Pre-labeling of diverse protein samples with a fixed amount of Cy5 for sodium dodecyl sulfate–polyacrylamide gel electrophoresis analysis

Accepted Manuscript Pre-labeling of diverse protein samples with a fixed amount of Cy5 for SDSPAGE analysis Erik Bjerneld, Johan Johansson, Ylva Laurin, Åsa Hagner-McWhirter, Ola Rönn, Robert Karlsson PII: DOI: Reference:

S0003-2697(15)00213-4 http://dx.doi.org/10.1016/j.ab.2015.04.036 YABIO 12069

To appear in:

Analytical Biochemistry

Received Date: Revised Date: Accepted Date:

22 January 2015 27 April 2015 29 April 2015

Please cite this article as: E. Bjerneld, J. Johansson, Y. Laurin, Å. Hagner-McWhirter, O. Rönn, R. Karlsson, Prelabeling of diverse protein samples with a fixed amount of Cy5 for SDS-PAGE analysis, Analytical Biochemistry (2015), doi: http://dx.doi.org/10.1016/j.ab.2015.04.036

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Pre-labeling of diverse protein samples with a fixed amount of Cy5 for SDSPAGE analysis

Erik Bjerneld, Johan Johansson, Ylva Laurin, Åsa Hagner-McWhirter, Ola Rönn, and Robert Karlsson

Affiliation: GE Healthcare Bio-Sciences AB, Björkgatan 30, SE-751 84 Uppsala, Sweden. Corresponding author: Erik Bjerneld GE Healthcare Bio-Sciences AB, Björkgatan 30, SE-751 84 Uppsala, Sweden. Phone: +46186121592 E-mail: [email protected]

Short Title: Cy5 labeling of proteins for SDS-PAGE analysis

1

Abstract A pre-labeling protocol based on Cy5 N-Hydroxysuccinimide (NHS) ester labeling of proteins was developed for 1-D SDS-PAGE analysis. We show that a fixed amount of sulphonated Cy5 can be used in the labeling reaction to label proteins over a broad concentration range - over three orders of magnitude. The optimal amount of Cy5 was found to be 50-250 pmol in 20 µl, using a Tris-HCl labeling buffer with pH 8.7. Labeling protein samples with a fixed amount of dye in this range balances the requirements of subnanogram detection sensitivity and low dye-to-protein (D/P) ratios for SDS-PAGE. Simulations of the labeling reaction reproduced experimental observations of both labeling kinetics and D/P ratios. 2-D electrophoresis was used to examine the labeling of proteins in a cell lysate, using both sulphonated and non-sulphonated Cy5. For both types of Cy5, we observed efficient labeling across a broad range of molecular weights and isoelectric points.

Keywords: Protein; labeling; Cy5; SDS-PAGE

Abbreviations used 1-D: one-dimensional, 2-D: two-dimensional, SDS-PAGE: sodium-dodecyl-sulfate polyacrylamide gel electrophoresis, NHS: N-hydroxysuccinimide, D/P ratio: dye-to-protein ratio, DIGE: difference gel electrophoresis, Tris: tris(hydroxymethyl)aminomethane, LOD: limit of detection, SNR: signal-tonoise ratio.

