Rapid Subpicogram Protein Detection on a Microchip without Denaturing Mari Tabuchi,*,† Yasuhiro Kuramitsu,‡ Kazuyuki Nakamura,‡,§ and Yoshinobu Baba†,| Department of Medicinal Chemistry, Faculty of Pharmaceutical Sciences, The University of Tokushima, Tokushima, Japan, CREST, Department of Biochemistry & Biomolecular Recognition, Central Laboratory for Biomedical Research and Education, Yamaguchi University School of Medicine, Ube, Japan, and Single-Molecule Bioanalysis Laboratory, National Institute of Advanced Industrial Science and Technology (AIST), Takamatsu, Japan Received February 10, 2003
Abstract: Protein size separation based on sodium dodecyl sulfate-gel electrophoresis (SDS-GE) requires denaturing, but we propose that denaturing is unnecessary for analysis by microchip electrophoresis (µ-CE). By omitting the protein denaturing process, we achieved not only shortened total analysis time, but also dramatically improved sensitivity without compromising size determination. The detection limit was improved to 0.1 ng/µL under conditions without denaturing and 600 pg (9.0 femtomol) of bovine serum albumin was detectable, which equals levels detectable by Silver stain, although a routine method by microchips in the Coomassie Blue detection level. Keywords: microchip electrophoresis • proteome analysis • high-sensitivity • Jurkat cell
1. Introduction Many SDS-GE protein analysis methods using capillary techniques or microchips have been recently assessed,but timeconsuming pre-1-3 or postcolumn labeling,4-6 and relatively low sensitivity (Coomassie blue levels) make them unsatisfactory. Picomolar detection limits were obtained using Sypro dyes5 and a more simplified labeling procedure was possible using dynamic labeling Nano Orange,7 but there was a high fluorescent background. The Agilent 2100 Bioanalyzer has recently been developed for practical usage,8 in which protein assay procedure steps of staining, destaining, separation, and detection are integrated in a microfabricated chip. The high fluorescent background was reduced by the destaining process. However, a denaturing process is not included in the integrated procedures, and so * To whom correspondence should be addressed. Phone & Fax: +81-88633-9507. E-mail:
[email protected]. † Department of Medicinal Chemistry, Faculty of Pharmaceutical Sciences, The University of Tokushima and CREST, Japan Science and Technology Corporation, JST. ‡ Department of Biochemistry & Biomolecular Recognition, Yamaguchi University School of Medicine. § Central Laboratory for Biomedical Research and Education, Yamaguchi University School of Medicine. | Single-molecule Bioanalysis Laboratory, National Institute of Advanced Industrial Science and Technology (AIST), Japan. 10.1021/pr034009m CCC: $25.00
2003 American Chemical Society
the analysts must perform the denaturing manually, which is troublesome. To construct a high-throughput screening (HTS) systems for protein analysis, this denaturing procedure also must be automated in an on-line channel by a special device with thermo control or be omitted. Size-based separation of complex protein mixtures is routinely performed using sieving matrixes, where, almost exclusively, a denaturing step is required. Most proteins are fully denatured by boiling in the presence of SDS and reducing agents.9 However, we speculated if a denaturing step is needless, a simpler assay would be attained. Hunt et al.10 investigated the various conditions of denaturing times and temperatures, but they could still found the denaturing to be required with a covalent dye. He et al.11 developed a rapid determination method of protein molecular weight and an HTS system by the Ferguson method and multiplexed capillary electrophoresis, but the denaturing procedure is necessary. In this report, we investigated the denaturing effect on intensity, migration, and reproducibility of details, based on which we concluded that denaturing was unnecessary and we developed a new assay method.
