Stoichiometry Determination of the MP1−p14 Complex Using a Novel

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Anal. Chem. 2009, 81, 10254–10261

Stoichiometry Determination of the MP1-p14 Complex Using a Novel and Cost-Efficient Method To Produce an Equimolar Mixture of Standard Peptides Johann Holzmann,*,† Peter Pichler,‡ Mathias Madalinski,† Robert Kurzbauer,† and Karl Mechtler†,§ Research Institute of Molecular Pathology (IMP), CD-Laboratory for Proteome Analysis, and Institute of Molecular Biotechnology (IMBA), 1030 Vienna, Austria Determination of protein complex stoichiometry can be achieved by absolute quantification of the interacting constituents based on isotope dilution mass spectrometry. Current available platforms for the generation of standard peptides are cost-intensive and deliver variable results concerning the equimolarity of the standard peptides. Here we describe a novel and cost-efficient method to generate an equimolar mixture of standard peptides, which we call the equimolarity through equalizer peptide (EtEP) strategy. The rationale of the strategy allows equalization of internal standard peptides and absolute quantification of any protein of interest via a single peptide, from which the exact amount was determined by amino acid analysis. This and the use of the mTRAQ reagent significantly decrease the costs of absolute quantification and stoichiometry determination. We used the EtEP strategy to determine the MP1-p14 complex stoichiometry of two different concentrations, one simulating a condition following tandem affinity purification. Absolute quantification of MP1-p14 was performed on two different mass spectrometers, and the 1:1 stoichiometry was confirmed with high accuracy and precision. On the basis of the quantification of MP1-p14, we demonstrate the importance to assess completeness of protein digestion and discuss the use of peptides containing labile amino acids and the choice of instrumentation. Proteins assemble into macromolecular complexes that form dynamic networks of high complexity to regulate fundamental cellular processes. Affinity purification coupled to tandem mass spectrometry (AP-MS) greatly advanced our understanding of the composition of protein complexes and their networks,1 but it so far has led to mostly qualitative data.2 To gain deeper understanding of the structural organization of a complex, it is * Corresponding author. Phone: +43-1-79044-4293. Fax: +43-1-79044-110. E-mail: [email protected]. † IMP. ‡ CD-Laboratory for Proteome Analysis. § IMBA. (1) Rigaut, G.; Shevchenko, A.; Rutz, B.; Wilm, M.; Mann, M.; Seraphin, B. Nat. Biotechnol. 1999, 17, 1030–1032. (2) Gingras, A.-C.; Gstaiger, M.; Raught, B.; Aebersold, R. Nat. Rev. Mol. Cell Biol. 2007, 8, 645–654.

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important to determine its stoichiometry. Successfully applied methods to achieve this are native MS and correlational quantification,3,4 yet another important and promising strategy is based on the absolute quantification of all interacting constituents. As mass spectrometry is not quantitative per se, the only strategy allowing for an accurate absolute quantification is to use isotopically labeled internal standards.5 This approach, referred to as isotope dilution mass spectrometry (IDMS), was only recently transferred to the absolute quantification of peptides.6,7 Nevertheless, different strategies have been developed to produce such labeled internal standard peptides. In the original strategy standard peptides are chemically synthesized and carry an isotopically labeled amino acid that was incorporated during synthesis. This method is referred to as the absolute quantification (AQUA) strategy.7 Alternatively, the isotopic label can be introduced after synthesis by chemical labeling (for an overview of the available chemical-labeling strategies see ref 5). Chemical labeling of internal standard peptides was successfully used to confirm the 1:1:1 stoichiometry of the human spliceosome U1 small nuclear ribonucleoprotein.8 However, in both strategies the exact concentration of all standard peptides has to be determined. Amino acid analysis (AAA) is widely regarded as the golden standard for the absolute quantification of peptides and proteins, although an average error of >10% was reported.9 Additionally, the analysis is time-consuming and costintensive. Furthermore, AQUA peptides are usually provided lyophilized, and resolubilization can be incomplete, potentially leading to erroneous absolute quantification.