2

Introduction Dyes and fluorophors have been used to covalently label proteins prior to electrophoresis since the early 70s [1]. The advantages of using fluorophors for covalent labeling of proteins prior to electrophoresis include high sensitivity, ability to monitor the electrophoresis run, and a simplified workflow [1]. An example is the pre-labeling of different protein samples with a set of charge- and size-matched dyes for two-dimensional difference gel electrophoresis (2-D DIGE). 2D-DIGE resolves proteins on the basis of molecular weights and isoelectric points and allow for quantitative comparisons of the amount of a protein in complex protein samples [2-6]. The advent of modern fluorescence imagers has enabled facile detection of pre-labeled proteins in gels and Western blot membranes. Although many imagers and scanners detect signals linearly over a wide dynamic range, the labeling reaction may vary from sample to sample as a result of differences in sample buffer and protein composition. Therefore, it is often difficult to use such data for quantitative analysis. Robust labeling protocols and quantitation strategies for diverse protein samples are much needed. Our objective was to develop a pre-labeling protocol for 1D SDS-PAGE which replaces the methods of post-staining gels after electrophoresis, e.g. silver or Coomassie staining. This requires a pre-labeling protocol for 1-D SDS-PAGE which provides quantitative data over a wide range of protein concentrations. In a typical 1-D SDS-PAGE experiment, many different samples are prelabeled and run on the same gel, e.g. to analyze purity. Preferably, there should be no need to do a protein concentration pre-determination step to adjust the amount of protein added to the labeling reaction. For the choice of dye, we used a bis-sulphonated Cy5. For SDS-PAGE there is no need to use the charge-matched dyes used for 2-D DIGE and the sulphonate groups increase water solubility and reduce quenching effects. The protein coverage of pre-labeling for 1-D SDS-PAGE should be as comprehensive as possible, on par with 2-D DIGE labeling, and all proteins with reactive amines should be labeled. NHS ester is one of the most common coupling reagents for the covalent modification of peptides and proteins [7-11]. It reacts with the amino (-NH2) groups of proteins: the N-termini and lysine residues. The reaction kinetics depends on parameters such as concentration of reactants, pH, 3

temperature, and composition of the sample buffer. It is critical to control the pH of the labeling reaction for quantitative comparisons of different samples. Tris or carbonate buffers are often used for this purpose. It is also necessary to take steps to minimize any differences in sample buffer composition (e.g., differences in salt and detergent concentrations) because they may have adverse effects on the labeling reaction. This can be attained by buffer exchange or sample dilution. In the protocol described here, samples are diluted ten-fold in a Tris based buffer (pH 8.7 at 25 °C), prior to labeling, in order to minimize sample buffer effects on the labeling reaction and to control the pH of the labeling reaction. It has been predicted that under certain conditions it is possible to label a protein to a particular D/P ratio without knowing the exact protein concentration in the labeling mixture [7]. For electrophoresis, an optimum D/P ratio must be low to avoid broad or multiple bands, but high enough to enable good sensitivity. For antibody-based detection methods it is common to optimize the labeling of the antibody for high fluorescence signal intensity per gram of protein without affecting the antibody function: this results in relatively high D/P ratios (D/P > 1). In one study, Cy3 and Cy5 dyes were used to label IgG over a wide range of D/P ratios and significant Cy5 quenching was observed at D/P ratios greater than two [8]. We have developed a new labeling protocol based on a fixed amount of sulphonated Cy5 NHS ester, which produce a linear response across a wide range of protein concentrations using 1-D SDSPAGE. The chosen Cy5 was compared to non-sulphonated DIGE Cy5. An optimal dye concentration in the labeling reaction, giving a suitable range of labeled protein D/P ratios with highest sensitivity for SDS-PAGE analysis, was determined. The performance of such a labeling protocol for protein quantitation was evaluated. Based on these results a model was developed to predict the degree of protein labeling. Finally, we examined the protein-to-protein variation for a small set of proteins and a cell lysate using both 1-D and 2-D electrophoresis.

4

Materials and methods Materials for Labeling and 1-D Electrophoresis α-lactalbumin, lysozyme, myoglobin, and transferrin were all obtained from Sigma-Aldrich Co., and dissolved in 50 mM Tris-Cl, pH 8.7, at a concentration of 10 mg/ml. Polyclonal rabbit anti-human transferrin came in a 0.1 M NaCl solution at a concentration of 8.5 mg/ml (Dako), L-lysine (SigmaAldrich Co.,), anhydrous DMSO (Sigma-Aldrich Co.,). LMW-SDS marker kit, Dithiothreitol (DTT), Chinese Hamster Ovary (CHO) cell extract prepared in Mammalian Protein Extraction Buffer, Amersham™ WB labeling buffer (120 mM Tris-HCl, 50 mM NaCl, 0.1% SDS, pH 8.7 at 25 °C) , Amersham WB loading buffer, Amersham WB Cy™5, and the Amersham WB molecular weight markers were all obtained from GE Healthcare. Unless otherwise stated, all equipment was obtained from GE Healthcare. The Amersham WB analyzer system was used for SDS-PAGE analysis and scanning of gels according to the manufacturer’s instructions. Typhoon™ FLA 9500 and Amersham Imager 600 were also used for gel imaging. For electrophoresis, 13.5 % homogeneous and 8-18 % gradient Amersham WB gel card 14 were used. The Amersham WB paper comb was used to remove excess dye from the wells when the limit of detection was measured. Gel images were analyzed with the Amersham WB evaluation software and ImageQuant™ TL (IQTL) software. Data was exported to Microsoft™ Excel™ 2010 for further analysis. Labeling Protocol Duplicate protein samples were diluted 10-fold with Amersham WB labeling buffer at room temperature. The samples were preheated (optional) at 95 °C for 1 min and allowed to cool to room temperature. One part Amersham WB Cy5 was added to 19 parts of protein sample in Amersham WB labeling buffer (e.g., 1 µl Cy5 to 19 µl protein sample). The samples were incubated at room temperature for 30 min followed by the addition of an equal volume (e.g., 20 µl) of Amersham WB loading buffer, which had been supplemented with 40 mM DTT. Samples were finally heated at 95 °C for 3 min and allowed to cool to room temperature. The samples were loaded onto an Amersham WB