2. Experimental Section Materials. A protein ladder (Agilent Technologies, Waldbronn, Germany) consisting of lysozyme (Mr 14.3 kDa), β-lactoglobulin (18.4 kDa), carbonic anhydrase (29.0 kDa), ovalbumin (43.0 kDa), serum albumin (68.0 kDa), phosphorylase B (97.4 kDa), and myosin (H-chain; 210.0 kDa), was used as the standard. Other proteins, bovine insulin (5.7 kDa), myoglobin (17.0 kDa), trypsin (23.0 kDa), and bovine serum albumin (BSA, 66.5 kDa) (Sigma Chemical, St. Louis, MO), were used as protein samples. A protein mixture extracted from human Jurkat cells, which are a T-lymphoblastic cell line (Dainippon Pharm, Suita, Japan), was used as a biological sample. Jurkat cells were cultured at 37 °C or 49 °C for 30 min in RPMI 1640 medium (Nissui, Tokyo, Japan).12,13 The cultured Jurkat cells were lysed in cellytic-M (Sigma-Aldrich, Tokyo, Japan) for 15 min. The cell lysate was centrifuged at 20 000 × g for 15 min and the supernatant was used. Proteins were denatured according to the manufacturer’s instructions. PBS buffer (Irvine Scientific), sodium dodecyl sulfate (SDS, Wako Pure Chemical Industries, Ltd), dithiothreitol (DTT, Sigma-Aldrich Japan, Tokyo, Japan), milli-Q water Journal of Proteome Research 2003, 2, 431-435
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Rapid Subpicogram Protein Detection
technical notes
Figure 1. µ-CE sensitivity of the routine method and the new method. (A) Bovine serum albumin (1 µg/µL, 66.5 kDa) was analyzed with heating a-d and without heating e-h. Protein was diluted in denaturing buffer (a, b, e, f) or in deionized water (c, d, g, h). DTT was used (a, c, e, g) or not used (b, f, d, h). Arrows show the peak of BSA. (B) The detection limit of the routine method (a) and the new method (b) of BSA (arrows). (C) Peak heights (a) and peak widths at half-height (b) of several concentrations of BSA by the routine method (open round symbols) and new method (red closed square symbols). Arrows show each detection limit. (D) Detection limits of the new assay method with low concentration marker (a) and addition of variable concentration marker (b).
(ICN Biomedicals, Aurora, OH), and fluorescent dye (Agilent Technologies, excitation/emission 650/680 nm, chemical name not available) were used. PBS-free, DTT-free, and no heating conditions were also tested as a new method. All chemicals were of analytical reagent grade and were used without purification. To further improve sensitivity, we used low concentration of markers (1 ng/µL; low-concentration marker assay) or suitable concentration markers (0.5 ng/µL for 0.15ng/µL of protein and 5 ng/µL for 5-50 ng/µL of protein; variable concentration marker assay using two or three markers). The µ-CE. Microchips in Protein Chips (Protein 200 Labchip; Agilent Technologies), made from soda lime glass, were used for separation. The sieving matrix (Agilent Technologies), destaining buffer (Agilent Technologies), and protein samples were loaded according to the instructions. Separation and detection were performed in an Agilent 2100 Bioanalyzer instrument (Agilent Technologies), which emits at 630 nm. Proteins were identified from databases (NCBI and SWISS PROT) and a Mass spectrometer (LCQ DUO, Thermoelecton, Tokyo, Japan).