(3) Heck, A. J. R. Nat. Methods 2008, 5, 927–933. (4) Wepf, A.; Glatter, T.; Schmidt, A.; Aebersold, R.; Gstaiger, M. Nat. Methods 2009, 6, 203–205. (5) Ong, S. E.; Mann, M. Nat. Chem. Biol. 2005, 1, 252–262. (6) Desiderio, D. M.; Kai, M. Biomed. Mass Spectrom. 1983, 10, 471–479. (7) Gerber, S. A.; Rush, J.; Stemman, O.; Kirschner, M. W.; Gygi, S. P. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 6940–6945. (8) Hochleitner, E. O.; Kastner, B.; Frohlich, T.; Schmidt, A.; Luhrmann, R.; Arnold, G.; Lottspeich, F. J. Biol. Chem. 2005, 280, 2536–2542. (9) Schegg, K. M.; Denslow, N. D.; Andersen, T. T.; Bao, Y.; Cohen, S. A.; Mahrenholz, A. M.; Mann, K. Tech. Protein Chem. VIII, [Symp. Protein Soc.], 10th, 1996 1997, 8, 207-216, 908. 10.1021/ac902286m  2009 American Chemical Society Published on Web 11/19/2009

In an alternative approach called QconCAT,10,11 and a closely related method termed PCS,12 isotopically labeled standard peptides are produced by fusing internal standard peptides to an artificial protein. After recombinant expression and purification, a trypsin digestion ideally results in an equimolar mixture of all standard peptides. Refined approaches successfully revealed the stoichiometry of the eIF2B-eIF2 complex12 and of the transducin complex.13 As a consequence of the rationale of QconCAT, erroneous concentration determination by AAA does not affect determination of stoichiometry, as long as equimolar amounts of standard peptides are generated by the digest. In practice, the limitations of the method are the high costs of the chemically synthesized artificial gene, potentially poor expression, and difficulties in purification of the protein as well as potentially incomplete digestion resulting in a nonequimolar mixture of standard peptides.14 Mirzaei et al. compared the AQUA and the QconCAT strategies for the production of an equimolar mixture of standard peptides. For their particular setting they found that the peptide mixture prepared with both strategies deviated from equimolarity. After optimization of the QconCAT construct and the trypsin digestion, the ratio of AQUA to QconCAT peptides was found to be 1.23 ± 0.68, demonstrating that generation of an equimolar peptide mixture is an important issue and requires optimization.14 Here we describe a novel and cost-efficient approach to produce an equimolar mixture of internal standard peptides of high accuracy, which we call the equimolarity through equalizer peptide (EtEP) strategy. In principle, the method can be seen as an intermediate between the AQUA and QconCAT approaches. The general outline of the strategy is illustrated in Figure 1. In brief, internal standard peptides are chemically synthesized including an N-terminal equalizer peptide (EP), which is a nonnaturally occurring tryptic peptide. After trypsinization and chemical labeling, an equimolar mixture of standard peptides is generated by normalizing all peptides to the same absolute amount of the EP. The method combines the benefits of AQUA and QconCAT. The fusion peptides are short enough to allow chemical synthesis as in the AQUA strategy, allowing flexibility in the study design and circumventing problems associated with generation and handling of the artificial QconCAT protein. Once equalized, the standard peptides are kept in solution to circumvent problems associated with resolubilization. In line with the rationale of the QconCAT strategy our method allows bringing the standard peptides to equimolarity, but in contrast to QconCAT, EtEP fusion peptides contain only one optimized trypsin cleavage site, alleviating complete and specific digestion. The major advantage over AQUA and QconCAT is based on the rationale of the EtEP strategy. As only one peptide (the light labeled EP) is requiredsfrom which the absolute concentration has to be determined by AAAsthe costs for absolute quantification can be significantly decreased. In fact, for the purpose of

