptides from the type 5 proteins with two 16O atoms at their C-termini, the set with the higher masses to peptides from the type 2 with two 18O atoms at their C-termini. Accurate monoisotope masses obtained for the peptides from adenovirus type 5 were used to identify the protein (Table 1). The corresponding sequences were used to calculate the theoretical isotope peak distribution for a particular peptide, using the MSIsotope at ProteinProspector web site.23 The ratios of the monoisotopic peaks for the peptides of type 2 and type 5 were calculated according to the following equation (eq 1), where I0, I2, and I4
I4 ratio 1 )
(
) (
)
M4 M2 M2 M2 1 I0 I2 I0 + I2 I M0 M0 M0 2 M0 0 I0
(1)
are the observed relative intensities for the monoisotope peak for (23) MS-Isotope, http://prospector.ucsf.edu.
the peptides without 18O label, the peak with masses 2 Da higher, and the peak with 4 Da higher masses, respectively; M0, M2, and M4 are the theoretical relative intensities for the monoisotope peaks for the type 5 peptides, the peaks with masses 2 Da higher, and the peaks with masses 4 Da higher, respectively, which are calculated using MS-Isotope.23 The natural isotope distribution pattern (relative intensity for each isotopic peak) for the 18Olabeled peptide was assumed to be the same as for the unlabeled. In the equation, the contribution from incomplete (single) incorporation of 18O is included as well. The calculated ratios of some of the peptides were summarized in Table 1 and have an overall average of 3.3 ( 0.6. In Table 1, the values of ratio 2 were equal to I4/I0. This kind of calculation is an approximate one, which does not account for either the differences among isotope distributions of different peptides or the single incorporation of a 18O atom at the C-termini of peptides. The overall average of ratio 2 is 3.2 ( 0.6. Unpaired Sets of Isotopic Peaks. In the Fourier transform ion cyclotron resonance mass spectra of the pooled peptide fractions, unpaired sets of isotopic peaks were also observed (Figure 5). At m/z 2099.20, the peptide (SLRPVASGNWQSTLNSIVGL, MH+calc 2099.12) is the C-terminus of protein VI in both Analytical Chemistry, Vol. 73, No. 13, July 1, 2001
2839
Figure 5. Partial high-resolution MALDI mass spectrum of pooled tryptic peptides from adenovirus type 2 and type 5. Inset a: Unlabeled molecular ion cluster from C-termini peptides from protein VI, identical in type 2 and type 5. Inset b: Molecular ion cluster from the type 2 peptide SDASSPFPSLIGSFTSTR-18O2, whose F (boldface) is mutated to L in type 5.
Figure 6. Nanospray tandem mass spectrum of a labeled and unlabeled tryptic peptide from adenovirus type 2 and type 5.
type 2 and type 5 (Figure 5a). In Figure 5b, the peak at m/z 1860.96 belongs to peptide SDASSPFPSLIGSFTSTR-18O2 (MH+calc 1860.91) from protein IIIa in adenovirus type 2. This peak does not have a counterpart 4 Da mass lower. MS/MS of Paired Isotope Clusters. With a low-resolution Q1 selection mode, a mass window encompassing both labeled and unlabeled isotope clusters was selected to provide precursor ions for MS/MS scans (Figure 6). The precursor ions were identified as peptide FTLAVGDNR from protein II. In Figure 6, major ions were marked with their ion types, as well as partial sequence. It can be seen that the presence of the isotope doublet readily distinguishes y ions from b ions and other ions. 2840
Analytical Chemistry, Vol. 73, No. 13, July 1, 2001
DISCUSSION Adenovirus Type 5 and Type 2 as a Model System. Proteins from two types of adenovirus virions were used as model proteomes for several reasons. They can be used as controls themselves, since the sequences and concentrations of virion proteins are known.18,22 The known genomes18 and proteomes22 of these two serotypes allow prediction of the proteolytic peptides and their masses. In this small set, matches in observed and calculated monoisotope masses for the peptides allow identification of their precursor proteins, without the need for sequence tags. Virion proteins from type 2 and type 5 were digested in silico by PeptideMass.20 A total of 909 tryptic peptides and amino acids are predicted, with no missing cleavages,18,21,22 among which 407 peptides have masses in the range of 700-3000 Da, the m/z range monitored in this study. Among these 407 peptides, 324 peptides (not including terminal peptides and reactive Cys-containing peptides) are expected to have the same sequence and mass in both types of adenovirus. When C-termini of type 2 peptides are labeled with 18O and those of type 5 with 16O, these peptides appear as paired isotope clusters 4 Da apart (Figure 3). A large number of peptides with matching sequences reflects high sequence homology in these closely related adenovirus. All the virion proteins, except protein IV, will yield