Absolute Quantitation of Proteins by a Combination of Acid Hydrolysis

Anal. Chem. 2004, 76, 3569-3575

Absolute Quantitation of Proteins by a Combination of Acid Hydrolysis and Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Olga A. Mirgorodskaya,† Roman Ko 1 rner,*,‡ Alexander Novikov,† and Peter Roepstorff§

Department of Molecular Biology, University of Southern Denmark, DK-5230 Odense, Denmark, and Institute of Cytology of the Russian Academy of Sciences, 4 Tikhoretsky Avenue, 194064 St. Petersburg, Russia

Quantitation by mass spectrometry is increasingly used to monitor protein levels in biological samples. Most of the current methods are based on the relative comparison of protein quantities but are not suited for the determination of the absolute amount of a given protein. Here we describe a method for the absolute quantitation of proteins that is based on amino acid analysis by matrix-assisted laser desorption/ionization mass spectrometry. Proteins are completely hydrolyzed by acid hydrolysis and then mixed with standards of isotopically labeled amino acids. For the presented study, lysine, leucine, and two different types of labeled arginine were examined as standards. Quantitation of proteins is then achieved by measuring the ratios of labeled and unlabeled amino acids. The method has a sensitivity down to the low-femtomole range and can be applied to quantitate proteins separated by gel electrophoresis. Furthermore, we demonstrate that a mixture of two proteins can be quantitated using two labeled amino acids simultaneously. Mass spectrometry (MS) is now the method of choice for protein identification and the characterization of posttranslational modifications and has driven much of the progress in biological sciences during the last years. However, besides the structural characterization of proteins, the monitoring of protein levels is also crucial to obtaining a complete picture of various biological processes. Large discrepancies between mRNA and protein levels have been reported by several groups,1-3 demonstrating the need for quantitative analysis on the protein level. Quantitation of proteins has been widely performed by comparing staining intensities of proteins separated by two-dimensional gel electrophoresis (2-DE). The best choice for protein * Corresponding author. Fax: +49 89 8578 3102. E-mail: rkoerner@ biochem.mpg.de. † Institute of Cytology of the Russian Academy of Sciences. ‡ Max-Planck Institute of Biochemistry, Department of Cell Biology, Am Klopferspitz 18a, D-82152 Martinsried, Germany. § University of Southern Denmark. (1) Anderson, L.; Seilhamer, J. Electrophoresis 1997, 18, 533-7. (2) Gygi, S. P.; Rochon, Y.; Franza, B. R.; Aebersold, R. Mol. Cell. Biol. 1999, 19, 1720-30. (3) Le Naour, F.; Hohenkirk, L.; Grolleau, A.; Misek, D. E.; Lescure, P.; Geiger, J. D.; Hanash, S.; Beretta, L. J. Biol. Chem. 2001, 276, 17920-31. 10.1021/ac035389y CCC: $27.50 Published on Web 05/15/2004

