An Identification Method for Altered Proteins in Tissues Utilizing

Fluorogenic Derivatization-Liquid Chromatography-Tandem Mass Spectrometric Proteome Method to Skeletal Muscle Proteins in Fast Thoroughbred Horses...
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Anal. Chem. 2003, 75, 3725-3730

An Identification Method for Altered Proteins in Tissues Utilizing Fluorescence Derivatization, Liquid Chromatography, Tandem Mass Spectrometry, and a Database-Searching Algorithm Chifuyu Toriumi† and Kazuhiro Imai*,†,‡

Laboratory of Bio-Analytical Chemistry, Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan, and Center for Research and Development, Kyoritsu College of Pharmacy, 1-5-30 Shibakoen, Minato-ku, Tokyo 105-8512, Japan

Two-dimensional polyacrylamide gel electrophoresis (2DPAGE) is now widely used as a tool for proteomic studies. For the sensitive determination of proteins in 2D-PAGE, fluorescence derivatization of primary amino moieties of proteins with cyanine dyes was recently developed. However, precipitation of the proteins could occur if completely derivatized because of the lower solubility of the resultant derivatives owing to the hydrophobicity of the reagents and the loss of the hydrophilic primary amino moieties. Thus, in this paper, a water-soluble and thiolspecific fluorogenic reagent, ammonium 7-fluoro-2,1,3benzoxadiazole-4-sulfonate, was adopted for the derivatization of proteins in tissues either with and without stimulation. Then, the method follows a separation of the derivatives by liquid chromatography with fluorescence detection, an isolation of only the altered proteins, an enzymatic digestion of the isolated proteins, and an identification of the proteins by liquid chromatography/ MS/MS with the database-searching algorithm. By using this method, we identified the altered expressions of five increased proteins (e.g., pancreatic polypeptide) as well as three decreased proteins (e.g., insulin 2) in the islets of Langerhans in Wistar rats 2 days after they were subcutaneously administered with dexamethasone. In the postgenome era, the identification of expressed proteins altered by a certain stimulation in a profiling proteomic study1-5 is one important challenge. One favorite approach in finding the altered proteins is to look for the difference in the amounts of the expressed proteins in tissues with and without stimulation. Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), * To whom correspondence should be addressed. Tel and Fax: +81-3-54002640. E-mail: [email protected]. † The University of Tokyo. ‡ Kyoritsu College of Pharmacy. (1) Anderson, N. L.; Anderson, N. G. Electrophoresis 1998, 19, 1853-1861. (2) Lopez, M. F. J. Chromatogr., B 1999, 722, 191-202. (3) Thornton, J. M. Science 2001, 292, 2095-2097. (4) Naaby-Hansen, S.; Waterfield, M. D.; Cramer, R. Trends Pharmacol. Sci. 2001, 22, 376-384. (5) Hancock, W. S.; Wu, S. L.; Shieh, P. Proteomics 2002, 2, 352-359. 10.1021/ac020693x CCC: $25.00 Published on Web 06/20/2003

© 2003 American Chemical Society

for example, is widely used.6,7 From the different patterns of the visualized spots on two gels, one can isolate the altered proteins, hydrolyze them enzymatically, determine the peptides fragments by tandem mass spectrometry (MS/MS), and identify the proteins by a database-searching algorithm.8,9 However, it is sometimes difficult to get reproducible patterns to identify the spots on 2DPAGE. To overcome the difficulty, two-dimensional differential gel electrophoresis has been developed. Unlu et al.10 used cyanine dyes and Tonge et al.11 used Cy dyes to derivatize the primary amino moieties of the proteins, and then they utilized 2D-PAGE to separate and pick up the altered proteins in different samples based on the comparison of different fluorescent color images. In addition, to determine the altered proteins by utilizing LC-MS/MS, isotope-coded affinity tags (ICAT)12 and 18O labeling13 methods have been developed. In those methods, each comparative experiment is internally standardized; i.e., the differences in two samples are determined from the same separation. However, the former adopted the incomplete derivatization condition (the dye-to-protein labeling ratio, 0.9994, n ) 2; Table 1), suggesting the precision of the derivatization. Unlike the methods utilizing Cy dyes,11 an excess amount of SBD-F (3.5 mM) against the proteins (less than 0.1 µM each) (17) Toriumi, C.; Imai, K. Anal. Chem. 2002, 74, 2321-2327.

