Peptide and Protein Quantitation by Acid-Catalyzed 18O-Labeling of

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Peptide and Protein Quantitation by Acid-Catalyzed 18O-Labeling of Carboxyl Groups Erik Haaf and Andreas Schlosser*,† Core Facility Proteomics, Center for Biological Systems Analysis (ZBSA), Habsburger Strasse 49, 79104 Freiburg, Germany

bS Supporting Information ABSTRACT: We have developed a new method that applies acidic catalysis with hydrochloric acid for 18O-labeling of peptides at their carboxyl groups. With this method, peptides get labeled at their C-terminus, at Asp and Glu residues, and at carboxymethylated cysteine residues. Oxygen atoms at phosphate groups of phosphopeptide are not exchanged. Our elaborated labeling protocol is easy to perform, fast (5 h and 30 min), and results in 95 97 atom % incorporation of 18O at carboxyl groups. Undesired side reactions, such as deamidation or peptide hydrolysis, occur only at a very low level under the conditions applied. In addition, data analysis can be performed automatically using common software tools, such as Mascot Distiller. We have demonstrated the capability of this method for the quantitation of peptides as well as for phosphopeptides.

A

large repertoire of methods using stable isotope labeling is now available for quantitative proteomics. The stable isotopes (e.g., 13C, 15N, and 18O) can be introduced either in vivo on the cellular level as for the metabolic labeling methods, such as SILAC1 or 15N-labeling,2 or in vitro on the peptide or protein level as for chemical tagging methods, such as iTRAQ,3 ICPL,4 or TMT.5 A comprehensive overview on the different quantitation strategies can be found in recent review articles.6,7 Enzyme-catalyzed 18O-labeling is a smart method that allows for the incorporation of stable isotopes at the peptide level without introducing a chemical tag.8 10 A comprehensive overview on this method is given in a recent review article by Fenselau et al.11 Proteases such as trypsin can be used to catalyze the exchange of the oxygen atoms at the C-terminal carboxyl group of peptides. The enzyme-catalyzed 18O incorporation can be performed either directly by digestion in 18O-labeled water or as an additional step in 18O-labeled water after digestion. Avoiding back exchange caused by residual protease activity is a major challenge for enzyme-catalyzed 18O-labeling, and various optimized protocols for avoiding enzyme-catalyzed back exchange have been published recently. It was shown that residual tryptic activity can be suppressed for example by low pH9 or by heating.12 However, the most efficient strategy for avoiding back exchange seems to be the application of immobilized trypsin.13 15 As two 18O atoms are incorporated at the C-terminus of the peptides, the mass difference between the light and the heavy peptides is 4 Da. For larger peptides this can cause overlapping isotopic patterns of the light and heavy peptides, which can hamper data analysis. Recently, the application of acid-catalyzed 18O-labeling as an alternative to enzyme-catalyzed 18O-labeling was suggested.16,17 In contrast to enzyme-catalyzed 18O-labeling, where only the r 2011 American Chemical Society

C-terminal carboxyl group is labeled, all carboxyl groups (i.e., at Asp, Glu, carboxymethylated Cys, C-terminal carboxyl group) are labeled by acidic catalysis. Niles et al. have shown that incubation of synthetic peptides with 50% H218O in the presence of 1% TFA leads to an exchange of 50% of the oxygen atoms of the carboxyl groups within 11 days.17 Liu et al. were able to demonstrate that the reaction time for the acid-catalyzed oxygen exchange can be dramatically reduced to about 15 min by using 2.5% FA in combination microwave irradiation.16 However, both laboratories have limited their analyses to labeling reactions in 50% H218O, conditions that cannot be directly applied for peptide quantitation, since the isotopic patterns of peptides labeled in 50% H218O are broad and in most cases overlap with the isotopic patterns of their corresponding light peptides. Jiang et al. applied acid-catalyzed 18O-labeling with 1% TFA at 37 °C for 85 h for labeling of an internal standard peptide.18 However, complete exchange of all oxygen atoms at all carboxyl groups by acid-catalyzed 18O-labeling in a reasonable reaction time has not been shown to be possible so far.