© 2004 American Chemical Society

stains in terms of sensitivity and linear dynamic range is fluorescent dyes4 or alternatively radioactive labeling of proteins prior to 2-DE. However, both of these methods require special equipment, and quantitation requires that no more than one protein is present in a given gel spot. Despite its high sensitivity, mass spectrometry is per se not well suited for quantitation since the ion signal of a peptide depends not only on its abundance but also on its sequence and suppression by other analytes.5,6 Therefore, internal standards with chemically very similar properties must be added to a sample for accurate quantitation by mass spectrometry. An excellent choice is standards that are chemically identical to the compound to be analyzed except that they are labeled with stable isotopes such as 2H, 13C, and 15N. Several labeling strategies have recently been developed for peptides that allow for the relative comparison of protein abundances between a labeled and an unlabeled sample. Stable isotopes may be introduced in vivo by growing cell cultures in 15N-enriched7 media or media containing isotopically labeled amino acids.8 Alternatively, peptides can be 18O-labeled at the C-terminus upon endoproteinase treatment in 18O-water9-14 and then quantitatively compared with a sample that was processed under identical conditions in unlabeled 16O-water. Several labeling strategies based on amino acid derivatization with a labeled versus an unlabeled reagent for relative quantitation have also been developed. Using (4) Berggren, K.; Chernokalskaya, E.; Steinberg, T. H.; Kemper, C.; Lopez, M. F.; Diwu, Z.; Haugland, R. P.; Patton, W. F. Electrophoresis 2000, 21, 250921. (5) Cohen, S. L.; Chait, B. T. Anal. Chem. 1996, 68, 31-7. (6) Kussmann, M.; Nordhoff, E.; Rahbek-Nielsen, H.; Haebel, S.; Larsen, M. R.; Jakobsen, L.; Gobom, J.; Mirgorodskaya, E.; Kristensen, A. K.; Palm, L.; Roepstorff, P. J. Mass Spectrom. 1997, 32, 593-601. (7) Oda, Y.; Huang, K.; Cross, F. R.; Cowburn, D.; Chait, B. T. Proc. Natl. Acad. Sci. U.S.A 1999, 96, 6591-6. (8) Ong, S. E.; Blagoev, B.; Kratchmarova, I.; Kristensen, D. B.; Steen, H.; Pandey, A.; Mann, M. Mol. Cell. Proteomics 2002, 1, 376-86. (9) Williams, D. C.; Whitaker, J. R. Biochemistry 1968, 7, 2562-94. (10) Schnoelzer, M.; Jedrzejwski, P.; Lehman, W. D. Electrophoresis 1996, 17, 945-53. (11) Mirgorodskaya, O. A.; Kozmin, Y. P.; Titov, M. I.; Ko ¨rner, R.; So ¨nksen, C. P.; Roepstorff, P. Rapid Commun. Mass Spectrom. 2000, 14, 1226-32. (12) Yao, X.; Freas, A.; Ramirez, J.; Demirev, P. A.; Fenselau, C. Anal. Chem. 2001, 73, 2836-42. (13) Heller, M.; Mattou, H.; Menzel, C.; Yao, X. J. Am. Soc. Mass Spectrom. 2003, 14, 704-18. (14) Yao, X.; Afonso, C.; Fenselau, C. J. Proteome Res. 2003, 2, 147-52.

Analytical Chemistry, Vol. 76, No. 13, July 1, 2004 3569

a biotinylated iodoacetamide derivative, Gygi and colleagues combined isotopical peptide labeling with the ability to perform affinity purification of the subset of cysteine-containing peptides based on biotin-avidin interaction.15 Other derivatization strategies target carboxyl16 and amino groups16-20 with isotopically labeled reagents. Even though the above-listed labeling approaches combine high sensitivity with the ability to be applied to protein mixtures, they are only suitable for relative quantitation because one sample set is used as internal standard without further knowledge of the absolute concentrations. Stemmann and colleagues have addressed this problem by adding isotopically labeled synthetic phosphopeptides to samples containing the unlabeled phosphopeptide for the absolute quantitation of phosphorylation sites.21 This strategy allowed them to quantify the dynamics of protein phosphorylation in a particular biological process but has the disadvantage that synthetic peptides have to be synthesized for each protein to be quantified. Another very accurate and well-developed method for absolute protein quantitation is amino acid analysis after acid hydrolysis of proteins. Amino acids are usually quantified by high-performance liquid chromatography after precolumn derivatization, which requires picomole amounts of proteins.22-27 Here we present a mass spectrometry-based method for amino acid analysis with femtomole sensitivity for absolute protein quantitation. Proteins are mixed with an isotopically labeled amino acid standard prior to acid hydrolysis. The amino acids are then analyzed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), and quantitation is achieved using the measured ratios of the labeled and the unlabeled amino acid together with the known amino acid composition of the protein. We show that most amino acids can be detected by MALDI-MS analysis without any derivatization step and without interference with matrix ions. Even though, one type of labeled amino acid is sufficient for quantitation, a standard composed of a mixture of different labeled amino acids results in a higher accuracy and an increased dynamic range of quantitation and can also be used to quantify the components of simple protein mixtures. Standards composed of two different labeled amino acids were examined in this study. (15) Gygi, S. P.; Rist, B.; Gerber, S. A.; Turecek, F.; Gelb, M. H.; Aebersold, R. Nat. Biotechnol. 1999, 17, 994-9. (16) Goodlett, D. R.; Keller, A.; Watts, J. D.; Newitt, R.; Yi, E. C.; Purvine, S.; Eng, J. K.; von Haller, P.; Aebersold, R.; Kolker, E. Rapid Commun. Mass Spectrom. 2001, 15, 1214-21. (17) Munchbach, M.; Quadroni, M.; Miotto, G.; James, P. Anal. Chem. 2000, 72, 4047-57. (18) Cagney, G.; Emili, A. Nat. Biotechnol. 2002, 20, 163-70. (19) Zhang, R.; Regnier, F. E. J. Proteome Res. 2002, 1, 139-47. (20) Peters, E. C.; Horn, D. M.; Tully, D. C.; Brock, A. Rapid Commun. Mass Spectrom. 2001, 15, 2387-92. (21) Stemmann, O.; Zou, H.; Gerber, S. A.; Gygi, S. P.; Kirschner, M. W. Cell 2001, 107, 715-26. (22) Negro, A.; Garbisa, S.; Gotte, L.; Spina, M. Anal. Biochem. 1987, 160, 3946. (23) Murthy, L. R.; Iqbal, K. Anal. Biochem. 1991, 193, 299-305. (24) Clarke, A. J. Anal. Biochem. 1993, 212, 344-50. (25) Sarwar, G.; Botting, H. G. J. Chromatogr. 1993, 615, 1-22. (26) Yan, J. X.; Wilkins, M. R.; Ou, K.; Gooley, A. A.; Williams, K. L.; Sanchez, J. C.; Golaz, O.; Pasquali, C.; Hochstrasser, D. F. J. Chromatogr., A 1996, 736, 291-302. (27) Horstmann, H. J. Anal. Biochem. 1979, 96, 130-8.