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Table 2. Precision and Reproducibility of the 2D-HPLC with Fluorescence Detection recovery (%) standards somatstatin insulin lactalbumin leptin

injection to IEC columna (pmol)

mean (n ) 3)

SD

CV (%)

2 20 2 20 2 20 2 20

74.5 78.7 85.3 96.7 72.9 73.2 64.6 68.2

14.2 11.2 3.7 10.4 2.8 12.6 16.7 4.0

19.1 14.2 4.3 10.8 3.8 17.2 25.9 5.9

a The solution of the mixture of the peptides and proteins was derivatized with SBD-F as described in the Experimental Section, and the corresponding amounts for each were injected to IEC.

Figure 2. Representative HPLC chromatogram of several proteins and peptides derivatized with SBD-F: 1, vasopressin; 2, oxytocin; 3, somatostatin; 4, calcitonin; 5, amylin; 6, insulin chain B; 7, R1-acid glycoprotein; 8, R-lactalbumin; 9, BSA; 10, leptin. Protein mixture (2 nM each) was derivatized with 3.5 mM SBD-F at pH 9.0 under 40 °C for 3 h in the presence of 1 mM TCEP, 2 mM EDTA, 10 mM CHAPS, and 6 M guanidine hydrochloride. Then, an aliquot of the mixture corresponding to 20 fmol each was injected onto the column. The detailed separation and detection conditions are described in the Experimental Section. Table 1. Detectability of Several Proteins and Peptides by the Proposed HPLC with Fluorescence Detection peptides and proteins

mol wt

no. of cysteine residues

detection limit (fmol)

calibration curve (r)

vasopressin calcitonin somatostatin oxytocin amylin leptin R1-acid glycoprotein insulin R-lactalbumin albumin (BSA)

1084 3418 1638 1007 3920 16014 21547 5808 16228 66385

2 2 2 2 2 2 4 6 8 35

5 6 1.8 1.3 1.2 3 1.3 0.7 0.5 0.2

0.9998 0.9994 0.9999 0.9997 0.9997 0.9999 0.9995 0.9999 0.9999 0.9999

could derivatize most of the thiol groups in the peptides and proteins. It should be noted that no precipitation occurred under the reaction with SBD-F because of the water solubility of the derivatives, owing to the sulfonic acid moiety of the SBD skeleton and the remaining intact primary amino moieties of the proteins. The detection limits for the proteins and peptides were 0.2-6.0 fmol. As expected, BSA, with 35 Cys residues, was sensitively detected. The detectability was superior to those of the stained spots on the electrophoregram.7 Separation of the Derivatized Peptides and Proteins Using 2D-HPLC. Separation of the derivatized peptides and proteins was then performed utilizing 2D-HPLC. The fluorescent peptide and protein mixture (somatostatin, insulin, leptin, R-lactalbumin) was first fractionated through IEC. The fractionation was performed with a stepwise sodium chloride gradient elution and the peptide and protein mixture was fractionated into four different fractions (the respective 0.04, 0.08, 0.12, and 0.3 M NaCl fraction for somatostatin, insulin, leptin, and R-lactalbumin). Each fraction was then further separated with a wide-pore-sized (30-nm pore diameter) RPLC column on the basis of their varying hydropho3728 Analytical Chemistry, Vol. 75, No. 15, August 1, 2003

bicities. The recovery of each derivative was almost constant regardless of the concentration with CV value of less than 26% (Table 2). Thus, the proposed method should be suitable for the quantitative identification of peptides and proteins. Identification of BSA Using HPLC, Tandem Mass Spectrometry, and a Database-Searching Algorithm. To evaluate the identification procedure, derivatized BSA (25 µM) with SBD-F was digested with trypsin and a part of the resulting peptide mixture was separated by RPLC and detected using a fluorescence detector. The rest of the mixture was subjected to the same RPLC with an ESI ion trap mass spectrometer to perform the MS/MS analysis of the separated peptide fragments. A direct connection of the fluorescence detector to the mass spectrometer is recommended for easier comparison of the peaks between fluorescence and base peak ion detection. In agreement with a theoretical expectation that BSA, by trypsin digestion, could generate 25 Cyscontaining peptides and 35 non-Cys-containing peptides of more than 4 amino acid residues, we have detected fluorometrically in the chromatogram more than 25 fluorescent peptides (Figure 3A). In comparison, however, our mass chromatogram (Figure. 3B) showed only 11 Cys-containing peptides. This suggested a more sensitive detection of the SBD-derivatives by fluorescence than by mass chromatographic detection. A more sensitive detection by MS can be achieved by using a fluorogenic reagent18 for a positively charged fragment. Figure 4 shows an example of the MS/MS spectrum obtained from collision-induced dissociation of the (M + 2H)2+ precursor ion, m/z ) 873.4 (marked with an arrow in Figure 3). Fragment ions in the spectrum represent mainly single-event preferential cleavage of the peptide bonds affording sequence information. The modified SBDs were confirmed to have attached to the cysteinyl residue. Utilizing the combined MS/MS spectra observed from Cys-containing peptides (m/z ) 520.2, 710.7, 780.9, 787.3, 792.7, 859.4, 873.3, 933.5, 1025.0) and non-Cys-containing peptides (m/z ) 501.8, 653.4, 700.4, 740.3, 784.5), the MASCOT,19 in which the fragment information of SBD structure was memorized, the protein was identified as BSA (sequence coverage, 27%). The amount of BSA needed can be reduced to subnanomolar levels (18) Stults, J. T.; Lai, J.; McCune, S.; Wetzel, R. Anal. Chem. 1993, 65, 17031708. (19) Perkins, D. N.; Pappin, D. J.; Creasy, D. M.; Cottrell, J. S. Electrophoresis 1999, 20, 3551-3567.