stoichiometry determination the absolute amount of the EP does not necessarily have to be determined, since equalization of standard peptides and calculation of peptide ratios are independent of the exact amount of this peptide species. A further decrease in costs comes with the usage of the mTRAQ reagent to introduce the isotopic label. mTRAQ is the nonisobaric version of the iTRAQ reagent, leading to a mass difference of 4 Da between light and heavy labeled peptide and thus allowing quantification based on the IDMS principle.15,16 We applied the new method to determine the protein stoichiometry of the endosomal adaptor complex MP1-p14. As stoichiometry determination by absolute quantification is dependent on the isolation of a single homogeneous complex, we chose to work with a recombinant protein complex of known stoichiometry.17 Thereby we avoided a situation with an unclear interpretation of protein stoichiometry, allowing the validation of our method. Using three different EPs, we confirmed the 1:1 stoichiometry of the recombinant MP1-p14 complex on two different MS platforms: a QTRAP 4000 hybrid triple-quadrupole/linear ion trap operated in SRM mode and an LTQ Orbitrap. To simulate a situation following affinity purification, we lowered the protein concentration to the submicromolar range and demonstrate that, only after adding ubiquitin as the carrier, protein digestion is complete and the correct stoichiometry can be determined. We show that stoichiometry determination via absolute quantification of protein subunits requires careful selection of standard peptides, implicating the importance of complete and unspecific digestion and an accurate method to produce an equimolar mixture of standard peptides.

(10) Pratt, J. M.; Simpson, D. M.; Doherty, M. K.; Rivers, J.; Gaskell, S. J.; Beynon, R. J. Nat. Protoc. 2006, 1, 1029–1043. (11) Rivers, J.; Simpson, D. M.; Robertson, D. H. L.; Gaskell, S. J.; Beynon, R. J. Mol. Cell. Proteomics 2007, 6, 1416–1427. (12) Kito, K.; Ota, K.; Fujita, T.; Ito, T. J. Proteome Res. 2007, 6, 792–800. (13) Nanavati, D.; Gucek, M.; Milne, J. L. S.; Subramaniam, S.; Markey, S. P. Mol. Cell. Proteomics 2008, 7, 442–447. (14) Mirzaei, H.; McBee, J. K.; Watts, J.; Aebersold, R. Mol. Cell. Proteomics 2008, 7, 813–823.

(15) Ross, P. L.; Huang, Y. N.; Marchese, J. N.; Williamson, B.; Parker, K.; Hattan, S.; Khainovski, N.; Pillai, S.; Dey, S.; Daniels, S.; Purkayastha, S.; Juhasz, P.; Martin, S.; Bartlet-Jones, M.; He, F.; Jacobson, A.; Pappin, D. J. Mol. Cell. Proteomics 2004, 3, 1154–1169. (16) DeSouza, L. V.; Taylor, A. M.; Li, W.; Minkoff, M. S.; Romaschin, A. D.; Colgan, T. J.; Siu, K. W. M. J. Proteome Res. 2008, 7, 3525–3534. (17) Kurzbauer, R.; Teis, D.; de Araujo, M. E. G.; Maurer-Stroh, S.; Eisenhaber, F.; Bourenkov, G. P.; Bartunik, H. D.; Hekman, M.; Rapp, U. R.; Huber, L. A.; Clausen, T. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 10984–10989.

EXPERIMENTAL SECTION General Chemicals and Reagents. All chemicals purchased were of the highest purity available. Trifluoroacetic acid (TFA), acetonitrile (ACN), 2-propanol, and tris(2-carboxyethyl)phosphine hydrochloride (TCEP) were purchased from Sigma-Aldrich (Steinheim, Germany). Formic acid (FA) was obtained from SAFC Biosciences (Andower, U.K.). Triethlyammonium bicarbonate (TEAB) and S-methyl thiomethanesulfonate (MMTS) were from Fluka (Buchs, Switzerland). Sequencing-grade modified trypsin was purchased from Promega (Madison, WI). Ultrapure 18 MΩ water was obtained from a Millipore Milli-Q system (Bedford, MA). Generation of Internal Standard Peptides. All peptides were synthesized by automated solid-phase Fmoc chemistry inhouse using a Syro peptide synthesizer (MultiSyntech, Bochum, Germany) in 96-well microtiter plates. Amino acids were purchased from Novabiochem (Laeufelfingen, Switzerland). Peptides were dissolved in 10% ACN, 0.1% TFA and purified on a Vision HPLC instrument (Applied Biosystems, Foster City, CA) equipped with a Phenomenex Jupiter C18 column (4.6 mm i.d. × 250 mm length, 5 µm particle size, 300 Å pore size) (Phenomenex,