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EXPERIMENTAL SECTION Reagents. Isotopically labeled L-arginine-guanido-15N2-HCl (98% atom 15N), L-arginine U-13C6 (98%)-HCl, and L-arginine U-13C6 (98%)U-15N4 (98%)-HCl were from Cambridge Isotope Laboratories (Andover, MA). Lysine-4,4,5,5-d4-2HCl (98% atom D) and L-leucine5,5,5-d3 (99% D) were obtained from Euriso-top (Gif-Sur-Yvette, France) and Aldrich (Milwaukee, WI), respectively. For acid hydrolysis, hydrochloric acid (30%, Suprapur grade), thioglycolic acid (pro analysi grade), and phenol (pro analysi grade) from VWR International (West Chester, PA) were used. As MALDI matrixes, 2,5-dihydroxybenzoic acid (DHB) from Aldrich and R-cyano-4hydroxylcinamic acid (CHCA) from Fluka (Buchs, Switzerland) were used. Modified sequencing grade trypsin was purchased from Promega (Madison, WI). Bovine serum albumin (BSA), bovine β-lactoglobulin, and chicken ovalbumin were obtained from Sigma (St. Louis, MO). In-Gel Digestion. Proteins separated by one-dimensional gel electrophoresis (1-DE) were in-gel digested following essentially the protocol of Shevchenko et al..28 After protein digestion, peptides were extracted by addition of 50 µL of acetonitrile and isotopically labeled amino acid standards (see Table 5) were added. Then gel pieces were removed, and the samples were dried in a vacuum centrifuge. Acid Hydrolysis. Proteins or protein digests were mixed with isotopically labeled amino acid standards (see Tables 3-6 for the amounts of proteins and standards) in a 0.5-mL plastic tube and lyophilized in a vacuum centrifuge. The vials were placed in 50mL glass vials, which were closed with a 24-mm mininert valve (Alltech, Deerfield, IL). A 800-µL mixture of 6 M HCl with 0.1% phenol and 0.1% thioglycolic acid was added to the bottom of the glass vial, which was then flushed with argon. The vial was evacuated to 1 mbar and placed in an oven at 107 °C for 18 h. The hydrolysates were centrifuged in a vacuum centrifuge for 1015 min to remove remaining traces of acid. Prior to analysis, samples were dissolved in 3.5 µL of 0.1% TFA. MALDI-TOF MS. Reflector-mode MALDI -TOF mass spectra were acquired in positive ion mode on a Voyager-DE Biospectrometry (Applied Biosystems, Framingham, MA) instrument equipped with delayed extraction. For sample preparations, the dried-droplet method was used: An 0.3-µL aliquot of the matrix solution (R-cyano-4-hydroxylcinnamic acid or 2,5-dihydroxybenzoic acid at a concentration of 20 mg/mL in 70% acetonitrile and 0.1% TFA) was mixed on the target with an equal volume of the sample (mixed with isotopically labeled amino acid standard before acid hydrolysis) and dried under vacuum. All spectra were averaged over 1000 laser shots. Lysine was measured using 2,5-dihydroxybenzoic acid as matrix, acquiring spectra in the mass range from 135 to 153 Da. Arginine was measured using R-cyanohydroxycinnamic acid as matrix, acquiring spectra in the mass range from 150 to 178 Da. Four spots for each sample were analyzed for comparison. Quantitation. Amounts of amino acids were calculated from the ratio of the spectral area of the first isotope of the natural amino acids and the isotopically labeled standards. Proteins were then quantified using the measured amounts of the selected amino acids and the known number of those amino acids in the analyzed (28) Shevchenko, A.; Wilm, M.; Vorm, O.; Mann, M. Anal.Chem. 1996, 68, 8508.