’ EXPERIMENTAL SECTION Materials and Reagents. All chemicals were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO) unless otherwise stated. Concentrated hydrochloric acid (37% HCl) was purchased from Carl-Roth GmbH (Karlsruhe, Germany). Trypsin was ordered from Promega (Madison, WI), thermolysin from R&D Systems Inc. (Minneapolis, MN), and elastase from MP Biomedicals Received: September 27, 2011 Accepted: November 14, 2011 Published: November 15, 2011 304

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Analytical Chemistry (Solon, OH). LC MS grade acetonitrile (ACN) was obtained from LGC Standards GmbH (Wesel, Germany). 18O-labeled water (99 atom % 18O) was purchased from Sigma-Aldrich and water with 97 atom % 18O was ordered from Euriso-Top (SaintAubin Cedex, France). Water was purified using an Elga Pure Lab Ultra System (Veolia Water Solutions & Technologies, Saint Maurice, France). [Glu1]-Fibrinopeptide B (Glu-Fib, EGVNDNEEGFFSAR) and the synthetic phosphopeptide RA-pS-PPLFQSR were purchased from Sigma-Aldrich. Dionex peptide mixture (P/N 161088) was ordered from Dionex (now Thermo Fisher Scientific, Waltham, MA). Dionex peptide mixture is a tryptic digest of the six proteins cytochrome C (bovine), lysozyme (chicken), alcohol dehydrogenase (yeast), serum albumin (bovine), apo-transferine (bovine), and β-galactosidase (Escherichia coli). The cysteine residues in this mixture are modified by alkylation with iodoacetic acid. The mouse cAMP-dependent protein kinase A catalytic subunit α was ordered from Biaffin GmbH & Co. KG (Kassel, Germany). Acid Catalyzed 18O-Labeling Procedure. Safety Consideration: Hydrochloric acid and HCl gas are extremely corrosive. Inhalation of vapor or gas can cause serious injury. Ingestion may be fatal. Liquid can cause severe damage to skin and eyes. Wear safety glasses or face mask and gloves when working with hydrochloric acid or HCl gas. All work has to be performed in a fume hood. HCl gas was generated in situ by dripping aqueous 37% HCl solution on anhydrous calcium chloride in a glass apparatus (see Figure S-1 in the Supporting Information), dried over phosphorus pentoxide, and then bubbled through 100 μL of 18 O-labeled water (99 atom % 18O) and for the control sample in parallel through H216O. This step was continued for 3 h until the solution was saturated. Then HCl in H218O and HCl in H216O were tempered for 15 min in an Eppendorf Thermomixer (Eppendorf AG, Hamburg, Germany) at 15 °C. Two 10 μL aliquots of Glu-Fib (5 pmol/μL) and two 4 μL aliquots of Dionex peptide mixture (100 fmol/μL) were dried in an Eppendorf Concentrator 5301 (SpeedVac), and 5 μL of HCl in H218O and HCl in H216O, respectively, were added to the dried peptides. Dry argon (additional drying was performed using a “big moisture trap” from Supelco (catalog no. 23991)) was added on top of the samples as a protective layer to keep air humidity away. The samples were incubated at 15 °C for 5 h and 30 min. Hydrochloric acid was subsequently removed by evaporation using a stream of dry nitrogen (additional drying was again performed using a “big moisture trap”) for 30 min. The last residues of hydrochloric acid were neutralized with 2 μL of a saturated solution of NH4HCO3 in H216O or H218O, respectively. Samples were then diluted, mixed in a ratio of 1:1, and acidified with formic acid (FA) for subsequent LC MS/MS analyses. Phosphoprotein Digest. In total, 20 pmol of mouse cAMPdependent protein kinase A catalytic subunit α were reduced with 1 μL of Nupage reducing agent from Invitrogen (Carlsbad, CA) for 10 min at 70 °C and carbamidomethylated with 50 mM iodoacetamide in the dark for 20 min at room temperature. Then the sample was run on a Nupage 4 12% gradient gel (Invitrogen) at 200 V for 40 min. Gel bands were excised and destained with 100 μL of 30% ACN in 100 mM NH4HCO3 pH 8 for 30 min. The supernatants were removed and bands were shrunk with 100 μL of ACN and dried in a SpeedVac for 30 min. Gel bands were resuspended in 30 μL of 100 mM NH4HCO3 pH 8 containing