Figure 3. Chromatogram with fluorescence detection of the trypsin digests of BSA (A) and the corresponding chromatogram detected by base peak ion in MS (B). The fluorescent peaks represented the Cys-containing peptides. The arrow shows a peak of the (M + 2H)2+ precursor, m/z ) 873.4, the MS/MS spectrum of which was shown in Figure 4.

Figure 4. Sequence identification of the digested peptide fragment (marked with an arrow in Figure 3) of BSA.

by use of a smaller diameter column (such as a few hundred micrometers) because of a more efficient separation.20,21 Application of the Method to the Identification of the Altered Proteins in Rat Pancreatic Tissues. The applicability (20) Link, A. J. Nat. Biotechnol. 1999, 17, 676-682. (21) Washburn, M. P.; Wolters, D.; Yates, J. R. 3rd. Nat. Biotechnol. 2001, 242247.

Figure 5. Representative chromatograms with fluorescence detection of the proteins obtained by the reversed-phase liquid chromatography of the islets of Langerhans from a rat without Dex treatment. The peaks corresponding to the altered proteins after Dex treatment were marked as follows: 12, protein P31; 15, dnaK-type molecular chaperone hsp72-psl; 24, pancreatic polypeptide; 29, insulin 2; 30, proinsulin 2; 36, 78-kDa glucose-regulated protein; 61, phosphatidylethanolamine binding protein; 121, thioredoxin.

of the method was tested in rat pancreatic tissues with or without Dex administration. Dex induces type 2 diabetes, a predominant type in human diabetes, through increase in hepatic glucose production and induction of insulin resistance.22,23 Two days after the Dex treatment, the blood glucose levels of the rat reached 310.5 mg/dL, significantly above the pretreatment value of 118.3 mg/dL (P < 0.001). However, as far as we know, there are no reports on the altered expression of proteins after the administration of Dex. In this experiment, the islets of Langerhans (∼60 islets) were collected from rats pretreated with or without Dex for two days and the tissue derivatized with SBD-F (n ) 3 each). The isolation of the altered proteins from the protein mixtures was then performed using the 2D-HPLC. 2D-HPLC was used to increase the chromatographic resolution and peak capacity.24-26 (22) McMahon, M.; Gerich, J.; Rizza, R. Diabetes Metab. Rev. 1988, 4, 17-30. (23) De Feo, P.; Perriello, G.; Torlone, E.; Ventura, M. M.; Fanelli, C.; Santeusanio, F.; Brunetti, P.; Gerich, J. E.; Bolli, G. B. Am. J. Physiol. 1989, 257, E3542. (24) Wagner, K.; Racaityte, K.; Unger, K. K.; Miliotis, T.; Edholm, L. E.; Bischoff, R.; Marko-Varga, G. J. Chromatogr., A 2000, 893, 293-305. (25) Davis, M. T.; Beierle, J.; Bures, E. T.; McGinley, M. D.; Mort, J.; Robinson, J. H.; Spahr, C. S.; Yu, W.; Luethy, R.; Patterson, S. D. J. Chromatogr., B. 2001, 752, 281-291. (26) Opiteck, G. J.; Jorgenson, J. W.; Arthur Moseley 3rd, M.; Anderegg, R. J. J. Microcolumn Sep. 1998, 10, 365-375.

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Table 3. Altered Proteins in Rat Islets of Langerhans after Dex Treatment for Two Days fluorescence intensity peak no.

Dex mean SD

control mean SD

12 15 24 29 30 36 61 121 n)3

36 85 1681 12963 799 178 153 141

77 218 805 24602 134 95 84 78

2 5 54 726 36 3 14 48

16 16 89 878 30 3 33 32

average ratio (Dex/control)

protein

Mw

database accession no.

