Comparison of SYPRO Ruby and Deep Purple Using Commonly Available UV Transilluminator: Wide-Scale Application in Proteomic Research Bulbul Chakravarti,* Wongrat Ratanaprayul, Neville Dalal, and Deb N. Chakravarti Keck Graduate Institute of Applied Life Science, 535 Watson Drive, Claremont, California 91711 Received November 8, 2007
Fluorescent stains are becoming increasingly useful in proteomics research involving protein expression as well as post-translational modification studies and are particularly useful for samples which are expensive and scarce. The fluorescent dyes Deep Purple and SYPRO Ruby are widely used in protein expression studies. Using UV transillumination and Charged Coupled Device (CCD) based imaging system, their relative sensitivity to detect proteins separated by two-dimensional polyacrylamide gel electrophoresis and downstream protein identification by liquid chromatography-tandem mass spectrometry (LC-MS/MS) was compared. Using mouse liver homogenate, we detected a greater number of spots using SYPRO Ruby over Deep Purple stain. However, the number of matched peptides and the percentage of amino acid residues identified for 21 different proteins were comparable suggesting their equivalency for LC-MS/MS identification. In spite of comparable MS compatibility, we recommend the use of SYPRO Ruby for expression proteomics due to its higher sensitivity in detecting protein spots. Keywords: SYPRO Ruby • Deep Purple • Proteomics
Introduction In the postgenomic era, proteomics is becoming the key technology in biological research. Fluorescent dyes are widely used for quantitative proteomics research. Because of their high sensitivity, rapid detection time, linearity over a wide dynamic range and relatively safe handling compared to traditional Coomassie Brillliant Blue, silver stain and/or autoradiography, these are becoming increasingly popular in spite of the fact that fluorescent dyes can be fairly expensive and requires a gel documentation system. Although fluorescent dyes have been used for multiple purposes, including multiplex analysis of protein samples where post-translational modification and expression can be determined on the same gel, such stains are most commonly used to study protein expression levels.1–7 In the present investigation, we have compared the staining efficiency of two commonly used fluorescent dyes (Deep Purple, absorption peak ∼360 nm and ∼520 nm, emission peak ∼610 nm; and SYPRO Ruby, absorption peak ∼280 nm and ∼470 nm, emission peak ∼610 nm) for proteins followed by the molecular characterization of the visually identified protein spots using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Two different types of electronic imaging systems can be used for the detection of fluorescent dyes: (a) laser based/ photomultiplier (PMT) system, and (b) broad spectrum, lampbased charged coupled device (CCD) system. However, the laser scanners are quite expensive and can be used for only those * To whom correspondence should be addressed. E-mail, bulbul_chakravarti@ kgi.edu; phone, (909) 607-9525; fax, (909) 607-9826. 10.1021/pr7007225 CCC: $40.75
2008 American Chemical Society
fluorophores whose excitation/emission spectra will match with the output of the available laser sources. In the present investigation, for comparative performance of the above two fluorescent dyes, we have used a broad spectrum, lamp-based CCD system which is suitable for multiwavelength application but is relatively less expensive and hence is affordable by a larger number of investigators.