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Figure 1. Overview of the EtEP strategy. (a) (1) Internal standard peptides are chemically synthesized with the EP concatenated to the N-terminus of the peptides. (2) A tryptic digest of each individual synthesized fusion peptide releases equimolar amounts of the EP and standard peptide. Peptides are then labeled with the heavy version of the mTRAQ reagent, targeting free primary amines at the amino termini and lysine side chains. After labeling, all different standard peptides are quantified via the EP and mixed accordingly to give an equimolar mixture. A spiked light labeled version of the EP, from which the concentration was determined by AAA, serves as an internal standard. (3) The protein complex of interest is digested with trypsin and labeled with the light version of the mTRAQ reagent. (4) The equimolar mixture of standard peptides is mixed with the light labeled tryptic peptides from the protein complex to determine the absolute abundance of the subunits. (5) Complex stoichiometry is calculated from the absolutely quantified subunits. (b) Three MP1 peptides and three p14 peptides selected for quantification were chemically synthesized concatenated to N-terminal equalizer peptides 1, 2, and 3, respectively.

Torrance, CA), and their identity was confirmed by MALDI-TOF (Bruker Daltonics Reflex III, Bremen, Germany). After lyophilization peptides were redissolved in 10% ACN and 500 mM TEAB at a concentration of 0.5 mg/mL and trypsinized for 2 h at 37 °C (1:100 trypsin to protein ratio). Next 10 µg of each was separately labeled with one-sixth of a unit of the heavy version of the mTRAQ reagent (Applied Biosystems) according to the manufacturer’s instructions. After 1 h peptides were diluted 5-fold with 1% TFA and stored at -80 °C until analysis. For equalization, approximately 5 pmol of each heavy labeled standard peptide were spiked with exactly 5 pmol of light labeled equalizer peptide and analyzed individually on the two mass analyzers to determine the absolute amount of the standard peptides via the EP. Mass spectrometric analysis was performed in triplicate as described below. Standard peptides were then combined according to the absolute quantification to give an equimolar mixture and stored at -80 °C. 10256

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Generation of the Light Labeled Equalizer Peptides. The three equalizer peptides ASALVNTIAR, GVTASVAGAR, and ASGVNTIAR were synthesized and purified as the fusion peptides described above, but on a larger scale. A 3 mg portion of each peptide was labeled with one-third of a 50 unit mTRAQ light reaction vial according to the manufacturer’s instructions. After lyophilization peptides were resolved in 5% ACN, 0.1% TFA and purified using a Vision HPLC equipped with a Phenomenex Jupiter C18 column (10 mm i.d. × 250 mm length, 5 µm particle size, 300 Å pore size) (Phenomenex). Purified and labeled equalizer peptides were sent to Thermo Fisher Scientific (Ulm, Germany) for amino acid analysis and aliquoting. Expression and Purification of MP1His6-p14. MP1His6-p14 was recombinantly expressed and purified essentially as described, except that an additional purification step was performed using an ion exchanger resin (MonoQ HR 5/5 from GE Healthcare, Piscataway, NJ) to remove small traces