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0.1 μg of protease. Trypsin, elastase, and thermolysin were used in parallel digests. Digests were carried out at 37 °C overnight. Peptides were extracted with 50% ACN, 0.1% FA from the gel slices and were unified with the corresponding supernatants. Each of the three different digests was split in a ratio of 1:1 and dried in a SpeedVac. Acid-catalyzed 18O-labeling was performed as described above, differentially labeled samples were pooled, and phosphopeptide enrichment was performed separately for each digest. Enrichment of Phosphopeptides Using TiO2. Phosphopeptide enrichment was performed essentially as previously described.19 Samples were dissolved in 5 μL of 50% ACN and acidified with FA to a pH of 2. Enrichment was carried out on a Dionex Ultimate 3000 system (Titanium) with an attached fraction collector (Probot). The TiO2-column was packed with Titansphere (1 cm length, 125 μm i.d.) from GL Sciences Inc. (Tokyo, Japan). Samples were loaded on the TiO2 column with 50% ACN, 0.1% FA at a flow rate of 5 μL/min, washed with 20 μL of 20% ACN, 2% FA, and eluted with 20 μL of 0.1 M ammonium citrate (pH 9.5) at a flow rate of 2 μL/min. The phosphopeptide-enriched fractions were acidified with 1 μL of formic acid and subsequently analyzed with nanoLC MS/MS. NanoLC MS/MS Analysis. NanoLC MS/MS analyses were performed on an Agilent 6520 Q-TOF mass spectrometer (Agilent Technologies, Santa Clara, CA). The instrument was coupled to a 1200 Agilent nanoflow system via a HPLC-Chip cube ESI interface. Peptides were separated on an HPLC-chip with an analytical column of 75 μm i.d. and 150 mm length and a 40 nL trap column both packed with Zorbax 300SB C-18 (5 μm particle size). Peptides were eluted with a linear acetonitrile gradient with 1%/min at a flow rate of 300 nL/min. The Q-TOF was operated in the 2 Ghz extended dynamic range mode. MS/MS analyses were performed using data-dependent acquisition mode. After a MS scan (2 spectra/s), a maximum of three peptides were selected for MS/MS (2 spectra/s). Singly charged precursor ions were excluded from selection. Internal calibration was applied using one reference mass. The precursor selection window was set to medium (width, 4 m/z). Data Analysis. Raw data was processed with Mascot Distiller 2.4 (Matrix Science Ltd., London, U.K.) essentially using provided standard settings for the Agilent Q-TOF. A protein database search was conducted with Mascot Server 2.3 (Matrix Science Ltd., London, U.K.) applying a custom database containing the proteins of interest. Protease specificity was set to “none” for all searches. The 18O-label was defined as a variable modification with the specific C-term (any C-term), D (anywhere), and E (anywhere). Neutral losses (satellite) of 2.004 246 Da (18O O( 1)) and 4.008 491 Da (18O(2) O( 2)) were defined for each specificity. Quantitative analysis was conducted using the Mascot Distiller 2.4 Quantitation Toolbox with an assumed incorporation rate of 95 atom % 18O. The complete quantitation method with all settings is provided as Supporting Information in the supplement. Theoretical isotopic patterns were calculated using IDCalc20 (Michael J. MacCoss, Department of Genome Sciences, University of Washington).

’ RESULTS AND DISCUSSION Acid-Catalyzed 18O-Labeling of Carboxyl Groups of Peptides and Phosphopeptides. We have used the synthetic peptide

[Glu1]-Fibrinopeptide B (Glu-Fib, EGVNDNEEGFFSAR) for 305

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Figure 1. Isotopic patterns of the peptide Glu-Fib (EGVNDNEEGFFSAR, 2+) containing five carboxyl groups. Experimental isotopic pattern before (A) and after (B) acid-catalyzed 18O-labeling of carboxyl groups. Calculated isotopic pattern without 18O incorporation (C) and with 97 atom % 18 O (D).

optimizing the conditions for acid-catalyzed 18O-labeling of carboxyl groups. This peptide contains five carboxyl groups (1 Asp, 3 Glu, and the C-terminal carboxyl group) and thus a total of 10 exchangeable oxygen atoms, which makes it a quite challenging peptide for complete 18O-labeling. For the optimization procedure, we dried 50 pmol aliquots of Glu-Fib in a SpeedVac and redissolved the peptides in 18O-labeled water (99 atom % 18O) together with different types of acids. First of all, we evaluated organic acids such as trifluoroacetic acid (TFA), trifluoromethanesulfonic acid (TFSA), and formic acid (FA) as catalysts for the oxygen exchange reaction. Applying similar conditions as described in a recent publication by Liu et al.16 (2.5% FA, microwave irradiation in a domestic microwave oven) did result in only minor oxygen exchange in our hands. The reason for this is not clear, but it might be that the domestic microwave oven that we have used had not enough power for efficient catalysis. Applying 10% TFA at 70 °C for 60 min was more successful and resulted in about 90% 18O incorporation at the carboxyl groups (data not shown). However, under these conditions a significant degree of peptide hydrolysis was observed. Higher concentrations of TFA (20% and 50%) in combination with lower temperatures (15 °C) and longer reaction times (overnight) did not significantly decrease the degree of peptide hydrolysis, and the 18O incorporation rate was even lower under these conditions (data not shown). We observed that increasing the TFA concentration to more than 10% always