Experimental Section Reagents. IPG strips, urea, thiourea, CHAPS, Destreak Rehydration Solution, 2D-clean up kit, 2D-Quant kit and Deep Purple were obtained from GE Healthcare Bio-Sciences Corp., Piscataway, NJ. Precast polyacrylamide gels, DTT and other reagents for electrophoresis as well as Bio-Safe Coomassie were purchased from Bio-Rad Laboratories, Hercules, CA. SYPRO Ruby was purchased from Molecular Probes, Eugene, OR. Sequencing grade tyrpsin was obtained from Promega Corporation, Madison, WI. Preparation of Sample for Two-Dimensional Gel Electrophoresis (2D-GE). The C57BL/6 male mice (about 5 months old) were obtained from Harlan Sprague-Dawley, Indianapolis, IN. The animals were euthanized by cervical dislocation, and livers were collected and frozen immediately in liquid nitrogen. The samples for 2D-GE were prepared by homogenizing the liver in lysis buffer (30 mM Tris-HCl/7 M urea/2 M thiourea/ 4% (w/v) CHAPS/40 mM DTT, pH 8.5) using a tissue grinder (Corning, Inc., Corning, NY) followed by centrifugation three times, each time at 500g for 10 min. The supernatant was cleaned up by using the 2D-clean up kit and the precipitate was dissolved in sample buffer (7 M urea/2 M thiourea/4% (w/v) Journal of Proteome Research 2008, 7, 2797–2802 2797 Published on Web 05/30/2008
research articles CHAPS/0.5% (v/v) IPG buffer, pH 3-10/40 mM DTT/ 0.002% (w/v) bromophenol blue) and centrifuged at 16 000g for 30 min to remove any insoluble material. The supernatant was collected and protein estimation was carried out using the 2DQuant kit. Samples were stored at -80 °C for further analysis. Isoelectric Focusing (IEF) and Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE). Sample for each stain was analyzed in quintuplicate by 2D-GE, that is, IEF followed by SDS-PAGE. Briefly, 15 µg of protein was applied to a 11 cm pH 3-10 Ready Strip IPG strip using the Ettan IPGphor II Cup Loading Manifold. The strips were rehydrated overnight with the Destreak Rehydration Solution (pH 3-10) and the samples were resolved according to their pIs using the IPGphor system (GE-Health care Bio-Sciences Corp.) (Step 1, 500 V for1 h; Step 2, 1000 V for 1 h; Step 3 (gradient), 6000 V for 2 h and Step 4, 6000 V for 0.3 h). Following reduction and alkylation of the above strips, further separation of the proteins was carried out by SDS-PAGE using linear gradient polyacrylamide gels (8%-16% (w/v) acrylamide) using a Criterion cell apparatus (Bio-Rad Laboratories). Following the seconddimension gel electrophoresis, the gels were stained either with SYPRO Ruby or Deep Purple according to the manufacturer’s instruction. If necessary, the gels were subsequently stained with Bio-Safe Coomassie to visualize the protein spots prestained with fluorescent stains, by the naked eyes. Gel Scanning and Spot Detection. For UV excitation and detection of the images of the gels stained with SYPRO Ruby or Deep Purple, a CCD based Bioimaging system (UVP, LLC, Upland, CA) containing UV transilluminator (300-340 nm) and emission filter (570-640 nm) was used. Images were captured using a Biospectrum AC dark room installed with BioChemi cooled 12 bit 1.4 mpx CCD camera and LabWorks 4.5 software (UVP, Inc., Upland, CA). Computer-assisted 2D gel image analysis was performed using the Image Master Platinum 2D software package, version 5.0 (GE-Health care Bio-Sciences Corp) for detection of protein spots. Where necessary, the spots were edited manually. To compare the staining efficiency of the two stains in different areas of the gel, the entire gel was divided into four regions (Region 1, molecular weight 25 000 Da and pI < 7.0 and Region 4, molecular weight >25 000 Da and pI > 7.0). The total number of spots detected in the whole gel as well as in four different regions was compared for the two stains. In-Gel Trypsin Digestion. In-gel trypsin digestion following excision of gel spots was carried out as described by Chakravarti et al.,.8 Briefly, the excised gel spots were destained with 50% (v/v) acetonitrile /0.2 M ammonium bicarbonate (pH 8.0), dried by adding acetonitrile and air drying followed by reduction and alkylation. Next, the gel spots were digested with trypsin (Promega, 20 µg/mL in 50 mM NH4HCO3, pH 8.0) overnight at 37 °C, the supernatant was collected, and acetonitrile/formic acid, 50:50 (v/v) (enough to immerse the gel) was added to the gel pieces. Following incubation at 60 °C and sonication, the solution around the gel bits was collected and combined with the supernatant obtained above. The acetonitrile present in the supernatant was evaporated. The tryptic peptides were stored at -20 °C. Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) of Tryptic Peptides, Database Searching, and Data Processing. LC-MS/MS of tryptic peptides, database searching and data processing were carried out using LCQ Deca XP ProteomX System (Thermo Electronics Corporation, CA) as 2798
Journal of Proteome Research • Vol. 7, No. 7, 2008
Chakravarti et al. 8
described by Chakravarti et al.,. One-dimensional liquid chromatography analysis of the tryptic peptides was performed using online reversed phase Picofrit column (BioBasic C18, 75 µm × 10 cm, tip ) 15 µm, New Objective, Woburn, MA). The tryptic digest was loaded and washed with solvent A (0.1% formic acid, v/v) for 20 min at a flow rate of 125 nL/min; the peptides were eluted by applying a gradient of 65% solvent B (0.1% formic acid, v/v in acetonitrile) over 25 min period using the same flow rate. Next, a gradient of 80% B was applied over a period of 5 min. Finally, the column was reset to 100% A over the next 1 min and equilibrated to 100% A over next 30 min before loading the next sample. The mass-spectrometer was set to acquire a full MS scan between 450 and 1800 m/z followed by a full MS/MS scan. Dynamic exclusion was enabled with two repeat counts, repeat duration of 30 s and 3 min exclusion duration unit. All MS/MS spectra were searched with the SEQUEST algorithm based Bioworks 3.3 (Thermo Finnigan) against a database created by extracting mouse entries from the NCBI ftp site (June 14, 2007). The searches were performed with enzyme constraints (trypsin) and a static modification of 57.05 Da for carboxyamidomethylation on the cysteine residue of each peptide. Two missed cleavages were allowed. The parent ion mass tolerance and the fragment ion mass tolerance were 1.4 and 0, respectively. The following stringent SEQUEST criteria were used to calculate the number of peptides for the purpose of identification of any particular protein: (i) Delta Cn score is at least 0.1, (ii) Rsp score is e3, (iii) Xcorr g1.5 for +1 charged peptides, (iv) Xcorr g2.0 for +2 charged peptides, (v) Xcorr g2.5 for +3 charged peptides. Spectra for all the hits were further verified manually. The following criteria were taken into account during the manual verification stage: (i) continuity of the b and y ion series, and (ii) good quality MS/MS spectrum, that is, the fragment ions should be clearly above the baseline. Proteins with three (at least with one of the fluorophores) or more unique spectra were accepted as positive identification.
Results and Discussion UV transillumination and CCD based imaging for detection of protein spots has been used in the present study and in general has several advantages such as (i) low cost compared to laser scanners, (ii) rapid exposure time (typically milliseconds to seconds), (iii) rapid multiplex analysis of proteins due to multiple fluorescent images captured as a result of single UV excitation and multiple emission spectra of the different dyes used to stain a single gel. As evident from Figure 1 and Table 1, when the gels were stained with SYPRO Ruby, a larger number of spots were detected compared to those stained with Deep Purple. Interestingly, when any particular gel was divided into four different regions depending on the molecular weights and isoelectric points, SYPRO Ruby was found to be more sensitive in detecting the total number of spots in any one of the four regions (see Table 2). However as observed earlier (Harris et al.),11 it was most sensitive in detecting the total number of protein spots in the region of the gels containing proteins with lower molecular weights and isoelctric points (molecular weight 7.0)
Da,
57 ( 6
24 ( 6
2.4
Da,
25 ( 4
13 ( 3
1.9
Da,
370 ( 43
232 ( 15
1.6
Da,
116 ( 12
62 ( 10
1.9
a The data represent the results obtained from five different gels stained with SYPRO Ruby (SR) and five different gels stained with Deep Purple (DP).