3. Results and Discussion 3.1 Protein Separation without the Denaturing Process. To simplify the assay procedure of protein analysis by µ-CE, we investigate whether it is possible to omit the denaturing step or not. Figure 1A, parts a-h, show the electropherograms of BSA (1 µg/µL) in several buffer conditions with or without 432
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denaturing. Figure 1A, parts g and h, clearly demonstrate that analysis without denaturing is possible, and 10 times higher sensitivity was obtained when the heating process was omitted and when water was used instead of denaturing buffer. This effect did not depend on the presence of DTT. On the other hand, under the conditions without a heating step, when the denaturing buffer was used, high intensity was not obtained (Figure 1A, parts e and f). However, even if the denaturing buffer was not used, in the case that heating was applied, the intensities were decreased (Figure 1A, parts c and d). Therefore, both the processes without heating and denaturing buffer seem to be significant for sensitivity. The migrations without heating process (Figure 1A, parts e-h) were 2 s faster than the routine method (Figure 1A, part a), and it did not depend on the presence of buffer or DTT, whereas migration with heating depended on the buffer conditions (Figure 1A, parts a and c). This may depend on the fact that folded proteins usually migrate faster than unfolded proteins.14,15 The real biological protein samples usually contain the culture solution or salts, which are sometimes different from the ladder protein’s conditions; therefore, mismatching of molecular sizing occurs with heating. In contrast, our data indicates that mismatching of molecular sizing of real biological proteins without denaturing will not occur using ladder proteins without denaturing. 3.2 Assay Detection Limit in the Absence of Denaturing. We investigated the detection limit of the new conditions. Figure 1B clearly shows that about 1 order improvement of the
technical notes
Tabuchi et al.
Table 1. Comparison Between Routine Method and New Method in Protein Analysis routine method
protein
lower marker lysozyme β-lactoglobulin carbonic anhydrase ovalbumin serum albumin phosphorylase myosin (upper marker) average
molecular size (kDa)
6.0 14.3 18.4 29.0 43.0 68.0 97.4 210
calculated size n ) 50a
new method
peak area n ) 50a (n ) 10)b
peak area n ) 50a (n ) 10)b
calculated size n ) 50a
average
RSD(%)
RE(%)
RSD(%)
average
RSD(%)
RE(%)
RSD(%)
13.6 18.0 28.4 42.0 68.4 97.7
4.1 1.7 2.7 1.2 1.5 0.96
-5.2 -2.0 -2.0 -2.4 0.6 0.27
27.3 (10.1) 25.9 (10.1) 35.9 (10.7) 27.3 (14.3) 36.1 (12.8) 28.6 (12.2) 26.4 (11.2)
14.4 18.2 28.6 41.9 67.5 97.7
3.0 2.1 1.3 1.2 1.0 0.87
0.63 -1.3 -1.3 -2.6 -0.77 0.33
16.6 (10.7) 16.6 (10.7) 19.8 (14.1) 28.0 (16.0) 28.3 (10.1) 26.9 (14.9) 25.3 (12.3)
2.03
-2.1
28.9 (11.6)
1.63
-1.20
22.8 (12.7)
a
b
Interchipanalysis: 50 channels; 5 chips were analyzed in 5 days with one chip (10 channels/1 chip) in each day. Intrachipanalysis: 10 channels(1 chip) were analyzed in a day.
detection limit was observed by the new method. The limit of BSA under the routine conditions was 20-50 ng/µL, as opposed to 5 ng/µL by the new method (S/N > 3). Figure 1C, part a, shows each detection limit measured by peak intensities in several concentrations and Figure 1C, part b, shows the peak widths at half-height. The detection limit by the new method certainly improved and it produced sharp peaks (Figure 1C). The same detection limits were also obtained for myoglobin or trypsin. The routine method includes two steps of a sample dilution procedure to decrease the ionic strength and SDS,8,16 whereas the new conditions include no dilution step because samples were dissolved in water (both SDS-free and buffer-free), therefore, our new method seems to have produced higher sensitivity than the routine method. In addition, the dispersed size of unfolded protein might have been produced by various levels of denaturing, resulting in broader peaks than those by the new method. The reproducibility of the new method was investigated using standard proteins (Table 1). The relative standard deviation (RSD) and relative error (RE) of sizing in the new method are satisfactory because almost all values from the new method were less than 3.0% and, in addition, were less than those of routine method. Because the new method produced satisfactory data, we concluded that the denaturing process is unnecessary. Omitting it can shorten analysis time by more than 5 min. 3.3 Low Concentration Marker Assay and Variable Concentration Marker Assay. By omitting the denaturing process and modifying the routine procedure, the detection limit dramatically improved to 0.2 ng/µL (S/N > 3) when the low concentration marker (1 ng/µL) was used for low-concentration determinations (Figure 1D, part a) or to 0.1 ng/µL (S/N > 3) when a variable concentration marker (0.5 ng/µL for 0.1-1 ng/ µL and 5 ng/µL for 5-50 ng/µL) was added (Figure 1D, part b) in the absence of denaturing. The low-concentration marker assay is available for low concentration samples because the scale can be magnified, but it can only be used for a narrow concentration range. The variable concentration marker is available for a dynamic range, because the scale can be magnified freely. The sensitivity by Coomassie Blue stain in gel electrophoresis is 50-100 ng/band,17 and that by Silver stain is 1-5 ng/band.18 In the best new method procedure, only 6 µL of 0.1 ng/µL of a protein is required, which is equal to about 600 pg of a protein,
Figure 2. Detection limit of proteins extracted from Jurkat cells of (A) the routine method and (B) new method. Protein mixture extracted from Jurkat cells was analyzed.