5

gel card (20 µl per well). The reactions were scaled up to final reaction volumes of 40 µ l - 100 µl. The pre-heating step is optional; it can be used to increase solubility in the labeling buffer and to efficiently denature proteins prior to labeling. However, it is not recommended for temperature-sensitive samples. Reaction kinetics Loss of dye activity in labeling buffer In order to determine the rate at which the dye loses coupling activity in the labeling buffer, we prepared duplicate reactions by mixing 2 µl of Amersham WB Cy5 (250 pmol/µ l) dye with 34 µl of Amersham WB labeling buffer. The samples were pre-incubated at room temperature at different durations (6, 10, 15, 20, 25, 30, and 60 min) before 4 µ l of protein sample (LMW marker dissolved in labeling buffer at a concentration of 1 mg/ml) was added. The samples were mixed and incubated for a further 30 min. A control reaction was performed in which the protein was added to the labeling buffer prior to the dye. An equal volume of Amersham WB loading buffer (supplemented with DTT) was added and the samples were heated at 95 °C prior to SDS-PAGE analysis. The Amersham WB system was used for electrophoresis and scanning. The sum of the integrated Cy5 signal intensity from all the protein bands was compared to the signal of the standard 30 min labeling reaction without preincubation of Cy5 in labeling buffer. Reaction kinetics of protein labeling The protein sample (LMW marker) was dissolved and diluted to 0.1 mg/ml in Amersham WB labeling buffer. Duplicate reactions were prepared. After addition of dye, the reaction was incubated for different times (5, 10, 15, 20, 25, 30, 60 min) at room temperature (23 °C). The reaction was stopped by addition of an equal volume of Amersham WB loading buffer (with 0.5 mM lysine) supplemented with DTT and the samples were subjected to heating and SDS-PAGE as described above. The sum of the Cy5 signal intensity from all the protein bands was calculated for each sample.

6

Dye-to-protein measurements Absorbance Method All proteins were weighed and dissolved in a 50 mM Tris-Cl pH 8.7 buffer at a final concentration of 10 mg/ml except for anti-transferrin which came in solution at a concentration of 8.5 mg/ml. The solutions of α-lactalbumin, lysozyme, myoglobin, transferrin, and anti-transferrin were diluted 10-fold with Amersham WB labeling buffer and each labeling reaction (duplicates) was stopped by the addition of 2 µl of 10 mM Lysine. PD Minitrap G-25 columns (GE Healthcare) were used to remove excess dye after labeling. U-2001 (Hitachi) and SpectraMax Plus 384 (Molecular Devices) spectrophotometers were used to measure protein and Cy5 absorbance at 280 and 650 nm, respectively. We prepared standard curves for the five proteins and Cy5 to measure the molar concentrations and determine D/P ratios after excess dye-removal. The D/P ratio is the ratio of molar concentrations, i.e., the average number of Cy5 molecules conjugated to each protein molecule. Finally, SDS-PAGE analysis was used to confirm protein purity specifications and excess dye removal. Fluorescence Method We also used a fluorescence-based method to determine D/P ratios. Duplicate protein samples were labeled and loaded, with excess dye, on Amersham WB gels for electrophoresis as previously described. Electrophoresis runs were stopped after 30 min to prevent the front from migrating out of the gel. Cy5 signals from both proteins and the excess dye front were measured for each protein sample with Amersham Imager 600 and Typhoon FLA 9500. We calculated the D/P ratio using the known amount of protein and dye loaded onto the gel plus the measured ratio of Cy5 fluorescence signal from protein relative to the total signal (protein and front). Varying the amount of dye in the labeling reaction Amersham WB Cy5 dye was diluted with anhydrous DMSO and standard labeling reactions were performed. Labeling of α-lactalbumin (0.5 to 1.0 mg/ml) was performed in a reaction volume of 40 µl and molar [Cy5]/[protein] concentration ratios was varied from 0.0004 to 4. The samples were