summarized in Table 3. In the Application note of Amersham Biosciences on the comparison of Deep Purple and SYPRO Ruby, higher sensitivity of Deep Purple over SYPRO Ruby was reported for the same protein samples. This may be due to plenty of lysine residues present in those proteins.9 In addition, similar sensitivity level was observed for Deep Purple while using 532 nm laser or 365 nm UV transilluminator. On the contrary, Hart et al. reported that using different excitation10 sources such as transillumination with UV and blue light (SafeImager) boxes as well as use of laser based instruments with 473 nm, 488 nm and 532 nm excitation, Deep Purple showed markedly lower signal and sensitivity with any source
research articles
other than 532 nm, whereas SYPRO Ruby stained gels can be imaged with similar signal and sensitivity.10 In fact, Harris et al.11 reported detection of comparable number of spots (slightly higher for SYPRO Ruby compared to Deep Purple) when excitation filters of 480 nm (bandwidth 30 nm) and 540 nm (bandwidth 40 nm) were used for SYPRO Ruby and Deep Purple, respectively. In the present study, we have used a commercially available instrument with the standard transilluminator as the source of excitation. In fact, using UV transillumination (300-340 nm) as the source of excitation, 570-640 nm emission filter and CCD based imaging system, we could detect higher number of spots for the same protein sample while using SYPRO Ruby compared to Deep Purple. As mentioned earlier, laser sources are expensive and restricted to fluorophores whose excitation/emission match with the available laser sources. Hence, broad lamp based CCD imaging was used which has multiwavelength applications and is relatively easily affordable for individual laboratories due to its lower price. As evident from Figure 1 and Table 1, a higher number of spots (1.7-fold) was detected for mouse liver homogenate separated by 2D-GE and stained with SYPRO Ruby compared to that stained by Deep Purple. We obtained similar results while using Escherichia coli extract (data not shown). Since robotic systems used for automatic excision of the fluorescently labeled proteins is prohibitively expensive for most laboratories, we excised the protein spots manually for identification by mass spectrometry. The fluorescently stained gels were subsequently stained with Bio-Safe Coomassie for visualization of the protein spots with naked eye. This helped in the manual excision of the spots compared to visualizing them under a hand-held UV illuminator (such as those sold by UVP, LLC) before excision. However, in this case, laboratory personnel should take adequate precautions for protection against UV light induced damage to the skin, such as wearing full sleeve laboratory coats and gloves and use of a UV blocking face shield. In fact, although one has to take some extra precaution for direct excision of protein spots following visualization under the UV lamp, the advantage of this procedure is the elimination of the cost of Bio-Safe Coomassie stain used. We also wanted to check the possibility that the reason for the dramatic difference in the number of spots defined between the two stains could be the inability to amplify the signal by UV transillumination as is possible using a laser-based photomultiplier system. In Figure 2, we have presented graphically the maximal pixel signal intensity for the brightest spot of different gels (top panel), as well as the total pixel intensity for different gels (bottom panel) stained with SYPRO Ruby and Deep Purple. As is evident from Figure 2, even when the brightest spot intensities of the two different gels (8 bit images, 0-255 gray scale value) stained with SYPRO Ruby and Deep Purple were comparable (such as 166 for Sypro Ruby and 167 for Deep Purple), the total intensity of the whole gel was higher (>2-fold) (such as 39, 740 for SYPRO Ruby and 17, 045 for Deep Purple) for the one stained with SYPRO Ruby compared to the one stained with Deep Purple. This indicates the inability of the UV transilluminator to amplify the signal as efficiently as the laser-based photomultiplier system. As evident from Table 3, for the majority of the proteins, the number of unique peptides identified for any particular protein stained with the above two stains was found to vary from 2 to 9. We believe that at least one of the reasons for the low number of peptides identified for spot no. 15 (2 peptides Journal of Proteome Research • Vol. 7, No. 7, 2008 2799