decreased the 18O incorporation rate. We assume that this effect is caused by an exchange of the oxygen atoms of TFA, which at higher TFA concentrations leads to a significant dilution of the 18 O-labeled water with H216O and thus generally limits the achievable 18O incorporation rate. This finding finally led us to the evaluation of strong inorganic acids without exchangeable oxygen atoms. The most favorable acid turned out to be hydrochloric acid (HCl). Hydrochloric acid in 18O-labeled water can be simply prepared (see Figure S-1 in the Supporting Information), and it can be completely removed after the labeling reaction by evaporation. For finding the optimal labeling conditions, we have incubated dried aliquots of Glu-Fib under a variety of different conditions with a solution of 18O-labeled water saturated with dry HCl gas. We were able to achieve 97% 18O incorporation at the carboxyl groups at 15 °C after 5 h and 30 min. Figure 1 shows the experimental isotopic patterns of Glu-Fib before and after the 18O-labeling procedure. The isotopic pattern observed after the oxygen exchange corresponds well to the isotopic pattern of Glu-Fib calculated with an 18O incorporation rate of 97% (Figure 1D). Incorporation of 18O at all carboxyl groups was confirmed by MS/MS (see Figure S-2 in the Supporting Information). In addition to intact Glu-Fib we were able to detect two fragments of Glu-Fib (FFSAR and GFFSAR), caused by hydrolysis of the peptide bonds on either side of Gly, pyro-Glu formation at the N-terminus, and deamidation of the Asn residue. However, all these side products are detected with 306

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Figure 2. Workflow for acid-catalyzed 18O-labeling procedure. The two samples to be compared are dried, incubated with concentrated hydrochloric acid for 5.5 h at 15 °C in either H216O or H218O, dried in a stream of dry nitrogen, and redissolved in NH4HCO3 pH 8. The two differentially labeled peptide samples are pooled and analyzed by nanoLC MS/MS.

Table 1. Quantitative Results for the Dionex Peptide Mixture (1:1 Mixture)a protein transferrin albumin alcohol dehydrogenase

mascot protein score

number of peptides

heavy/light ratio

77 703 69 248

3590 3082

36 40

0.99 ( 0.09 0.97 ( 0.17 1.02 ( 0.12

36 800

802

11

116 409

459

3

1.27 ( 0.11

lysozyme

16 228

497

3

0.98 ( 0.09

cytochrome C

11 697

353

5

1.10 ( 0.05

β-galactosidase

a

molecular mass

The protein H/L ratios are the calculated mean of the corresponding peptide H/L ratios.

only minor peak intensities (base peak chromatograms seen in Figure S-3 in the Supporting Information) in the range of a few percent. Especially the extent of deamidation was much lower than expected (about 1 2%). Deamidation was recently recognized as a side reaction of acid-catalyzed 18O-labeling.16,21 As recognized by others before, the back exchange of the 18Olabel at carboxyl groups is sufficiently slow under standard LC MS conditions (e.g., 0.1% FA).16,17 We incubated labeled peptides in 5% FA overnight at room temperature and were not able to detect any change in the isotopic pattern of the peptides (data not shown). During method optimization we experienced that the most critical step influencing the 18O incorporation rate is the evaporation of the HCl-saturated 18O-labeled water after peptide labeling. It turned out that evaporation in a SpeedVac is highly inappropriate for this step, since peptide hydrolysis seems to be strongly promoted under the conditions in a SpeedVac. As alternative methods we have tested evaporation by a stream of dry nitrogen and lyophilization. We found that evaporation by a stream of dry nitrogen was the best method with only very moderate additional peptide hydrolysis and no measurable oxygen back exchange (see Figure S-4 in the Supporting Information). Since we intend to apply acid-catalyzed 18O labeling for the relative quantitation of phosphorylation sites, we next explored the applicability of this method for phosphopeptides. Figure S-5 in the Supporting Information shows the experimental isotopic pattern of the phosphopeptide RA-pS-PPLFQSR before and after labeling with 18O. As expected, two oxygen atoms (at the C-terminus of the peptide) get exchanged by 18O. The 18O incorporation rate was determined again to 97%. It turned out that the phosphate residue does not interfere with our applied labeling conditions. We neither found any sign of dephosphorylation nor of 18O incorporation at the phosphate moiety. Side products (deamidation at Gln, hydrolysis on the N-terminal side of Ala) were detected again with very low intensities (base peak chromatograms see Figure S-6 in the Supporting Information).

Relative Quantitation of Peptides and Proteins by AcidCatalyzed 18O-Labeling of Carboxyl Groups. Having optimized