of unbound MP1His6.17 The purity was assessed by SDS-PAGE, and the concentration was determined from the absorption at 280 nm using the specific absorption coefficient. Trypsin Digestion and Labeling of the MP1-p14 Complex. MP1-p14 was diluted to a final concentration of 0.5 mg/ mL (17.8 µM) in 500 mM TEAB, reduced for 30 min at 56 °C with a final concentration of 1 mM MMTS, and alkylated with a final concentration of 2 mM TCEP for 30 min at room temperature in the dark. To 20 µL of MP1-p14 were added different amounts of trypsin (800, 400, and 200 ng), and the ratio of trypsin to protein was therefore 1:12.5, 1:25, and 1:50, respectively. For the experiments where a denaturant was added, MP1-p14 was diluted to 0.5 mg/mL with a final concentration of 2 M urea, 80% ACN, and 0.1% RapiGest (Waters, Milford, MA), respectively. The final TEAB concentration in the 80% ACN solvent was 150 mM. The incubation time and temperature were varied as described in the Results and Discussion. For the simulation of a condition following affinity purification, MP1-p14 was diluted after reduction and alkylation to a concentration of 25 µg/mL (890 nM) using 500 mM TEAB. Different amounts of trypsin were added to result in a trypsin to protein ratio of 1:25, 1:12.5, and 1:5, respectively. Samples were incubated for 16 h at 43 °C. Immediately after the incubation period an aliquot was analyzed on a Dionex Ultimate Switchos equipped with a 200 µm i.d. monolithic column (PepSwift from Dionex, Amsterdam, The Netherlands) and separated with a gradient from 5% ACN, 0.1% TFA to 80% ACN, 10% TFE, 0.08% TFA over 25 min. Completely digested samples (10 µg each) were labeled with one-sixth of a unit of mTRAQ light reagent according to the manufacturer’s instructions. After 1 h samples were diluted 5-fold with 1% TFA and stored at -80 °C until analysis. Nano-RP-HPLC. Immediately prior to mass spectrometric analysis digested MP1-p14 was spiked with the standard peptide mixture at a ratio of approximately 1:1 and treated with a final concentration of 0.5% hydrogen peroxide for 20 min. Oxidation was quenched by addition of dithiotreithol. Samples were then diluted to a 2-propanol concentration of 99%. A list of three SRM transitions per light and heavy peptide was generated for quantification on the QTRAP instrument (Table S-2, Supporting Information). The digested and labeled MP1-p14 complex was spiked with the three different mixtures of internal standard peptides, and approximately 200 fmol of MP1-p14 was loaded onto the column. No unspecific transitions were observed, and light and heavy labeled peptides eluted simultaneously. LCSRM chromatograms are shown in Figure 4a for EP2 and in Figures S-7a and S-8a (Supporting Information) for EP1 and EP3, respectively. The measured absolute amounts of MP1-p14 peptides differed on average by 2.7% with an SD of 1.4% between the two instruments (Figure S-10, Supporting Information). The average MP1:p14 ratio calculated over the three EPs and the two instruments was 1.00 with an average SD of 0.05 excluding the labile p14 peptide NGNQAFNEDSLK and 0.94 with an average SD of 0.10 including this peptide (Table 1, top section); hence, inclusion of this partially deamidated p14 peptide introduced an error in the determination of stoichiometry. While the peptide scattering (i.e., the relative standard deviation of the absolute amounts measured for the different peptides) was 3.3% over the three EPs and the two MS platforms excluding the peptide NGNQAFNEDSLK, the scattering increased to 7.6% including this peptide (see Figure S-10). The stronger deviation of the NGNQAFNEDSLK peptide from the average can most likely be explained by differences in the ionization efficiencies of the native and deamidated forms. As the degree of deamidation differed between the light and the heavy labeled peptide, an error is introduced when the areas of the two forms are summed. However, if the labile peptide is excluded, the degree of scattering is well below the average error of AAA as well as the scattering of QconCAT peptides, demonstrating the power of our method to generate an equimolar mixture of standard peptides.9,14 The accuracy of our method is also illustrated by the fact that the scattering of measured absolute peptide levels within each experiment using an individual EP (EP1, 3.8%; EP2, 2.8%; EP3, 3.4%sexcluding NGNQAFNEDSLK) is significantly less than the standard deviation of the measured absolute peptide levels calculated using data from all the experiments with the three different EPs together (9%). As the EPs are the only peptides from which the absolute amount was determined by AAA, the differences obtained most probably reflect the error in absolute quantification by AAA. While the average difference between peptide amounts quantified via EP2 and EP3 was within the

Figure 4. LC-SRM traces of MP1-p14 peptides. Absolute quantification using internal standard peptides equilibrated via EP2. (a) LCSRM traces of approximately 200 fmol of the MP1-p14 complex (concentration of 17.8 µM at digestion) spiked with 200 fmol of internal standard peptides. The signal intensity of quantified peptides was at least 2000 cps. (b) UV chromatogram of the experiment described in (a). (c) LC-SRM traces of approximately 100 fmol of the MP1-p14 complex (concentration of 890 nM at digestion) spiked with 100 fmol of internal standard peptides. No unspecific transitions were detected. The signal intensity of quantified peptides was at least 1000 cps. (d) UV chromatogram of the experiment described in (c). As ubiquitin is added in a 20-fold excess, the UV chromatogram is dominated by ubiquitin peaks.