indicating that subpicogram quantities of a protein can be detected by the new method. The value is equal to the sensitivity obtained by Silver stain, and sub-picogram (femtomol of BSA) detection is also possible by µ-CE. By contrast, the routine method using the Agilent 2100 analyzer µ-CE requires at least 80 ng (4 µL of 20 ng/µL; subpicomol of BSA) of a protein, staying in Coomassie Blue range. Although picomolar assay sensitivity3 for protein separations using capillary was reported, the practical method by the microchip remained in Coomassie Blue range.8 3.4 Analysis of Real Proteins Extracted from Jurkat Cells. We also confirmed this new method effect for a real biological protein mixture, the intracellular soluble proteins extracted from Jurkat cells, which are a human T lymphoblastic cell line. The electropherograms from Figure 2 show the comparison of detection limit (total proteins) between the routine (Figure 2A) and the new method (Figure 2B) of proteins from Jurkat cells cultured under standard conditions. More than 20 peaks were recognized by both methods using the µ-CE of the Agilent 2100 Bioanalyzer. About 1.2 µg (0.3 µg/µL × 4 µL) of total protein Journal of Proteome Research • Vol. 2, No. 4, 2003 433
technical notes
Rapid Subpicogram Protein Detection Table 2. Peak Sizing and Concentration of Proteins from Jurkat Cells conc (RSD ( %)a 37 ° C size kDa (RSD peak no.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 Total
routine
6.0 (-) 8.9 (4.9) 11.3 (1.9) 14.7 (7.0) 18.5 (27.5) 24.7 (9.3) 29.9 (5.5) 34.8 (6.6) 38.2 (6.8) 42.6 (5.9) 49.4 (6.3) 52.2 (8.1) 58.0 (4.0) 70.9 (1.3) 78.9 (2.4) 86.2 (6.3) 108.4 (4.9) 113.8 (1.1) 125.0 (1.1) 139.3 (0.5) 153.5 (0.2) 156.0 (0.4) 167.0 (0.7) 210.0 (3.9)
%)a new
routine Figure 3A
49 ° C new Figure 3C
routine Figure 3B
new Figure 3D
*(I)d
6.0 (0) 8.3 (5.3) 14.3 (2.3) 17.4 (5.2) 19.8 (4.7) 24.0 (4.4) 28.3 (3.9) 31.1 (3.8) 36.3 (3.3) 40.3 (1.7) 47.0 (3.1) 55.2 (3.1) 62.9 (1.5) 71.9 (3.6) 86.8 (3.3) 91.6 (2.7) 115.0 (1.7) 127.0 (1.1) 134.0 (1.6) 145.0 (2.2) 151.0 (1.2) 210.0 (2.0)
marks in Figure 3
1.1 (72.9) 0.06 (40.4) 5.9 (10.8) 4.0 (44.6) 5.9 (62.6) 7.2 (24.2) 5.5 (18.9) 28.1 (15.3) 8.7 (59.2) 3.4 (16.3) 2.0 (39.4) 4.1 (25.6) 4.4 (49.0) 13.2 (50.5) 0.8 (56.5) 1.5 (48.8) 2.3 ( 9.2) 0.8 ( 8.5) 2.6 (18.9) 0.36 (13.8) 2.5 (13.2) (34.5)
1.7 +b(33.9) 2.3 -(26.3) 3.0 (18.4) 3.9 (20.7) 3.0 -(6.6) 2.9 -(14.7) 11.6 + (5.5) 23.0 -( 7.3) 3.0 -(10.4) 5.8 + ( 6.3) 4.2 + (20.0) 4.9 + (12.9) 19.6 + (16.2) 0.6 -(10.0) 2.9 +(15.8) 2.1 (41.9) 1.5 + (34.2) 0.2 + (33.4) 0.2 (15.0) (19.9)
1.9 (50.5) 3.1 +b(38.5) 6.4 + (18.6) 3.0 -(20.5) 7.4 +(18.5) 9.3 +(21.1) 0.6 -(18.8) 29.4 + (20.5) 10.0 + (25.2) 8.4 + (15.6) 3.0 + (20.6) 11.0 + (25.5) 0.5 (20.4) 2.4 -(35.5) 1.8 + (20.6) 0.7 -(10.3) 1.5 -( 8.5 ) 0000(24.9)