Figure 1. MALDI-MS spectrum of 500-fmol hydrolysates of BSA. R-Cyanohydroxycinnamic acid was used as matrix. Different mass ranges of the same spectrum are shown in (a) and (b). Amino acids marked with an asterisk interfere with matrix ions of R-cyanohydroxycinnamic acid.

Figure 2. MALDI-MS spectra of the matrix ions of (a) R-cyanohydroxycinnamic acid and (b) 2,5-dihydroxybenzoic acid.

proteins. In cases where the labeled amino acid standard contains a substantial amount of the unlabeled amino acid, the relative amount of the unlabeled amino acid can be subtracted. RESULTS AND DISCUSSION MALDI-TOF MS Analysis of Amino Acids. MALDI -TOF MS was chosen for the analysis of protein hydrolysates because of its high speed of data acquisition and the ease to automate sample preparation and processing. Since amino acid monoisotopic masses lay in the interval of 74-204 Da, their signals may overlay with MALDI matrix signals in MALDI-TOF MS analysis. To evaluate the feasibility of MALDI-TOF MS for amino acid analysis, a BSA acid hydrolysate was analyzed using CHCA as MALDI matrix (Figure 1). The figure shows that most amino acid signals can be clearly detected and distinguished from CHCA signals, even though CHCA is present in large molar excess in the sample. Due to the presence of the CHCA-related signal at m/z 147, this matrix is not suitable when lysine and its stable isotope analogue are used for quantitation (Figure 2a). If lysine detection is required, another MALDI matrix has to be used. DHB, for example, does not show any matrix signals in the m/z range of Lys, but on the other hand, its matrix ions interfere in the m/z region of arginine at m/z 175 (Figure 2b). Thus, the choice of MALDI matrix for quantitative amino acid analysis depends on which amino acids are selected for quantitation. CHCA was chosen for the quantitative analysis of arginine and leucine, whereas DHB was chosen for the quantitation of lysine. Lysine and arginine were chosen for quantitation experiments since they are well represented in protein sequences. In addition, being basic amino acids, they are expected to have good ionization efficiencies in the positive ion mode. D4-Lysine and 15N2-arginine were chosen as their stable isotope analogues. Figure 3 shows spectra of Lys, D4Lys, and their equimolar mixture acquired with DHB as MALDI matrix. As can be seen from Figure 2b, no matrix signal interference is observed in the area of interest. The small peak at mass of 147.1 in Figure 2b (labeled Lys) is most likely due to a contamination with unlabeled Lys. Generally, labeled amino acids cannot be supplied in 100% purity. Such contaminations with

Figure 3. MALDI-MS spectrum of (a) 300 fmol of lysine, (b) 300 fmol of deuterium-labeled D4-lysine, and (c) a 1:1 mixture of 300 fmol of lysine and 300 of fmol D4-lysine. 2,5-Dihydroxybenzoic acid was used as matrix.

unlabeled compounds can be corrected for during the calculation of the peak ratios of unlabeled and labeled compounds. The tiny signal at mass 151.1 in Figure 2a (unlabeled Lys) may be due to chemical or electronic noise in the baseline. Since signals of low intensity may be partially “hidden” in the noise, a good signal-tonoise ratio (better than 10) is desirable for accurate quantitation. Mixtures containing different molar ratios of lysine and D4-lysine were also analyzed (Table 1). Using the areas of the observed monoisotopic signals, lysine concentrations were calculated using D4-lysine as internal standard and the obtained values were compared with expected data (Table 1). The line of best fit of measured versus expected amount of lysine is shown in Figure 4. The average error was 4.1%, and errors were generally larger with increased molar ratios between lysine and D4-lysine (see Table 1). In addition, the error increased with decreasing absolute Analytical Chemistry, Vol. 76, No. 13, July 1, 2004