research articles
Chakravarti et al. a
Table 3. Identification of Protein Spots Stained with SYPRO Ruby or Deep Purple spot no./ staining reagent
1/SR
GenInfo identifier no.(gi no.)
mol. wt. (a) exp. (b) theor.
pI (a) exp. (b) theor.
no. of peptides matchedb
% of amino acid residues covered
protein probability
(a) 59, 582 (b) 68, 648 (a) 59, 582 (b) 164, 515 (a) 62, 132 (b) 68, 648 (a) 62, 132 (b) 164, 515 (a) 45, 839 (b) 33, 387 (a) 46, 514 (b) 33, 387 (a) 38, 590 (b) 39, 162 (a) 38, 590 (b) 39, 162 (a) 52, 120 (b) 56, 503 (a) 53, 564 (b) 56, 503 (a) 49, 571 (b) 46, 996 (a) 49, 571 (b) 47, 112 (a) 50, 530 (b) 46, 996 (a) 50, 530 (b) 47, 112 (a) 46, 810 (b) 47, 658
(a) 5.3 (b) 5.7 (a) 5.3 (b) 6.5 (a) 5.3 (b) 5.7 (a) 5.3 (b) 6.5 (a) 5.1 (b) 5.0 (a) 5.1 (b) 5.0 (a) 4.9 (b) 5.7 (a) 4.9 (b) 5.7 (a) 5.8 (b) 7.5 (a) 5.8 (b) 7.5 (a) 5.8 (b) 6.8 (a) 5.8 (b) 6.4 (a) 5.8 (b) 6.8 (a) 5.8 (b) 6.4 (a) 5.8 (b) 6.1
11
24.8
1.00 × 10-10
3
3
1.00 × 10-8
14
29.1
3.00 × 10-10
5
5.6
8.00 × 10-8
4
21.7
5.00 × 10-8
3
17.4
1.00 × 10-9
5
18.9
3.00 × 10-10
4
14.9
6.00 × 10-9
9
24.7
2.00 × 10-10
8
25.2
6.00 × 10-11
3
11.1
4.00 × 10-11
4
13.6
2.00 × 10-9
3
11.1
5.00 × 10-10
4
13.6
3.00 × 10-10
7
21.1
4.00 × 10-10
21431841
(a) 47, 985 (b) 47, 658
(a) 5.8 (b) 6.1
6
20.6
6.00 × 10-10
9506589
(a) 42, 329 (b) 36, 890 (a) 42, 714 (b) 36, 890 (a) 39, 318 (b) 36, 446 (a) 39, 504 (b) 36, 446 (a) 53, 273 (b) 61, 299 (a) 55, 365 (b) 61, 299 (a) 41, 638 (b) 39, 840 (a) 42, 224 (b) 39, 840 (a) 37, 460 (b) 32, 655 (a) 38, 072 (b) 32, 655 (a) 32, 616 (b) 29, 349 (a) 32, 833 (b) 29, 349 (a) 57, 232 (b) 54, 434 (a) 54, 805 (b) 54, 434 (a) 48, 794 (b) 44, 993
(a) 5.7 (b) 6.1 (a) 5.7 (b) 6.1 (a) 5.6 (b) 5.9 (a) 5.6 (b) 5.9 (a) 6.3 (b) 7.9 (a) 6.3 (b) 7.9 (a) 6.3 (b) 9.2 (a) 6.3 (b) 9.2 (a) 6.4 (b) 7.2 (a) 6.4 (b) 7.2 (a) 6.5 (b) 7.0 (a) 6.5 (b) 7.0 (a) 7.3 (b) 7.7 (a) 7.3 (b) 7.7 (a) 7.3 (b) 7.8
4
21.3
4.00 × 10-8
5
21.6
3.00 × 10-9
4
17.9
4.00 × 10-7
5
24
2.00 × 10-12
6
10.8
4.00 × 10-9
6
10.8
4.00 × 10-8
3
7.3
9.00 × 10-9
5
14.7
1.00 × 10-10
3
12.6
9.00 × 10-10
4
18.4
3.00 × 10-7
6
31.9
2.00 × 10-8
6
35.4
3.00 × 10-8
8
25.9
2.00 × 10-10
6
17.8
7.00 × 10-9
7
32.4
1.00 × 10-12
2/SR
ALBU_MOUSE Serum albumin precursor carbamoyl-phosphate synthetase 1 ALBU_MOUSE Serum albumin precursor carbamoyl-phosphate synthetase 1 regucalcin
2/DP
regucalcin
3/SR
put. beta-actin (aa 27-375)
49868
3/DP
put. beta-actin (aa 27-375)
49868
4/SR
6753036
5/SR
aldehyde dehydrogenase 2, mitochondrial aldehyde dehydrogenase 2, mitochondrial enolase 3 beta, muscle
70794816
5/DP
hypothetical protein LOC433182 enolase 3 beta, muscle
70794816
7/SR
hypothetical protein LOC433182 SAHH_MOUSE Adenosylhomocysteinase (S-adenosyl-L-homocyteine hydrolase) (AdoHcyase)(Liver copper-binding protein) (CUBP) SAHH_MOUSE Adenosylhomocysteinase (S-adenosyl-L-homocyteine hydrolase) (AdoHcyase)(Liver copper-binding protein) (CUBP) fructose biphosphatase 1
7/DP
fructose biphosphatase 1
8/SR
31982178
9/SR
malate dehydrogenase 1, NAD (soluble) malate dehydrogenase 1, NAD (soluble) glutamate dehydrogenase 1
9/DP
glutamate dehydrogenase 1
6680027
10/SR
ornithine transcarbamylase
762985
10/DP
ornithine transcarbamylase
762985
11/SR
glycine N-methyl transferase
6754026
11/DP
glycine N-methyl transferase
6754026
12/SR
carbonic anhydrase 3
31982861
12/DP
carbonic anhydrase 3
31982861
13/SR
aldehyde dehydrogenase family 1, subfamily A1 aldehyde dehydrogenase family 1, subfamily A1 betaine_homocysteine methyl transferase
85861182
1/DP
4/DP
6/SR
6/DP
8/DP
13/DP 14/SR
2800
protein name
5915682 124248512 5915682 124248512
Journal of Proteome Research • Vol. 7, No. 7, 2008
6677739 6677739
6753036 6679651
6679651
21431841
9506589
31982178 6680027
85861182 7709990
research articles
Comparative Evaluation of SYPRO Ruby and Deep Purple Stains Table 3. Continued spot no./ staining reagent
14/DP
protein name
GenInfo identifier no.(gi no.)