4.0 ++c(19.8) 5.7 ++(14.0) 3.5 (28.6) 3.0 -(30.6) 8.7 ++(10.6) 9.5 + (12.6) 0.6 - -( 2.1) 27.1 ++( 5.5) 7.5 + (10.6) 9.0 + (9.1) 3.0 (10.6) 12.0 ++(12.1) 2.2 - -(15.1) 2.1 + (11.7) 0.5 - (10.1) 1.5 -(5.5) 000
II
III
protein
lower marker system peak PKC inhibitor stathmin HSP28 hnRNP 26S proteasomee hnRNP HSP60 HSP70
IV
VCPf HSP90
*
upper marker
(14.7)
a Three experiments of cell culture and three analyses of each. b +: increment or -: decrement against 37 °C with routine method. c +: increment, -: decrement,++: amplifiedincrement,or- -: amplifieddecrementagainst37°Cwithnewmethod. d I: Thymosinβ4notbeobserved. e1 MPELAVQKVVVHPLVLLSVVDHFNRIGKVG30 sequence coverage 48%. f VCP; Transitional endoplasmic reticulum ATPase; 20KNRPNRLIVDEAINEDNSVVS40 sequence coverage 56%.
was the limit of detection for the routine method, whereas 80 ng (0.02 µg/µL × 4 µL) of total proteins were detectable using the new method (S/N > 3). These data clearly demonstrate that even in the real protein samples from organism could be analyzed with higher-sensitivity by new method. Although nanogram to picogram detection limits were also achieved by 2-DE,10,19,20 submicrograms of total proteins must be loaded in a gel and several hours of complicated procedures are required. In contrast, only subpicogram amounts of a pure protein and nanograms of total proteins from biological sample were required for our method, and only 30 min was required for 10 samples by µ-CE. The targeted proteins in biological samples are sometimes present in low concentrations; the method we developed is promising for proteome analysis. 3.5 Application of the New Method to Analysis of Protein Expression during Heat Shock. The electropherograms of Figure 3 show protein expression during heat-treatment at 49 °C for 30 min. Cellular responses to stress signals, especially heat shock signals, have been extensively studied, and studies on the expression of proteins during heat shock and their functional regulation are topics of whole research fields of cell biology. Jurkat cells possess target proteins (7 kDa; thymosin and 18 kDa; stathmin), which usually increase after heattreatment.12,13 The increment of the concentration of the peak at 18 kDa was confirmed by both the routine method and the new method (peak 5 in Table 2), although it is unclear in Figure 3. Unfortunately, the increment of the peak at 7 kDa could not be observed by the Agilent 2100 Bioanalyzer (Figure 3), because the migration of this target protein overlapped with the lower marker and system peak (6-9 kDa). However, the other increased peaks (heat shock proteins; HSP) and new decreased 434
Journal of Proteome Research • Vol. 2, No. 4, 2003
Figure 3. Electropherograms of proteins extracted from Jurkat cells, (A) cultured at 37 °C or (B) heat-treated at 49 °C by the routine method and (C) cultured at 37 °C or (D) heat-treated at 49 °C by the new method. The symbols I, II, III, and IV have the same meanings as in Table 2.