the conditions for 18O-labeling of carboxyl groups of peptides, we next applied this labeling strategy for the relative quantitation of peptides and proteins. The general workflow is shown in Figure 2. The two samples to be compared by relative quantitation are consecutively dried (SpeedVac), redissolved in either 16O- or 18 O-water saturated with dry HCl gas and incubated for 5 h and 30 min at 15 °C, dried in a stream of dry nitrogen, and finally redissolved in bicarbonate solution. The two samples treated in this way are pooled and analyzed by nanoLC MS/MS. By treating both samples in the same way under the same conditions, the influence of side reactions, such as deamidation or peptide hydrolytic cleavage, on the quantitation results is minimized. For evaluating the overall performance of the acid-catalyzed 18 O-labeling for the relative quantitation of peptides and proteins, we have used a mixture of peptides generated by tryptic digest of six proteins (Dionex peptide mixture). About 800 fmol of this peptide mixture were split in a ratio of 1:1, and both samples were treated as described in Figure 2. A part of this sample was analyzed with nanoLC MS/MS. An 18O incorporation rate of about 95% was observed for the heavy peptides. The peptide H/L ratios for all identified peptides were calculated from the LC MS/MS raw data using Mascot Distiller. All Metand Thr-containing peptides (except those with N-terminal Thr) were manually excluded (see the discussion below). The results for the relative quantitation are shown in Table 1. All six known proteins of the Dionex peptide mixture could be identified with high Mascot protein scores, and all proteins could be quantitated with at least three peptides. The protein H/L ratios are the calculated mean of the peptide H/L ratios. The proteins serotransferrin and serum albumin were quantitated with 36 and 40 peptides, respectively. The overall distribution of the H/L ratios for all 98 peptides used for quantitation is shown in Figure 3. The calculated mean H/L ratio of all peptides is 1.01 ( 0.14. 307

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Since the cysteine residues of the peptides in the Dionex peptide mixture have been modified by carboxymethylation, an additional carboxyl group is added by each Cys so that some peptides have up to 10 carboxyl groups with a total of 20 exchangeable oxygen atoms. Although the isotopic patterns of peptides with many carboxyl groups get somewhat broadened, the experimental isotopic patterns generally correspond well to the calculated isotopic patterns, and consistent ratios were calculated for these peptides. As an example, Figure S-7 in the Supporting Information shows the isotopic patterns of two different peptides from the Dionex peptide mixture, one peptide with one carboxyl group and one peptide with eight carboxyl groups. A closer look at the peptide MS/MS spectra of the 18O-labeled peptides revealed that these often contain fragments with satellite

peaks at 2 Da and 4 Da, respectively. These satellite peaks appear either when the isotopic patterns of the light and the heavy peptide are very close to each other (e.g., 4 Da mass difference for peptides with only one carboxyl group) and the window for the precursor selection is medium or wide (e.g., as shown in Figure S-8a in the Supporting Information) or for peptides with many carboxyl groups (as shown in Figure S-8b in the Supporting Information). In both cases the Mascot scores for most peptides can be somewhat improved by defining satellite neutral losses of 2 and 4 Da in the Mascot modification configuration (for details see Experimental Section). With these settings, the satellite peaks are ignored during peptide score calculation (the complete comparison with vs without these settings is shown in Table S-1 in the Supporting Information). The detailed analysis of the heavy peptides isotopic patterns revealed that several peptides appear to have a partial incorporation of one additional 18O atom. Figure 4 shows the isotopic pattern of the peptide LVTDLTK as an example. We recognized that peptides with such isotopic patterns more often than not have at least one threonine residue (on the other hand, not all threoninecontaining peptides show this irregularity). Analyzing the MS/ MS spectrum of the peptide LVTDLTK revealed that all observed fragment ions from y3 to y6 show satellite peaks caused by the additional incorporation of one 18O atom, whereas y1 and y2 do not show such satellite peaks (see Figure S-9 in the Supporting Information). This data, as well as the data from several other Thrcontaining peptides with disturbed isotopic patterns, suggests that the additional 18O atom gets incorporated into the backbone amide bond on the N-terminal side of the threonine residue. We assume that the side chain hydroxyl group of threonine residues can catalyze the oxygen exchange at the adjacent N-terminal peptide bond. A proposed mechanism is shown in Figure S-10 in the Supporting Information. The isotopic patterns of some Sercontaining peptides show also evidence for additional incorporation of 18O, however to a much lesser extent. In principle, the partial incorporation of additional 18O atoms into Thr-containing

Figure 3. Distribution of H/L ratios for all 98 peptides of the Dionex peptide mixture used for quantitation. All ratios have been calculated automatically using Mascot Distiller. Met- and Thr-containing peptides have been excluded manually. The calculated mean H/L ratio of all peptides is 1.01 ( 0.14.

Figure 4. Isotopic patterns of the peptide LVTDLTK indicating the exchange of a backbone amide oxygen. (A) Experimental isotopic patterns of the light (monoisotopic m/z 395.239) and heavy (monoisotopic m/z 399.248) form (1:1 mix) showing the additional incorporation of one atom 18O (monoisotopic m/z 400.250). (B) Theoretical isotopic patterns for the light and heavy form of the peptide LVTDLTK calculated with an 18O incorporation rate of 95 atom % 18O. 308