one or more tryptic peptides, which are expected to appear in pairs in the m/z range of 7003000. Many of these were indeed observed in high-resolution mass spectra. Adenovirus serotype 2 and 5 are closely related, and the counterpart proteins in both types of virions have the same copy numbers.21 Thus, relative concentrations of virion proteins are only dependent on the mixing ratio of two types of virions.21 In other words, all the peptides with the same sequences are expected to have the same ratio (relative amount) as that for virions, and any deviation from this ratio for a particular peptide should have an experimental origin. This provides a solid basis for examining the ability of proteolytic labeling to provide relative quantitation. The dynamic range in protein concentrations in adenovirus is ∼600-fold. The major proteins in the mature virions are protein II, copy number 720, weight percentage 62%; protein III, copy number 60, weight percentage 3%; protein IIIa, copy number 60, weight percentage 3%; protein IV, copy number 40, weight percentage 2%; protein V, copy number 157, weight percentage 5%; protein VI, copy number 360, weight percentage 8%; protein VII, copy number 833, weight percentage14%; protein VIII, copy number 26, weight percentage 0.3%; protein IX, copy number 240, weight percentage 3%; and terminal protein, copy number 2, weight percentage 0.1%. The terminal protein has only two copy numbers in each virion and is attached to DNA. Without the aid of DNAase, this protein was digested by trypsin, and the resulting peptides were detected after fractionation of the peptide mixtures. Quantitation of protein VIII was a challenge, since this protein counts for only less than 0.3% of total protein in weight, and the proteins from the two adenoviruses have only one tryptic peptide (QAILTLQTSSSEPR) in common in the range of 700-3000 Da. No peptides were identified as originating from protein IV (fiber protein), which is known to be readily lost in the isolation procedure.24 In real systems, some proteins are more difficult to denature for proteolysis than others, and some are more resistant to proteolysis. The well-folded capsid proteins in adenovirus provide another opportunity to validate further the proteolytic labeling (24) Stannard, L. http://www.virology.net/Big_Virology/BVDNAadeno.html.
Scheme 1
approach. No peptides from protein II were detected after trypsin digestion. However, peptides were obtained if the virion protein mixture was first incubated with Lys-C protease in urea-containing buffer, and then with trypsin. 18O Incorporation into the C-Termini of Peptides. Previously, 18O incorporation into peptide C-termini has been used to facilitate C-terminal sequencing via MS. The incorporated 18O atoms are reported to be stable during most chemical manipulations. Only under extreme acidic and basic conditions, the labile 18O-C bond in a carboxylate can be broken and back-exchanged with 16O in water.17 The 18O-C bonds in peptides have been shown to be sufficiently stable to survive most common peptide mixture separation procedures and ionization processes in mass spectrometry.16 We did observe partial loss of 18O labeling when HPLC fractions containing TFA were dried by SpeedVac, although the label survives as long as the fractions remain in solution. A simple solution was found for this problem: HPLC eluates containing TFA were collected in solutions of volatile buffers, such as ammonia bicarbonate and ammonia acetate. The incorporation of two 18O atoms into a peptide shifts the mass of the peptide 4 Da higher. With this mass difference, the isotope peaks from the labeled and unlabeled peptides, which are lower than ∼3000 Da, can be easily distinguished in a FT-ICR instrument or other types of high-resolution mass spectrometers such as quadrupole time-of-flight mass spectrometers. The distribution of tryptic peptides decreases exponentially as mass increases,25 and proteins can generally be quantified based on tryptic peptides with masses lower than 3000 Da. It is worthwhile to comment on the incorporation of two 18O atoms in the C-termini of peptides with catalysis by trypsin, LysC, or Glu-C. To facilitate C-terminal sequencing of peptides by MS, proteins have traditionally been digested by trypsin in buffers containing a mixture of H216O and H218O at a ratio of 50:50.13-15 For statistical reasons, single 18O incorporation at C-termini is typically the major product under these conditions. In such experiments, it would appear that trypsin can incorporate only one 18O atom, and peptides and their fragments containing C-termini are present as doublets 2 Da apart in the mass spectra. However, when highly enriched 18O water (>95%) is used, (25) Fenyo, D.; Qin, J.; Chait, B. T. Electrophoresis 1998, 19, 998-1005.