technical variance, absolute amounts determined via EP1 were on average 18% less than those measured with EP2 and EP3, respectively (Table 1 and Figure S-10). Anyhow, a standard deviation of 9% for the three EPs is in good agreement with the average error reported for AAA. To assess the linearity of quantification, we spiked different amounts of equalized standard peptides to the trypsinized MP1-p14 complex. Figure S-9 (Supporting Information) shows that absolute quantification on both instruments is independent of the ratio between light and heavy in the range tested. The same stoichiometry was determined when the amounts of spiked standard peptides were in the range of 1/5 to 5× the amount of native MP1-p14 peptides, which indicates the linearity and robustness of our method. Analytical Chemistry, Vol. 81, No. 24, December 15, 2009

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Table 1. Absolute Quantification of MP1-p14 and Calculation of the Complex Stoichiometrya excluding NGNQAFNEDSLK EP1 EP1 EP2 EP2 EP3 EP3

QTRAP Orbitrap QTRAP Orbitrap QTRAP Orbitrap

including NGNQAFNEDSLK

amt of MP1 (fmol)

amt of p14 (fmol)

MP1:p14

amt of MP1 (fmol)

amt of p14 (fmol)

MP1:p14

189.32 ± 8.25 182.25 ± 8.75 226.34 ± 8.10 223.83 ± 7.67 222.81 ± 6.81 222.51 ± 5.27

186.95 ± 9.19 185.82 ± 3.25 219.41 ± 4.15 223.89 ± 1.40 226.05 ± 6.27 225.05 ± 6.27

1.01 ± 0.07 0.98 ± 0.05 1.03 ± 0.04 1.00 ± 0.03 1.00 ± 0.07 0.99 ± 0.04

189.32 ± 8.25 182.25 ± 8.75 226.34 ± 8.10 223.83 ± 7.67 222.81 ± 6.81 224.51 ± 5.27

200.51 ± 24.38 194.70 ± 15.55 232.47 ± 22.81 233.22 ± 16.19 242.84 ± 29.45 241.11 ± 26.47

0.94 ± 0.12 0.94 ± 0.09 0.97 ± 0.10 0.96 ± 0.07 0.93 ± 0.12 0.92 ± 0.11

excluding NGNQAFNEDSLK EP1 EP1 EP2 EP2 EP3 EP3

QTRAP Orbitrap QTRAP Orbitrap QTRAP Orbitrap

including NGNQAFNEDSLK

amt of MP1 (fmol)

amt of p14 (fmol)

MP1:p14

amt of MP1 (fmol)

amt of p14 (fmol)

MP1:p14

93.01 ± 4.39 90.18 ± 3.45 111.30 ± 2.41 109.75 ± 5.39 111.22 ± 5.07 112.46 ± 5.10

94.13 ± 1.19 90.95 ± 2.45 112.22 ± 5.98 110.83 ± 5.59 110.45 ± 9.90 112.23 ± 3.30

0.99 ± 0.05 0.99 ± 0.05 0.99 ± 0.06 0.99 ± 0.07 1.01 ± 0.10 1.00 ± 0.05

93.21 ± 4.39 90.18 ± 3.45 111.30 ± 2.41 109.75 ± 5.39 111.22 ± 5.07 112.46 ± 5.10

102.63 ± 16.48 99.04 ± 14.53 119.42 ± 13.18 117.51 ± 12.24 121.91 ± 22.76 120.98 ± 15.34

0.91 ± 0.15 0.91 ± 0.14 0.93 ± 0.10 0.93 ± 0.11 0.91 ± 0.18 0.93 ± 0.13

a Quantification was carried out using two different instruments. Data are the mean ± SD of triplicate measurements. Three different standard peptide mixtures were used for quantification (equalization via EP1, EP2, and EP3, respectively). Complex stoichiometries are given, which were calculated from the absolutely quantified subunits MP1 and p14, either excluding NGNQAFNEDSLK or including NGNQAFNEDSLK. (Top section) MP1-p14 was digested at a concentration of 17.8 µM, and approximately 200 fmol was analyzed on the column. (Bottom section) MP1-p14 was digested at a concentration of 890 nM, and approximately 100 fmol was analyzed on the column.