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Figure 5. Analysis of the extent of peptide hydrolysis caused by the treatment with concentrated hydrochloric acid. The Dionex peptide mixture was incubated at 15 °C for 5 h and 30 min with concentrated hydrochloric acid. Trace A gives the base peak chromatogram of the nanoLC MS/MS analysis; trace B gives the extracted ion chromatograms of semitryptic peptides originating from bovine serum albumin as a surrogate for the peptides generated by HCl-hydrolysis. The intensities of the semitryptic peptides (trace B) is more than 1 order of magnitude lower than the intensities of the tryptic peptides (trace A).

peptides could be taken into account by the quantitation software simply by adding the peak intensity of the satellite peak (with one additional 18O atom) to the peak intensity of the corresponding peptide with the correct number of 18O atoms. Unfortunately, the current version of Mascot Distiller was not able to correct the ratios of the affected peptides, so that we manually excluded all Thrcontaining peptides from quantitation. The H/L ratios of a number of Thr-containing peptides are somewhat shifted to smaller H/L ratios (see Figure S-11b in the Suppporting Information). The mean H/L ratio for all Thr-containing peptides is 0.81 ( 0.16. The next release of Mascot Distiller should allow for the necessary correction for the Thr-containing peptides (personal communication John Cottrell, Matrix Science). In addition, we recognized that the ratios of some Metcontaining peptides, no matter whether they are oxidized on methionine or not, exhibit large variations (see Figure S-11c in the Supporting Information). For that reason, we also excluded all Metcontaining peptides from quantitation. Although the two samples have been processed in parallel under (as far as possible) the same conditions, it seems that the degree of methionine oxidation was different for the light and the heavy sample. If the quantitation of Met-containing peptides is for some reason necessary, it would probably be helpful to perform complete oxidation of all methionine residues (e.g., by treatment with H2O2) in both samples before 18O-labeling. Finally, we have analyzed the extent of peptide hydrolysis caused by the treatment with concentrated hydrochloric acid. For that purpose we have incubated the Dionex peptide mixture at 15 °C for 5 h and 30 min with concentrated hydrochloric acid. As a control, the same amount of Dionex peptide mixture was incubated under the same conditions with water. Both samples where then analyzed by nanoLC MS/MS. Protein database searching without protease specificity revealed a total of 44 additional

semitryptic peptides in the HCl-treated sample that do not appear in the control sample, preferentially cleaved at glycine, serine, or threonine residues, demonstrating that peptide hydrolysis under the applied acidic conditions happens. However, analyzing the peak intensities of the semitryptic peptides shows that the extent of peptide hydrolysis is only in the range of a few percent (as already shown for GluFib and for the phosphopeptide RA-pS-PPLFQSR (Figures S-3 and S-6 in the Supporting Information)). Figure 5 shows the extracted ion chromatograms of the semitryptic peptides for serum albumin compared to the base peak chromatogram. Acid-catalyzed peptide hydrolysis was shown to happen only to a minor extent under the applied conditions and did not significantly deteriorate the quantitation results of the samples analyzed in this study. However, the impact of acid-catalyzed peptide hydrolysis on the quantitation results of highly complex samples with very different protein concentrations has not been explored in this study. Although acid-catalyzed 18O-labeling might also be well applicable to highly complex samples, the main area of application is expected to be for samples with medium or low complexity, such as samples obtained after affinity purification. Relative Quantitation of Phosphopeptides by AcidCatalyzed 18O-Labeling of Carboxyl Groups. Finally we evaluated our new 18O-labeling strategy for the relative quantition of phosphorylation sites. For that purpose we used the phosphoprotein protein kinase A catalytic subunit α. We digested the protein in parallel with the proteases trypsin, elastase, and thermolysin. Then we split each digest in a ratio of 1:1, performed the 18O-labeling procedure as described in Figure 2, pooled the heavy and the light peptides for each digest, performed the enrichment of phosphopeptides with titanium dioxide, and analyzed the phosphopeptideenriched fractions by nanoLC MS/MS. We again observed an 18 O-labeling rate of about 95% for the heavy peptides. This indicates that all the different proteases are completely 309

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Table 2. H/L Ratios for All Detected Phosphopeptides of Protein Kinase A Catalytic Subunit α Digested with Trypsin, Elastase, and Thermolysin phosphorylation site