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Table 1. Quantitation of Lys Using D4-Labeled Lys as Internal Standard amount of D4labeled Lys (pmol) 10.65 4.2 1.3 0.21 0.084 0.017

amount of Lys (pmol) measureda expected 1.45 ( 0.04 2.88 ( 0.08 1.2 ( 0.04 0.14 ( 0.01 0.053 ( 0.05 0.013 ( 0.001

1.4 2.9 1.2 0.14 0.057 0.015

error (%) 3.6 0.7 0.0 0.0 7.0 13.3

a For the measured values, the averages and standard deviations are given using four independent measurements.

Figure 4. Line of best fit of measured versus expected amount of lysine as given in Table 1. Table 2. Quantitation of Arg Using Internal Standard amount of 2-Arg (pmol)

15N

1.5 0.75 0.30 0.15 0.15 0.075 0.030

15N -Labeled 2

amount of Arg (pmol) expected measureda 1.56 ( 0.04 0.72 ( 0.02 0.16 ( 0.01 0.15 ( 0.01 0.31 ( 0.01 0.079 ( 0.01 0.054 ( 0.001

1.5 0.75 0.15 0.15 0.30 0.075 0.056

Arg as

error (%) 4.0 4.0 6.3 0.0 3.3 5.3 3.6

a For the measured values, the averages and standard deviations are given using four independent measurements.

amounts of the amino acids (see Table 1) but did not exceed 10% at 30 fmol of lysine. Sensitivities of quantitation on the protein level, however, are higher due to the usually good representation of lysine in protein sequences. A corresponding analysis was performed for arginine and its 15N analogue using CHCA as MALDI matrix (Table 2). The 2 obtained average error for the amount of measured Arg, using 15N -arginine as internal standard, was 3.8%. The overall lower 2 calculation errors for arginine compared to lysine can be attributed 3572 Analytical Chemistry, Vol. 76, No. 13, July 1, 2004

Table 3. Quantitation of Leu and Ile Using D3-Labeled Leu as Internal Standard

amino acid Leu Ile Leu & Ile

amount of D3-Leu (pmol) 17 1.7 17 1.7 17 1.7

amt of unlabeled amino acid on MALDI target (fmol) measureda expected 10.8 ( 0.3 1.02 ( 0.03 12.4 ( 0.7 1.21 ( 0.05 23.8 ( 1.3 2.3 ( 0.16

11 1.1 12 1.2 23 2.3

error (%) 1.8 7.3 3.3 0.8 3.5 0.0

a For the measured values, the averages and standard deviations are given using 4 independent measurements.

to the generally observed fact that CHCA results in a more homogeneous sample preparation compared to DHB, and consequently, CHCA should give better spot-to-spot statistics. Isoleucine and leucine are abundant amino acids in protein sequences and are therefore a logical choice for protein quantitation. The feasibility of their use for quantitative analysis was evaluated using D3-leucine as internal standard (Table 3). Isoleucine and Leucine cannot be distinguished based on their molecular mass; thus, both amino acids would contribute to the observed molecular ion abundance at m/z of 132, increasing sensitivity of the analysis. On the other hand, to be able to obtain correct values for the added amounts of isoleucine and Leu, it should be shown that calculations based on the stable isotope Leu analogue give the same correlation between the observed ion peak intensities and the amount present in the sample for both leucine and isoleucine. Therefore, each amino acid and their mixtures were analyzed, and corresponding concentrations were calculated using D3-leucine as internal standard for all three cases (Table 3). The obtained data showed that, despite the chemical difference between leucine and isoleucine, the error between measured and expected amounts was similar for both amino acids as well as for their mixture, thus proving that one standard can be used for the calculation of the added amounts of isoleucine and leucine. Other amino acids with masses that do not overlap with matrix ions, such as alanine, serine, proline, naline, and threonine (see Figure 1), may also be excellent choicees for protein quantitation but were not tested in the course of this study. Application for the Determination of Protein Concentrations. Standard proteins of known concentrations were used to evaluate the suggested approach of using their amino acids for quantitative analysis. Proteins (BSA, ovalbumin) were subjected to acid hydrolysis to release amino acids and were analyzed in the presence of D4-lysine and 15N2-arginine. For this experiment, the amount of standards added to the sample was chosen to be in equimolar ratio with expected (calculated) amounts of lysine and arginine based on the BSA sequence (59 lysine and 23 arginine residues). The results of quantitation experiments for both BSA and ovalbumin with either D4-lysine or 15N2-arginine as internal standard are shown in Table 4, sections a and b, respectively. The average errors (3.1 and 4.6%, respectively) between measured and expected amounts were slightly higher compared to the previously described amino acid analysis but were still below 10%. Protein amounts of 5-10 fmol could be analyzed with this method, improving clearly the sensitivity compared to