Simulation of a Condition Following Tandem Affinity Purification. Using a bottom-up approach to determine the stoichiometry of an affinity-purified complex, two issues need special attention: complete digestion of low- to submicromolar protein concentrations and homogeneity of the purified complex. As homogeneity is a matter of complex stabilization and optimization of the purification procedure, this issue was not addressed in this study. Therefore, we have chosen to continue to work with MP1-p14, but dilute the complex 20-fold to 890 nM (25 ng/µL), a concentration we typically find after tandem affinity purification of high-copy-number complexes from human cells. Trypsin digestion of low to submicromolar protein concentrations is known to be problematic, since the digestion rate is limited by the substrate concentration.21 Digestion using the optimized protocol for MP1-p14 as described above resulted in an incomplete release of peptides as judged by monolithic chromatography (data not shown). Denaturants were already tested in the initial optimization stage without success. A change in trypsin to protein ratio from 1:25 to 1:5 resulted in a chromatographic elution profile on a monolithic column similar to the one obtained with the completely digested MP1-p14 complex at a concentration of 17.8 µM (compare parts c and d of Figure S-1, Supporting Information). However, when we analyzed approximately 100 fmol of digested MP1-p14, we observed a large variance of the absolute values measured for the different peptides (Figure 5), and LC-MS/MS experiments performed on the Orbitrap instrument identified several missed cleavage sites, indicating incomplete digestion. Next we determined whether the addition of a carrier protein could stabilize trypsin and lead to a complete digestion of our model complex. Ubiquitin is a small protein which releases only a small number of peptides upon trypsinization and therefore increases sample complexity to only a minor extent (Figure 4d, Figures S-7d and S-8d, Supporting Information). Ubiquitin was spiked in a 20-fold excess to the MP1-p14 complex, and digestion (21) Quadroni, M.; James, P. Electrophoresis 1999, 20, 664–677.

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Figure 5. Absolute quantification of MP1-p14 peptides via EP1. MP1-p14 was diluted to a concentration of 890 nM and trypsinized. Quantification was carried out on two different instruments. Data are the mean ( SD of triplicate measurements. Quantification of MP1 and p14 peptides showed a strong scattering when digestion was performed with a trypsin to protein ratio of 1:5 (white and light gray bars). Scattering of peptides was reduced to 3.8% (excluding peptide NGNQAFNEDSLK) and 10.4% (including peptide NGNQAFNEDSLK) when digestion was performed in a 20-fold excess of ubiquitin relative to MP1-p14, indicating complete digestion and thus allowing correct determination of the complex stoichiometry.

was performed according to our optimized protocol (trypsin to protein ratio of 1:25 for 16 h at 43 °C). Quantification on both instruments was carried out in triplicate. UV and SRM traces are shown in Figure 4 for EP2 and in Figures S-7 and S-8 for EP1 and EP3, respectively. Absolute amounts of approximately 100 fmol of MP1-p14 peptides measured with both instruments were again in good agreement and differed on average by 3.0% with an SD of 2.4%. Using ubiquitin as the carrier protein, we determined the MP1:p14 ratio to be 0.99 with an average SD of 0.06 calculated over the three EPs and the two instruments excluding NGNQAFNEDSLK. Again inclusion of this peptide increased the average peptide scattering from 3.8% to 10.4% and introduced an