products with two 18O atoms are predominant.16 Apparently, the incorporation of a second 18O atom results from enzymatic use of the products from the initial reaction as pseudosubstrates (Scheme 1). A common feature of trypsin, Glu-C, and Lys-C is that they are all involved in electrostatic interactions with the amino acid side chains of proteins and peptides, in contrast to other endoproteases, which use weaker hydrophobic interactions. MALDI and ESI Analysis of Mixtures of Labeled and Unlabeled Peptides. It has long been recognized that peptides are differentially ionized during the MALDI process. This differential ionization effect has been attributed to differences in peptide-matrix interactions26 and the basicities of argininecontaining peptides and lysine-containing peptides.27,28 In our experiment, 90% of the peptides detected contain arginine. This is consistent with previous MALDI-TOF studies, in which argininecontaining peptides have been reported to have signal intensities more than 5 times higher than those of lysine-containing peptides in MALDI-TOF spectra.29 Electrospray ionization generates doubly charged ions of tryptic peptides. On a mass spectrometer of a quadrupole timeof-flight configuration, tandem mass spectra of these ions were readily obtained. If the quadrupole selection window is wide, both monoisotope peaks for doubly charged ions of labeled (two 18O) and unlabeled peptides can pass, because their masses differ only by 4 Da (2 units when z ) 2), and enter in the collision cell to fragment. In Figure 6, N-terminal ions such as a and b ions are singlets, while y ions containing C-terminus are present as doublets. The unique isotope patterns of the y ion doublets allow easy and unambiguous interpretation of MS/MS data to obtain a partial sequence (TLAV) from the peptide for protein identification. Relative Quantitation of Proteins from Paired Sets of Isotopic Peaks. An average ratio (ratio 1) of 3.3 ( 0.6 for relative intensities was obtained for the set of peptides in Table 1. The absolute concentrations for virions of type 2 and type 5 adenovirus were not directly measured because of the limited amount of material. Instead, the concentrations of tryptic peptides from virion (26) Zhu, Y.; Lee, K.; Tang, K.; Allman, S.; Taranenko, N.; Chen, C. Rapid Commun. Mass Spectrom. 1995, 9, 1315-1320. (27) Harrison, A. Mass Spectrom. Rev. 1997, 16, 201-217. (28) Wu, Z.; Fenselau, C. Rapid Commun. Mass Spectrom. 1994, 8, 777-780. (29) Krause, E.; Wenschuh, H.; Jungblut, P. Anal. Chem. 1999, 71, 4160-4165.
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proteins of both serotypes were determined by UV absorbance at 216 nm. The ratio of absorbance of type 2 peptides to type 5 peptides was calculated to be 3.1 ( 0.5, in a good agreement with the average (3.3 ( 0.6) based on mass spectrometric analysis. The UV ratio for peptides from both types of adenovirus should give a reliable measurement of relative quantities of virion proteins, since each counterpart virion protein has the same copy number in both types of virions,21 and these proteins are highly homologous in their sequences,18 providing very similar UV extinction coefficients at 216 nm. This agreement suggests that mass spectrometric characterization of 18O-labeled peptides is a useful tool for relative quantitation of peptides and their precursor proteins and for comparison of relative protein expression in different proteomes or different cell states as well. It is of interest to examine the relative concentrations determined for proteins of different copy number and abundance. Protein II, protein VII, protein VIII, and protein IX account for 60, 14, 3, and 0.3% of the total virion proteins by weight. The relative concentrations determined for protein II, protein VII, protein VIII, and protein IX, are 2.9 ( 0.3 (average of six peptides), 3.7 ( 0.4 (average of four peptides), 3.3 ( 0.5 (a single peptide), and 3.0 ( 0.5 (average of four peptides) similar to the overall average ratio (3.3 ( 0.6). The similarity among those ratios is consistent across the range of adenoviruses. A comparison of the ratios for individual proteins with the overall one suggests that proteolytic 18O labeling supported by mass spectrometry has a precision better than 25% in relative quantitation of proteins, when multiple peptides are used to quantify their precursor protein. The evaluation of 18O proteolytic labeling with the adenovirus model system requires enzymatic digestion of structured virions and folded proteins. This uncertainty in quantitation should reflect what may be expected when 18O proteolytic labeling is applied to study complicated protein mixtures and proteomes. In addition, although capsid proteins, such as protein II, are harder to digest than some others, there was no significant