mol. wt. (a) exp. (b) theor.
pI (a) exp. (b) theor.
no. of peptides matchedb
% of amino acid residues covered
protein probability
(a) 50, 607 (b) 44, 993 (a) 11, 933 (b) 15, 700 (a) 11, 797 (b) 15, 700 (a) 58, 521 (b) 57, 108
(a) 7.3 (b) 7.8 (a) 6.8 (b) 7.5 (a) 6.8 (b) 7.5 (a) 4.7 (b) 4.7
8
34.4
9.00 × 10-9
2
17.8
2.00 × 10-7
3
18.5
6.00 × 10-9
8
19.5
2.00 × 10-9
129729
(a) 58, 521 (b) 57, 108
(a) 4.7 (b) 4.7
7
14.5
2.00 × 10-9
19526790
(a) 53, 483 (b) 43, 482 (a) 53, 483 (b) 43, 482 (a) 46, 204 (b) 34, 787 (a) 46, 204 (b) 34, 787 (a) 46, 990 (b) 40, 066
(a) 5.3 (b) 5.4 (a) 5.3 (b) 5.4 (a) 5.9 (b) 6.6 (a) 5.9 (b) 6.6 (a) 6.0 (b) 6.6
3
9.6
2.00 × 10-6
4
13.4
6.00 × 10-6
5
22.9
6.00 × 10-9
4
19.5
1.00 × 10-5
3
10.9
4.00 × 10-5
25108890
(a) 46, 990 (b) 40, 066
(a) 6.0 (b) 6.0
3
10.4
2.00 × 10-4
38142460
(a) 35, 466 (b) 27, 607
(a) 7.5 (b) 8.2
2
14.9
7.00 × 10-11
38142460
(a) 35, 466 (b) 27, 607
(a) 7.5 (b) 8.2
3
16.5
1.00 × 10-5
31980648
(a) 55, 009 (b) 56, 266
(a) 4.9 (b) 5.1
6
14.7
3.00 × 10-7
31980648
(a) 55, 009 (b) 56, 266
(a) 4.9 (b) 5.1
5
13
1.00 × 10-3
7709990
15/SR
betaine_homocysteine methyl transferase beta-1-globin
15/DP
beta-1-globin
4760590
16/SR
18/SR
PDIA1_MOUSE Protein disulfide-isomerase precursor (PDI)(Prolyl 4 hydroxylase subunit beta)(Cellular thyroid hormone binding protein) PDIA1_MOUSE Protein disulfide-isomerase precursor (PDI)(Prolyl 4 hydroxylase subunit beta)(Cellular thyroid hormone binding protein) Methionine adenosyltransferase I, alpha Methionine adenosyltransferase I, alpha Arginase 1, liver
18/DP
Arginase 1, liver
19/SR
DHSO_MOUSE Sorbitol dehydrogenase (L-iditol 2-dehydrogenase) DHSO_MOUSE Sorbitol dehydrogenase (L-iditol 2-dehydrogenase) Electron transferring flavoprotein, beta polypeptide Electron transferring flavoprotein, beta polypeptide ATP synthase, H+ transporting mitochondrial F1 complex, beta subunit ATP synthase, H+ transporting mitochondrial F1 complex, beta subunit
16/DP
17/SR 17/DP
19/DP
20/SR
20/DP
21/SR
21/DP
4760590
129729
19526790 7106255 7106255 25108890
a DP and SR represent Deep Purple and SYPRO Ruby, respectively. Spots 1-21 were excised from three gels for each stain and each gel was loaded with 15 µg of protein. The gels were stained with Bio-Safe Coomassie before excision of spots. The trypsin digestion and LC-MS/MS were carried out as described in Experimental Section. (a) Exp. and (b) theor. represent the experimental and theoretical values, respectively, for molecular weight as well as pI. b The number denotes the number of unique peptides and not the total number of peptides identified for each protein.