0.5 0.4 2.1 0.5 6.0 1.9 1.8 1.8

protein P31 dnaK-type molecular chaperone hsp72-psl pancreatic polypeptide insulin 2 proinsulin 2 78-kDa glucose-regulated protein phosphatidylethanolamine binding protein thioredoxin

13 284 70 884 10 968 5 797 12 331 72 302 20 788 12 854

CSRT31 S31716 NP_036758 NP_062003 NP_062003 P06761 NP_058932 NP_446252

A guard column, a C8 minicolumn, was attached in front of a DEAE column to help adsorption of the fluorescent peptides and proteins. Without the minicolumn, the loss of many proteins occurred, because the high ionic strength of the surfactant and denaturant adopted eluted out the peptides and proteins from the DEAE column. As shown in Figure 5, there were almost 3-50 peaks in each RPLC chromatogram obtained from the sample derived from the nontreated rat while there were 129 overall peaks in 2D-HPLC. The peak capacity, a theoretical measure of the performance of HPLC as n ) L/(4σ), where L is the total time over the analysis and 4σ is peak width,27 was calculated to be 40 per each RPLC and the overall peak capacity including the five steps of the IEC-RPLC was ∼200. The separation ability of the present 2D-HPLC is inferior to that of 2D-PAGE, which usually visualizes more than 1500 spots using 25-cm-square 2D gel.28 To detect more species of proteins, the peak capacity should be increased by increasing the sodium chloride gradient steps for IEC and the separation efficiency in RPLC. Figure 5 shows the entire fluorescent peaks on the RPLC chromatograms obtained from the nontreated rats. To find the difference of peak intensity with or without Dex treatment, each peak area was calculated, and they were compared based on the ratio (peak area in Dex-treated rats)/(peak area in nontreated rats). In consequence, it was found that Dex treatment increased five fluorescent peaks more than 1.8 times and halved three fluorescent peaks (n ) 3, Table 3). These proteins were isolated separately and digested with trypsin as described above. The peptide mixtures were then subjected to the conventional-pore RPLC (12-nm pore diameter) with MS/MS. Accordingly, the peaks of the increased area after Dex treatment (respective peak area with or without Dex treatment, mean ( SD, n ) 3) were identified with more than 4% sequence coverage as pancreatic polypeptide (1681 ( 54, 805 ( 89), proinsulin 2 (799 ( 36, 134 ( 30), 78-kDa glucose-regulated protein (178 ( 3, 95 ( 3), phosphatidylethanolamine binding protein (153 ( 14, 84 ( 33), and thioredoxin

(141 ( 48, 78 ( 32), respectively. The peaks of the decreased area after Dex treatment (respective peak area with or without Dex treatment, mean ( SD, n ) 3) were identified as protein P31 (36 ( 2, 77 ( 16), dnaK-type molecular chaperone hsp72-psl (85 ( 5, 218 ( 16), and insulin 2 (12963 ( 729, 24602 ( 878), respectively. The physiological meaning for these altered proteins remains to be elucidated. In this study, although only the altered proteins were the main targets for identification, by using the same procedure, other proteins appeared on the chromatogram (Figure 5) and can also be identified comprehensively. For the more popular use of the method, we should increase the detectability of the proteins. As suggested above, we are now trying to adopt a LC column of a small diameter (such as a few hundred micrometers) before MS/ MS to increase the peak capacity which could easily identify the low-abundance proteins. The proposed method is simple and not cumbersome as compared with the 2D-PAGE method. It is also more straightforward to identify the proteins as compared with other 2D-HPLC methods including the ICAT method,12 which needs prior digestion of proteins with the enzyme, resulting in the loss of some critical sequence information before LC/MS/MS. In conclusion, by use of the proposed method, we have successfully isolated and identified the altered proteins in the islets of Langerhans in rats treated with Dex. Because of the severe reaction condition to derivatize proteins and the water solubility of SBD-F, the method can be applicable to any kind of tissue and cell samples and should be a useful tool for identifying altered proteins following stimulations to the tissues.

(27) Wolters, D. A.; Washburn, M. P.; Yates, J. R. 3rd. Anal. Chem. 2001, 73, 5683-5690. (28) Gygi, S. P.; Corthals, G. L.; Zhang, Y.; Rochon, Y.; Aebersold, R. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 9390-9395.

Received for review November 8, 2002. Accepted April 29, 2003.

3730 Analytical Chemistry, Vol. 75, No. 15, August 1, 2003

ACKNOWLEDGMENT The authors thank Dr. Keiho Lee for his kind suggestions and reviews. We acknowledge Tosoh Co. for kindly supplying of TSKgel DEAE 5PW column and also thank Imtakt Co. for kindly supplying of Cadenza TC-C18 and Presto FT-C18 columns.

AC020693X