deviation of the average ratio (2.9 ( 0.3) for protein II from the overall one (3.3 ( 0.6). The calculations based on eq 1 take into account both the incomplete incorporation of 18O and the differences among the natural isotope envelopes of individual peptides, based on knowledge about individual sequences. An approximate but straightforward calculation was also made of the ratios of precursor proteins of the tryptic peptides, simply by using relative intensities (ratio 2) of monoisotopic peaks of the labeled and unlabeled peptides. An average of ratio 2 of 3.2 ( 0.6 was obtained, in good agreement with the average (3.3 ( 0.6) of ratio 1.The agreement of these two ratios reflects the fact that single incorporation of 18O atom was less than ∼10%. When labeled and unlabeled peptides are present in equivalent amount and single incorporation of 18O atom is smaller than 10%, theoretical calculations suggest that this simplification in quantitation is a good approximation (data not shown). Importance of Unpaired Sets of Isotopic Peaks. In the MS spectra of the 16O- and 18O-labeled adenovirus peptides, there are also unpaired sets of isotopic peaks (Figure 4). There are typically three cases where paired sets (4 Da apart) of isotope peaks will not be observed in a comparative proteomics experiment using this labeling strategy. (1) Carboxyl-terminus peptides of proteins are not labeled, for example, the C-terminal peptide for protein VI (SLRPVASGNWQSTLNSIVGL) as seen in Figure 5a. (2) There
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are mutations, or different posttranslational modifications, in proteins from different cell populations. An example is shown in Figure 5b, where the counterpart peptide from protein IIIa in type 5 does not appear at the position 4 Da lower. In the type 5 peptide (SDASSPFPSLIGSLTSTR), residue L has replaced F in type 2 (SDASSPFPSLIGSFTSTR-18O2). (3) There are extreme changes in expression levels of the corresponding proteins from different pools. In this case, a reverse labeling experiment will be valuable. The cases in which there is a mutation or posttranslational modification of a peptide, or an extreme change in protein expression level, are of interest since they can provide mechanistic information to understand biological processes, identify new drug targets, etc. Overall Considerations of Proteolytic Labeling for Comparative Proteomics. Stable isotope tags provide a very sound strategy for quantitative comparisons of protein abundances. Ideally the label should be introduced prior to cell lysis and sample manipulation8-10 to compensate for any differential loss of material. However, cost and flexibility have motivated the development of chemical methods for postlysis labeling.1,11,12 Proteolytic labeling shares with these the risk that proteins may be differentially lost before the samples are pooled. In this case, proteolysis and labeling are carried out in single, highly specific reactions catalyzed by enzymes, which, ideally, must be driven to completion in order to achieve uniform, global labeling of all peptides. Since proteolytic 18O labeling causes only minimal structural modification at the negatively charged C-termini of peptides, labeled and unlabeled peptides coelute chromatographically (data not shown). The differential isotope labels at the C-termini simplify deduction of sequences from CID spectra and thus facilitate identification of precursor proteins via bioinformatics tools. Design of proteolytic 18O labeling strategy for proteomic studies is modular, in contrast, for example, to integrated design of the ICAT approach.1 The quantitation module (based on 16O/18O codes at C-termini of peptides) and the separation module (based on individual peptide sequences) are separate in the proteolytic labeling method. This modular design allows customized integration of chromatographic, electrophoretic, or affinity-based separations to reduce the complexity of peptide mixtures and to identify maximal numbers of relevant protein targets. Although not demonstrated here, any group of peptides with quantitation codes can be isolated from the same mixture, for instance, cysteinecontaining peptides, phosphopeptides, or glycopeptides, by affinity chromatography. Thus, proteolytic 18O labeling enables a quantitative shotgun approach for proteomic studies. This approach is attractive for proteomic studies of problematic proteins, such as small, large, hydrophobic, or basic molecules.1,2 ACKNOWLEDGMENT The authors thank Y. Hathout and J. Bundy for helpful discussions. Financial support from NIH (GM 21248) is gratefully acknowledged. Presented in part at the 48th Annual Conference of the American Society for Mass Spectrometry, Long Beach, CA, June 2000. Received for review November 30, 2000. Accepted March 26, 2001. AC001404C