in case of staining with SYPRO Ruby and 3 peptides while using Deep Purple) and spot no. 20 (2 peptides in case of staining with SYPRO Ruby and 3 peptides while using Deep Purple) was due to their low molecular weight thereby generating fewer tryptic peptides to identify. Generation of hydrophobic peptides following tryptic digestion can also lead to low peptide recovery. In general, while using two different stains, the variation in the number of unique peptides identified was found to be none to 1 for any one particular protein spot (except spot nos. 1, 10 and 13, the variation was found to be 2 to 3 peptides). In fact, the variation observed was marginal and not due to higher MS compatibility of any of the above two dyes. For example, for spot no. 1, which was found to be a mixture of two proteins, we were able to detect higher number of peptides while using Deep Purple compared to SYPRO Ruby for both ALBU MOUSE Serum albumin precursor (14 peptides for Deep Purple and 11 peptides for SYPRO Ruby) and
carbamoyl-phosphate synthetase 1 (5 peptides for Deep Purple and 3 peptides for SYPRO RUBY). On the contrary, for SYPRO Ruby and Deep Purple stained spot no. 4, we were able to detect 9 and 8 peptides, respectively, for aldehyde dehydrogenase 2, mitochondrial. Again for spot no. 9, we were able to detect same number of peptides, that is, six peptides for glutamate dehydrogenase while using Deep Purple as well as SYPRO Ruby. A close correlation between experimental and theoretical molecular weight and pI (e15%) for the majority of the proteins provided additional confirmation of the identification of the protein spots (cf. Table 3). One of the reasons for the larger discrepancy observed may be proteolysis of the original protein thereby shifting the molecular weight and pI. As explained above, in the present study, we have undertaken LC-MS/MS approach for the identification of protein spots following in-gel trypsin digestion. Earlier, Tannu et al.12 have used MALDI-TOF MS to compare the MS compatibility of Journal of Proteome Research • Vol. 7, No. 7, 2008 2801
research articles
Chakravarti et al. the same size gel is slightly cheaper for Deep Purple (15-20% cheaper for Deep Purple compared to Sypro Ruby).
Conclusion In summary, in spite of slightly higher price range of the SYPRO Ruby stain, greater sensitivity in detecting the total number of protein spots, as well as comparable MS compatibility, suggests advantage of SYPRO Ruby over Deep Purple while using a standard UV transilluminator and CCD based imaging system. Although the excitation wavelength is not optimal, it is a reasonable compromise due to the expensive nature of the laser photomultiplier system with optimum source of excitation which is not affordable by most laboratories. Hence, we recommend SYPRO Ruby for expression proteomics studies.
Figure 2. Comparison of the maximal pixel intensity of the brightest spot and total pixel intensity of the whole gels (8 bit images, 0-255 gray scale value) stained with SYPRO Ruby (9) or Deep Purple (0). Fifteen micrograms of mouse liver proteome was analyzed by 2D-GE. The top panel presents the maximal pixel intensity of the brightest spot observed for five different gels (1-5) as well as the average maximal pixel intensity of the brightest spots (A) of these five different gels which were stained with SYPRO Ruby or Deep Purple. The bottom panel represents the total pixel intensity for five different gels (1-5) as well as the average total pixel intensity (A) for these five different gels which were stained with SYPRO Ruby or Deep Purple.