Table 4. Quantitation of Proteins in Solution

sample BSA BSA ovalbumin ovalbumin

sample BSA BSA ovalbumin ovalbumin

(a) D4-Labeled Lys as Internal Standard amt of protein (fmol) amt of D4Lys (fmol) measureda expected 880 550 490 120

17.9 ( 0.7 8.68 ( 0.08 20.5 ( 0.3 5.65 ( 0.44

18.0 9.0 21.0 6.0

(b) 15N2-Labeled Arg as Internal Standard amt of protein (fmol) amt of 15N2Arg (fmol) measureda expected 385 180 480 120

16.6 ( 0.37 8.18 ( 0.63 21.6 ( 0.6 5.6 ( 0.22

17.5 8.5 21.0 6.0

Table 5. Quantitation of Proteins from an In-Gel Digest

error (%) 0.6 3.6 2.4 5.8

error (%) 5.1 3.8 2.9 6.7


liquid chromatography-based quantitation methods for amino acid analysis.9,22-25,27 Amino acid sequences of proteins to be quantified have to be known beforehand or have to be determined in a second experiment by mass spectrometrical identification in protein sequence databases. The application of the described quantitation method was also evaluated for gel-separated proteins. SDS-PAGE-separated BSA and ovalbumin were reduced, alkylated, and subjected to tryptic digestion. The resulting trypsin hydrolysate was divided into two parts and one part was used for peptide mapping to control the tryptic digestion. The second part was subjected to acid hydrolysis after adding amino acid standards. The resulting hydrolysates were lyophilized and redissolved in 0.1% TFA for MALDI-TOF analysis. Based on the intensities of native lysine and arginine and their corresponding analogues, protein concentrations were calculated (Table 5). The accuracy of the quantitation of gelseparated proteins depends on the ability to cut spots precisely. Furthermore, it is complicated by the fact that proteins cannot be completely recovered from gels. The data in Table 5 are consistent with an extraction efficiency of ∼75% from gel slices, and the measured protein concentrations may be weighted using this value. Different proteins, however, may have different extraction efficiencies, so it will be more accurate to calculate the extraction efficiencies based on a known amount of the specific protein. If such samples of known concentrations are not available, the separation of protein mixtures by (1D or 2D) liquid chromatography may give more accurate results compared to gel-based separation techniques. Although the use of single amino acid standards was shown to be sufficient for protein quantitation, a mixture of two different amino acid standards should improve precision of the analysis. The results of the quantitation analysis for BSA (Table 6, n ) 1) and β-lactoglobulin (Table 6, n ) 2) using two internal standards, D3-Leu and 15N2-Arg, are shown in Table 6. Even though each individual internal standard resulted in an accurate quantitation of the two protein standards, a mixture of different isotopically labeled amino acids may increase the confidence level for difficult (for example, low amounts of) samples.