peaks (about 36 and 87 kDa; marked III and IV with reversed triangles in Figure 3B) were observed after heat-treatment, and the latter peak was identified as important heat-shock related proteins (26S proteasome and VCP, respectively; Table 2). In addition, the decrease in the levels of these proteins were amplified by the new method (Figure 3, parts C and D). The mean value of molecular size, concentration, and RSD (%) of each protein are summarized in Table 2. We can recognize the lower RSD values of size and concentration for new method. Protein samples with the routine method were dissolved in ionic buffer. Since the sensitivity of the method is dependent on the ionic strength of the protein sample, the routine method utilized the dilution of the sample in order to amplify the peak intensity.8 However, some peaks would be amplified beyond
technical notes the intrinsic peak intensity due to the stacking effect,16 and some other peaks would be decreased by a real dilution effect. This is the reason some calculated concentrations were mismatched between the routine method and the new method in the control sample (37 °C). However, we think that the new method reveals more natural data because proteins were dissolved in water and the intensities by our new method are independent of the stacking effect. Because the protein sizing separation was achieved even in the absence of a denaturing process, the concentrations of SDS and proteins in a microchannel seem to have been sufficient for forming the negative charged protein and compounding the SDS-protein-dye complex. The very low concentration of protein usually gives a denaturing effect. The fact that µ-CE requires only a small concentration of proteins may help remove the need for the denaturing procedure. In contrast, the high concentration and high amounts of total proteins in 2-DE produce the aggregation of each protein, which can cause serious sizing mistakes without denaturing. From these analyses, we confirmed that the new method without denaturing is effective for a real biological protein sample in terms of rapid detection of protein expression. Although folded conformations, which are natural proteins, or unfolded conformations, which are caused by denaturing, are serious for diagnosis,15 we propose that, for the purpose of sizing and detection of some important proteins, omitting a denaturing process does not give serious errors and will give more stable data for some biological samples by µ-CE.
4. Concluding Remarks To achieve high sensitivity and short analysis time by µ-CE analysis, we omitted the protein denaturing process. When proteins were dissolved in water and when proteins were not heated for denaturing, the sensitivity increased by an order of 10-100. The new method without denaturing is fully competitive with the analysis of a real biological protein mixture from Jurkat cells, and the expressed proteins can be readily analyzed by the new method. In conclusion, we propose that the denaturing process is unnecessary for protein sizing analysis by µ-CE and diagnosis of some proteins. This new method will give effective advantage for constructing the HTS systems.
Acknowledgment. This work was partially supported by a Grant funding of Core Research for Evolutional Science
Tabuchi et al.
and Technology (CREST) from the Japan Science and Technology Corporation (JST), by a Grant from the New Energy and Industrial Technology Development Organization (NEDO) of the Ministry of Economy, Trade and Industry, Japan, a Grantin-Aid for Scientific Research from the Ministry of Health, Labor, and Welfare, Japan, a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Technology, Japan, and by the Single-molecule Bioanalysis Laboratory, National Institute of Advanced Industrial Science and Technology (AIST), Japan.
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PR034009M
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