SYPRO Ruby and Deep Purple. Although LC-MS/MS can be more expensive compared to peptide mass fingerprinting by MALDI-TOF MS, the former method, which provides mass and the amino acid sequence information of the peptide identified, is more definitive in the identification of an unknown protein compared to MALDI-TOF MS based peptide mass fingerprinting, which provides information only on the mass of the peptides identified. Using MALDI-TOF MS, Tannu et al.12 have observed that Deep Purple can result in increased peptide recovery compared to SYPRO Ruby and hence can improve MSbased identification of lower intensity protein spots. Compared to the studies by Tannu et al.,12 we have analyzed fewer number of protein spots (21) for their identification by LC-MS-MS. Even with 21 protein spots analyzed, we detected greater number of peptides for certain protein spots stained with SYPRO Ruby, and for some other protein spots, we detected greater number of peptides stained with Deep Purple and for certain protein spots, we detected the same number of peptides regardless of their staining with Deep Purple or SYRPO Ruby (cf. Table 3). In other words, there was not a common pattern of higher peptide recovery of one stain compared to the other. Also, some of the protein spots (such as spot no. 20) were of relatively lower intensity compared to high intensity spots (such as spot no. 15). While using UV transillumination and CCD based imaging, we were able to detect proteins stained with SYPRO Ruby or Deep Purple at femtomole levels; mass spectrometric identification was possible with proteins present at picomole levels under the conditions used. Another concern for the researchers is obviously the price of the reagent to be used. While comparing between the above two stains, we found that the price of the two stains for staining
2802
Journal of Proteome Research • Vol. 7, No. 7, 2008
Acknowledgment. The authors acknowledge the generous support provided by the Keck Graduate Institute of Applied Life Sciences, Claremont, CA, The Arnold and Mabel Beckman Foundation and the Ralph M. Parsons Foundation. This work is supported in part by National Science Foundation grants FIBR 0527023. N.D. is an undergraduate student and worked on the project as part of the outreach program of the above NSF grant. The authors also acknowledge Pamela Fathy for technical help. References (1) Patton, W. F. A thousand points of light: the application of fluorescence detection technologies to two-dimensional gel electrophoresis and proteomics. Electrophoresis 2000, 21, 1123–1144. (2) Patton, W. F. Detection technologies in proteome analysis. J. Chromatogr., B: Anal. Technol. Biomed. Life Sci. 2002, 771, 3–31. (3) Lopez, M. F.; Mikulskis, A.; Golenko, E.; Herick, K.; Spibey, C. A.; Taylor, I.; Bobrow, M.; Jackson, P. High-content proteomics: fluorescence multiplexing using an integrated, high-sensitivity, multiwavelength charge-coupled device imaging system. Proteomics 2003, 3, 1109–1116. (4) Hart, C;.; Schulenberg, B.; Steinberg, T. H.; Leung, W. Y.; Patton, W. F. Detection of glycoproteins in polyacrylamide gels and on electroblots using Pro-Q Emerald 488 dye, a fluorescent periodate Schiff-base stain. Electrophoresis 2003, 24, 588–598. (5) Adam, G. C.; Sorensen, E. J.; Cravatt, B. F. Chemical strategies for functional proteomics. Mol. Cell. Proteomics 2002, 1, 781–790. (6) Adam, G. C.; Sorensen, E. J.; Cravatt, B. F. Trifunctional chemical probes for the consolidated detection and identification of enzyme activities from complex proteomes. Mol. Cell. Proteomics 2002, 1, 828–835. (7) Steinberg, T. H.; Agnew, B. J.; Gee, K. R.; Leung, W. Y.; Goodman, T.; Schulenberg, B.; Hendrickson, J.; Beechem, J. M.; Haugland, R. P.; Patton, W. F. Global quantitative phosphoprotein analysis using Multiplexed Proteomics technology. Proteomics 2003, 3, 1128–1144. (8) Chakravarti, B.; Oseguera, M.; Dalal, N,.; Fathy, P.; Mallik, B.; Raval, A.; Chakravarti, D. N. Proteomic profiling of aging in the mouse heart: Altered expression of mitochondrial proteins. Arch. Biochem. Biophys. 2008, in press. (9) Comparison of Deep Purple Total Protein Stain and Sypro Ruby in 1-D and 2-D gel electrophoresis, Application note, 2003, Amersham Biosciences, 18-1177-44, 2003-09, pp 1-5. (10) Hart, C.; Ahnert, N.; Hajivandi, M.; Lindsey, A.; Harwood, S. H. Comparative performance of fluorescent total protein stains on one- and two- dimensional PAGE gels. J. Biomol. Tech. 2006, 17, 5. (11) Harris, L. R.; Churchward, M. A.; Butt, R. H.; Coorsen, J. R. Assessing detection of methods for gel-based proteomic analyses. J. Proteome Res. 2007, 6, 1418–1425. (12) Tannu, N. S.; Sanchez-Brambila, G.; Kirby, P.; Andacht, T. M. Effect of staining reagent on peptide mass fingerprinting from in-gel trypsin digestions: A comparison of SyproRuby TM and DeepPurpleTM. Electrophoresis 2006, 27, 3136–3143.
PR7007225