(a) D4-Labeled Lys as Internal Standard amt of protein on MALDI target (fmol) amt of D4Lys (fmol) measureda expected

sample amt of protein in gel (pmol)

BSA (2) BSA (1) BSA (0.5) BSA (0.5) ovalbumin (2) ovalbumin (1) ovalbumin (0.5)

720 720 900 420 900 300 60

21.2 ( 0.9 8.0 ( 0.3 4.5 ( 0.1 4.4 ( 0.1 18.2 ( 0.9 9.7 ( 0.2 4.8 ( 0.3

24 12 6 6 24 12 6

(b) 15N2-Labeled Arg as Internal Standard amt of protein on sample amt of MALDI target (fmol) protein in gel amt of15N4(pmol) Arg (fmol) measureda expected BSA (2) BSA (1) BSA (0.5) ovalbumin (2) ovalbumin (1) ovalbumin (0.5)

240 180 180 360 120 36

22.5 ( 0.4 8.9 ( 0.2 4.5 ( 0.2 20.1 ( 0.2 6.8 ( 0.9 4.5 ( 0.1

calcd extractn effic (%) 88.3 66.7 75.0 73.3 81.3 75.8 80.0

calcd extractn effic (%)

24 12 6 24 12 6

93.8 74.2 75.0 83.8 56.7 75.0


Another advantage of using a mixture of different amino acid standards is that this opens the stage for protein mixture analysis. If the number and the sequences of different proteins in a mixture are known (for example, by LC-MS/MS analysis29,30), the corresponding number of internal amino acid standards can be chosen. The number of internal amino acid standards should be equal to or larger than the expected number of proteins in a mixture. Thus, for N protein in a mixture, the individual protein concentrations can be calculated by solving a set of at least N linear equations. This principal is demonstrated for a simple mixture analysis of two protein standards, BSA and β-lactoglobulin, of known concentrations (Table 6, n ) 3 to n ) 6). For these proteins, the molar amounts of arginine are 23 and 3, and the added molar amounts of isoleucine and leucine are 75 and 32, respectively. Therefore, we can write the following set of equations:

Argmeasured ) CBSA × 23 + CLac × 3

(1)

Ile/Leumeasured ) CBSA × 75 + CLac × 32

(2)

where Argmeasured and Ile/Leumeasured are the measured concentrations of these amino acids using a mixture of isotopically labeled arginine and leucine. CBSA and CLac are two unknowns, corresponding to the amounts of BSA and β-lactoglobulin in the mixture, respectively. These concentrations can now be easily determined by solving the set of equations. The results of these calculations for a mixture of BSA and β-lactoglobulin are shown in Table 6 (n ) 3 to n ) 6). The average error of this mixture (29) Link, A. J.; Eng, J.; Schieltz, D. M.; Carmack, E.; Mize, G. J.; Morris, D. R.; Garvik, B. M.; Yates, J. R., III. Nat. Biotechnol. 1999, 17, 676-82. (30) Aebersold, R.; Mann, M. Nature 2003, 422, 198-207.


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Table 6. Quantitation of BSA and β-Lactoglobulin in Individual Solutions (Samples 1-2) and in a Mixture (Samples 3-6) Using Mixtures of D3-Labeled Leu and 15N2-Arg as Internal Standard amt of protein on MALDI target (pmol)

stds on MALDI target (pmol)

BSA

15N -Arg 2

measureda

expected

5.7

0.15

0.10 ( 0.00 (D3-Leu) 0.09 ( 0.00 (15N2-Arg)

0.10

2

5.8

0.15

3 4 5 6

5.6 11.2 6.1 8.4

2.5 1.95 2.4 1.9

n

D3-Leu

1

a

0.067 ( 0.001 0.043 ( 0.001 0.025 ( 0.001 0.021 ( 0.001

measureda

expected

BSA

β-Lact

0.0

0.70 ( 0.01 (D3-Leu) 0.70 ( 0.01 (15N2-Arg) 0.19 ( 0.01 0.33 ( 0.01 0.40 ( 0.08 0.48 ( 0.01

0.066 0.046 0.030 0.023

0. 69

1.4

0.23 0.32 0.43 0.50

1.5 6.5 16.7 8.7

17.4 3.1 7.0 4.0

For the measured values, the averages and standard deviations are given using four independent measurements.