error in stoichiometry determination (MP1:p14 ratio of 0.92 with an average SD of 0.13). Choice of Instrumentation. Both instruments performed equally well with respect to accuracy and precision and responded linearly over the dynamic range tested. The measured absolute amounts of MP1-p14 peptides differed on average by 3.0% with an SD of 2.0% only between the two instruments over all measurements conducted. The relative SD of triplicate measurements over the three mixtures of standard peptides was 3.0% on the QTRAP 4000 instrument and 1.4% on the LTQ Orbitrap instrument. Triple-quadrupole instruments operated in SRM mode are due to their wide dynamic range and their high sensitivity considered the instrumentation of choice for quantitative analysis.22 The QTRAP 4000 offers the unique possibility to be operated either as a triple quadrupole in SRM mode for quantitative analysis or as a linear ion trap allowing the collection of full scan and fragment spectra for peptide identification using an MS/MS search engine.23 However, a high-precision instrument such as the LTQ Orbitrap facilitates the identification of missed and unspecific cleavage sites and chemical modifications, which is an important task in the initial phase when internal standard peptides have to be selected. EtEP versus Full-Length Protein Standards. Although EtEP, AQUA, and QconCAT are different platforms to generate a mixture of standard peptides, they share a common feature; quantification is based on a limited number of peptides. Their measurement does not necessarily reflect the absolute amount of protein, such as in the case of incomplete digestion or the occurrence of a modification/unspecific cleavage within the quantified peptide. This obvious obstacle for quantification can be partially circumvented by the use of full-length isotopically labeled proteins as internal standards.24-26 However, generation of labeled recombinant standard proteins is an expensive and laborious task, and successful expression is not guaranteed. On the other hand, proteins have been quantified using a single internal standard peptide.13,27,28 These results have to be interpreted with caution as the native peptide might not have been released completely or might have been partially cleaved in an (22) Lange, V.; Picotti, P.; Domon, B.; Aebersold, R. Mol. Syst. Biol. 2008, 4, 222. (23) Hopfgartner, G.; Varesio, E.; Tschappat, V.; Grivet, C.; Bourgogne, E.; Leuthold, L. A. J. Mass Spectrom. 2004, 39, 845–855. (24) Brun, V.; Dupuis, A.; Adrait, A.; Marcellin, M.; Thomas, D.; Court, M.; Vandenesch, F.; Garin, J. Mol. Cell. Proteomics 2007, 6, 2139–2149. (25) Hanke, S.; Besir, H.; Oesterhelt, D.; Mann, M. J. Proteome Res. 2008, 7, 1118–1130. (26) Singh, S.; Springer, M.; Steen, J.; Kirschner, M. W.; Steen, H. J. Proteome Res. 2009, 8, 2201–2210. (27) Cheng, D.; Hoogenraad, C. C.; Rush, J.; Ramm, E.; Schlager, M. A.; Duong, D. M.; Xu, P.; Wijayawardana, S. R.; Hanfelt, J.; Nakagawa, T.; Sheng, M.; Peng, J. Mol. Cell. Proteomics 2006, 5, 1158–1170. (28) Bondar, O. P.; Barnidge, D. R.; Klee, E. W.; Davis, B. J.; Klee, G. G. Clin. Chem. 2007, 53, 673–678.

unspecific fashion. Quantifying at least two or three peptides per subunit provides a good compromise between the ability to detect inconsistencies by means of calculating peptide scattering and effort/cost efficiency. CONCLUSION Generation of an equimolar mixture of standard peptides is an important issue, and the current available peptide production platforms for absolute quantification deliver variable results.14 We have shown that our method has the power to produce an equimolar mixture of internal standard peptides of high accuracy as evidenced by the low degree of peptide scattering observed in our experiments. Equally important, using our strategy, we could significantly reduce the costs for absolute quantification and stoichiometry determination. To successfully label the complete set of 18 standard peptides and the native MP1-p14 peptides used in this study, we required 3 units of heavy and light mTRAQ reagent, respectively, corresponding to costs of ∼$500. The rationale of our strategy allows the absolute quantification of every protein of interest via one equalizer peptide. Expenses for the generation of 1 mg of each of the three labeled, amino acid analyzed, and prealiquoted equalizer peptides were about $700. Stored as aliquots, this amount of equalizer peptides is sufficient for the absolute quantification of a multitude of complexes. Furthermore, the recent launch of an mTRAQ triplex reagent offers the unique possibility of absolute quantification and stoichiometry determination of two samples simultaneously. Taken together, the EtEP strategy serves as a cost-efficient tool in quantitative proteomics and is ideally suited for stoichiometry determination of protein complexes. ACKNOWLEDGMENT We are grateful to our colleagues from the Mechtler Group: Michael Schutzbier, Michael Mazanek, and Ines Steinmacher for technical assistance and Elisabeth Roitinger and Andreas Schmidt for useful discussions and critical reading of the manuscript. This work is supported by the Austrian Proteomics Platform (APP) within the Austrian Genome Research Programme (GEN-AU) and by the Special Research Program (SFB-F3402) Chromosome Dynamics from the Austrian Science Fund (FWF). SUPPORTING INFORMATION AVAILABLE Additional results and discussion, monolithic chromatograms, details on the quantification of the labile peptides, SRM lists, and MS/MS spectra. This material is available free of charge via the Internet at http://pubs.acs.org.

Received for review October 9, 2009. Accepted November 11, 2009. AC902286M

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