S-11

phosphopeptide sequence

H/L

AAKKGpSEQESVKEFLA AAKKGpSEQESVKEFLA

1.01 0.91

elastase elastase

AKKGpSEQESVKEFLA

0.91

elastase

AKKGpSEQESVKEFLA

0.92

elastase

KKGpSEQESVKEFLAK

1.05

trypsin

KKGpSEQESVKEFLAK

1.13

trypsin

KKGpSEQESVKEFLAKA

0.94

elastase

KKGpSEQESVKEFLAKA

0.90

elastase

KKGpSEQESVKEFLA KKGpSEQESVKEFLA

0.88 0.88

elastase elastase

KGpSEQESVKEFLAK

1.06

trypsin

KGpSEQESVKEFLAK

1.14

trypsin

KGpSEQESVKEFLAK

1.12

trypsin

GpSEQESVKEFLAK

0.98

elastase

GpSEQESVKEFLAK

1.08

trypsin

GpSEQESVKEFLAK

1.08

trypsin

pSEQESVKEFLAK pSEQESVKEFLAK

1.11 1.10

trypsin trypsin

pSEQESVKEFLA RIGRFpSEPHAR RIGRFpSEPHAR

S-140

S-339

S-339

protease

0.97

elastase

1.17

trypsin

1.20

trypsin

IGRFpSEPHARF

1.23

thermolysin

IGRFpSEPHAR

1.15

trypsin

IGRFpSEPHAR

1.14

trypsin

IGRFpSEPHAR GRFpSEPHARFYAA

1.11 1.12

thermolysin elastase

GRFpSEPHARFYA

additional

threonine

modifications

containing

1.13

elastase

RFpSEPHAR

1.23

trypsin

RVpSINEKCGKEF

1.12

elastase

RVpSINEKCGKEF

1.13

elastase

carbamidomethyl (C)

IRVpSINEKCGKE

1.12

thermolysin

carbamidomethyl (C)

IRVpSINEKCGKE

carbamidomethyl (C)

carbamidomethyl (C)

1.18

thermolysin

FKGPGDTSNFDDYEEEEIRVpSINEK FKGPGDTSNFDDYEEEEIRVpSINEK

0.87 0.86

trypsin trypsin

+ +

FKGPGDTSNFDDYEEEEIRVpSINEK

0.87

thermolysin

+

FKGPGDTSNFDDYEEEEIRVpSINEK

0.79

thermolysin

+

GPGDTSNFDDYEEEEIRVpSINEK

0.88

trypsin

+

RVpSINEKCGKEFTEF

0.81

elastase

carbamidomethyl (C)

+

RVpSINEKCGKEFTEF

0.81

elastase

carbamidomethyl (C)

+

RVpSINEKCGKEFTEF

0.76

elastase

carbamidomethyl (C)

+

RVpSINEKCGKEFT

0.79

elastase

carbamidomethyl (C)

+

inactivated by the treatment with concentrated hydrochloric acid, so that no back exchange at the C-terminal carboxyl groups happens. In addition this result shows that no significant back exchange during the enrichment of the phosphopeptide happens, although the phosphopeptides are exposed to acidic conditions during the loading and washing step (2% FA, pH 2) and to basic conditions during the elution step (pH 9.5). Figure S-12 in the Supporting Information shows the isotopic patterns of the phosphopeptide RIGRF-pS-EPHAR in its light and heavy version as an example. Table 2 summarizes the quantitative results for all detected phosphopeptides. All H/L ratios of the phosphopeptides

generated with different proteases are consistent and show only minor deviations, except for the Thr-containing peptides that again show a small systematic shift to lower ratios. The H/L ratios of the overlapping phosphopeptides can be averaged and used to increase the quantitation accuracy for each phosphorylation site.

’ CONCLUSIONS Here we present a new protocol for acid-catalyzed 18Olabeling of peptides at their carboxyl groups. With this protocol it is possible to achieve 95 97% 18O-labeling within 5 h and 310

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Analytical Chemistry 30 min, which makes this quantitation method readily combinable with standard workflows for quantitative proteomics. Acidcatalyzed 18O-labeling can be applied for any peptide with at least one carboxyl group, which makes this strategy applicable for peptides where the well-established enzyme-catalyzed 18O-labeling strategy fails, e.g., for C-terminal peptides, for peptides with a modified C-termini, for peptide mixtures that are not generated by digest with a single protease (e.g., peptides isolated from serum or cerebrospinal fluid), or for peptides generated by chemical cleavage (e.g., BrCN-cleavage). As already pointed out by others,16,17 acid-catalyzed 18O-labeling has the advantage that most peptides have a mass difference larger than 4 Da, which reduces the risk of overlapping isotopic patterns and makes data analysis easier and more reliable. There are no special requirements for data analysis, and common quantitation tools, such as Mascot Distiller, can be used for quantitation. Back exchange of oxygen atoms by residual protease activity is a fundamental challenge in enzyme-catalyzed 18O-labeling, and various strategies for avoiding back exchange after enzymecatalyzed 18O-labeling have been developed.12,13 With the acid-catalyzed 18O-labeling strategy, proteases are completely deactivated during the incubation in concentrated hydrochloric acid, thus avoiding protease-catalyzed back exchange of oxygen atoms without the need of any additional precautions. In addition we have shown that no significant back exchange happens when peptides are handled under conditions typically used for peptide LC MS analysis (e.g., 0.1% FA) or phosphopeptide enrichment (e.g., 2% FA). We have shown here that acid-catalyzed 18Olabeling can be applied for unmodified peptides as well as for phosphopeptides, and we assume that this strategy is also applicable to peptides with other kinds of covalent modifications except for acid-labile modifications. In contrast to chemical tagging strategies (e.g., iTRAQ, ICPL, TMT) that are often applied in quantitative proteomics, acidcatalyzed 18O-labeling does not introduce any chemical group, and thus does not alter any peptides property, such as hydrophobicity, ionization efficiency, or peptide fragmentation behavior. The acid-catalyzed 18O-labeling procedure is simple to perform without the need for any additional cleanup procedure. The hydrochloric acid used for isotopic labeling can be easily removed by evaporation. Finally, acid-catalyzed 18O-labeling is costefficient, especially when water with 97 atom % 18O is used. Our analyses did not indicate any significant advantage using water with 99 atom % 18O. In summary, with the new protocol presented here, acid-catalyzed 18O-labeling becomes a versatile tool for quantitative proteomics that should find various applications in the future.