Table 7. Quantitation of Arg Using Different Concentrations of 13C 15N -Labeled Arg as Internal Standards 6 4

15N -Labeled 2

n

15N -Arg 2

13C

6-Arg

Arg,13C6-Labeled Arg, and

amt of Arg (pmol) measd using the following stdsa

amt of stds (pmol) 13C 15N -Arg 6 4

1

5.2

5.25

5.0

2

0.85

0.40

6.5

3

0.1

3.1

25.0

a

error (%)

β-lactoglobulin

15N

2-Arg

6.1 ( 0.07 (error 1.6%) 1.9 ( 0.04 (error 5.0%) 1.2 ( 0.04 (error 14.3%)

13C

6-Arg


13C

6-Arg


expected 6.0 2.0 1.4

For the measured values, the averages and standard deviations are given using four independent measurements. Errors are added in parentheses.

analysis was 8.1%, which is somewhat higher than in the case of the quantitation of individual proteins, but still acceptable for many applications. If more then two proteins are present in a mixture, additional amino acid standards have to be added to be able to increase the number of equations in the system to the number of unknowns to be calculated. A limitation of the described mixture analysis, however, is the fact, that it cannot be applied to proteins with very similar sequences such as protein isoforms. An important issue of quantitation using internal standards is the dynamic range of the method. Generally, the accuracy of quantitation is optimal if the internal standard is added at a concentrations that is close to the concentration of the analyte. In line 1 of Table 1, we show that 1.4 pmol of lysine could be quantified using 10.65 pmol (internal standard-to-analyte ratio of 7.6) of deuterium-labeled lysine with an accuracy of 3.6% (average of 4 independent measurements). Higher dynamic ranges, however, may be desirable if a rough estimate of the concentration of the analyte cannot be made. In such cases, the analyte can be mixed with the internal standard in different ratios (for example, varying by a factor of 10), and finally, the sample with the standard concentration that turns out to be closest to the concentration of the analyte is used for quantitation. The drawback of this method is that the sample has to be analyzed several times in parallel, resulting in an higher sample consumption. Alternatively, a mixed standard composed of differently labeled derivates of the same 3574


amino acid can be employed as an internal standard. In Table 7, we have examined a mixed standard composed of 15N2-Arg (mass shift 2 mass units), 13C 6-Arg (mass shift 6 mass units), and 13C 15N -Arg (mass shift 10 mass units). In line 1 of Table 7, we 6 4 show that all three arginine derivatives can be used with excellent accuracy (better than 2%) for the quantitation of arginine if they are mixed with the analyte at close to equal ratios. Other ratios of the internal standard-to-analyte concentrations were examined in line 2 and line 3 of Table 7. As expected, the general trend shows increased quantitation errors for wider internal standardto-analyte ratios. For the quantitation of 1.4 pmol of arginine using 25 pmol of 13C615N4-arginine (ratio 1:17.8, line 3 of Table 7), for example, an average error of 7.1% was observed, whereas the average error for the quantitation of 1.4 pmol of arginine mixed with 0.1 pmol of 15N2-arginine (ratio 14:1, line 3 of Table 7) as internal standard was 14.3%. The key advantage of the employed mixed standard is that a wide dynamic range can be covered without diminishing the quantitation accuracy. In the example shown in line 3 of Table 7, the three internal labeled arginine standards in amounts of 0.1, 3.1, and 25 pmol, respectively, were employed in a mixture to quantify 1.4 pmol of arginine. As expected, the standard that was closest in concentration to the concentration of the measured arginine (the 3.1-pmol standard) resulted in the best accuracy of quantitation (0% deviation). Since even more then three labeled amino acid derivatives may be

employed in a mixture, special standards may be designed to cover very broad dynamic ranges (more then 3 orders of magnitude).

complexes, the quantitation of posttranslationally modified amino acids, and the automation of the method.

CONCLUSIONS The present results demonstrate that a combination of acid hydrolysis and MALDI-TOF MS can be used for the absolute quantification of proteins. Lysine, leucine, and arginine were successfully examined, but other labeled amino acids may as well be employed. Due to the excellent sensitivity of MALDI-TOF MS, proteins can be quantified down to the 5-100-fmol range using stable-isotope amino acid analogues. Importantly, the dynamic range of the method can be extended to several orders of magnitude using mixed internal standards composed of different derivatives of the same amino acid. Future work will concentrate on the determination of the stoichiometry of simple protein

ACKNOWLEDGMENT We thank Ekaterina Mirgorodskaya for translation, critical reading, and fruitful discussion of the manuscript. Furthermore, we gratefully acknowledge Søren Andersen for technical assistance with gel electrophoresis experiments. This work was partly supported by the Danish National Research Council (Grant SNF 21-01-0533).

Received for review November 24, 2003. Accepted April 2, 2004. AC035389Y


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Absolute Quantitation of Proteins by a Combination of Acid Hydrolysis

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