ARTICLE

’ ACKNOWLEDGMENT We thank Stephanie Lamer and Ulrike Lanner for technical support. ’ REFERENCES (1) Ong, S. E.; Blagoev, B.; Kratchmarova, I.; Kristensen, D. B.; Steen, H.; Pandey, A.; Mann, M. Mol. Cell. Proteomics 2002, 1, 376–386. (2) Krijgsveld, J.; Ketting, R. F.; Mahmoudi, T.; Johansen, J.; ArtalSanz, M.; Verrijzer, C. P.; Plasterk, R. H. A.; Heck, A. J. R. Nat. Biotechnol. 2003, 21, 927–931. (3) Hunt, T.; Huang, Y.; Ross, P.; Pillai, S.; Purkayastha, S.; Pappin, D. Mol. Cell. Proteomics 2004, 3, S286–S286. (4) Schmidt, A.; Kellermann, J.; Lottspeich, F. Proteomics 2005, 5, 4–15. (5) Hamon, C.; Thompson, A.; Schafer, J.; Kuhn, K.; Kienle, S.; Schwarz, J.; Schmidt, G.; Neumann, T. Anal. Chem. 2003, 75, 1895– 1904. (6) Bantscheff, M.; Schirle, M.; Sweetman, G.; Rick, J.; Kuster, B. Anal. Bioanal. Chem. 2007, 389, 1017–1031. (7) Yao, X. Anal. Chem. 2011, 83, 4427–4439. (8) Schnolzer, M.; Jedrzejewski, P.; Lehmann, W. D. Electrophoresis 1996, 17, 945–953. (9) Stewart, I. I.; Thomson, T.; Figeys, D. Rapid Commun. Mass Spectrom. 2001, 15, 2456–2465. (10) Mirza, S. P.; Greene, A. S.; Olivier, M. J. Proteome Res. 2008, 7, 3042–3048. (11) Fenselau, C.; Yao, X. D. J. Proteome Res. 2009, 8, 2140–2143. (12) Petritis, B. O.; Qian, W. J.; Camp, D. G.; Smith, R. D. J. Proteome Res. 2009, 8, 2157–2163. (13) Bezstarosti, K.; Ghamari, A.; Grosveld, F. G.; Demmers, J. A. A. J. Proteome Res. 2010, 9, 4464–4475. (14) Fenselau, C.; Brown, K. J. J. Proteome Res. 2004, 3, 455–462. (15) Bundy, J. L.; Sevinsky, J. R.; Brown, K. J.; Cargile, B. J.; Stephenson, J. L. Anal. Chem. 2007, 79, 2158–2162. (16) Liu, N.; Wu, H. Z.; Liu, H. X.; Chen, G. N.; Cai, Z. W. Anal. Chem. 2010, 82, 9122–9126. (17) Niles, R.; Witkowska, H. E.; Allen, S.; Hall, S. C.; Fisher, S. J.; Hardt, M. Anal. Chem. 2009, 81, 2804–2809. (18) Jiang, H.; Ramos, A. A.; Yao, X. D. Anal. Chem. 2010, 82, 336– 342. (19) Schlosser, A.; Vanselow, J. T.; Kramer, A. Anal. Chem. 2005, 77, 5243–5250. (20) Kubinyi, H. Anal. Chim. Acta 1991, 247, 107–119. (21) Wang, S.; Bobst, C. E.; Kaltashov, I. A. Anal. Chem. 2011, 83, 7227–7232.

’ ASSOCIATED CONTENT

bS Supporting Information. Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org. ’ AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected]. Present Address †

Rudolf Virchow Center for Experimental Biomedicine, University of Wuerzburg, Josef-Schneider-Str. 2, 97080 Wuerzburg, Germany. 311

dx.doi.org/10.1021/ac202561m |Anal. Chem. 2012, 84, 304–311