7

divided and run on two Amersham WB electrophoresis gels. One gel was stopped after 30 min, to measure the fluorescence of both the protein band and the dye front for D/P ratio calculations, and the second gel was run under normal conditions. Protein quantitation A series of diluted protein samples (LMW marker proteins, myoglobin, and CHO cell lysate) in Amersham WB labeling buffer was prepared followed by a standard labeling reaction in duplicate at a fixed reaction volume and constant concentration of dye. The samples were run on a gel and the protein bands were analyzed using IQTL. For analysis of the CHO cell lysate a minimum profile background subtraction was performed. The dynamic range was evaluated using both linear and logarithmic (log-log) plots. The signal-to-noise (SNR) ratio was calculated for the weaker bands, a SNR greater than 3 was considered to be the limit of detection (LOD). 2-D Electrophoresis materials and methods CHO cell extract prepared in Mammalian Protein Extraction Buffer (containing 10 µl/ml Protease Inhibitor Mix) at a final protein concentration of 20 mg/ml was used for 2-D electrophoresis. Aliquots containing 40 µ g of protein were pre-labeled with 250 pmol of Amersham WB Cy5 (1 µ l) in a final volume of 20 µ l of Amersham WB labeling buffer or 250 pmol of Cy5 DIGE Fluor minimal dye (Cy5 DIGE) in a urea-based buffer (Protein Extraction Buffer II) supplemented with 30 mM TrisHCl, pH 8.5. The labeling reaction was performed at room temperature for 30 min and terminated by the addition of 1 µl 10 mM lysine (Sigma). Samples were prepared in duplicate and prior to loading onto the first dimension separation, each Cy5-labeled sample was mixed with a Cy3-DIGE labeled sample (Cy3 DIGE Fluor minimal dye) plus 450 µl of sample buffer (Protein Extraction buffer II, 0.5 % IPG buffer 3-11NL, and 20 mM DTT) followed by a concentration step with Vivaspin500 (MWCO 5000) to an approximate volume of 100 µl. The Cy3 DIGE labeled sample was used to keep track of the pI shift introduced by negatively charged Amersham WB Cy5. The concentrated samples were supplemented with a trace of BFB tracking dye and 8 µl of unlabeled CHO cell lysate (160 µg protein) for detection with Coomassie™ staining. The samples were loaded onto IPG DryStrip pH 3-11 NL (24 cm) re-hydrated in DeStreak Rehydration Solution by anodic cup loading. Isoelectric focusing (IEF) 8

was performed with the Ettan™ IPGphor™ system according to the manufacturer’s instructions. The IPG strips were subsequently reduced and alkylated for 15 min per step by addition of DTT (65 mM) and iodoacetamide (135 mM) to the equilibration solution respectively. The second dimension SDSPAGE was performed using precast Ettan™ DALT Gel (12.5 %) and DALT Buffer kit in Ettan DALTsix separation unit according to the manufacturer’s instructions. After electrophoresis, the gels were fixed in 15 % ethanol and 7.5 % acetic acid followed by scanning in FLA9500 imaging system for the acquisition of the Cy5 and Cy3 images. The gels were stained with Coomassie (SimplyBlue™ SafeStain, Invitrogen) and imaged with Amersham Imager 600. Spot detection and image analysis were performed with ImageMaster™ 2D Platinum v7.0 software. Spots from the acidic edge of the gel, next to the sample application zone (anodic cup loading), were excluded for calculation of the cumulative volume sum with respect to pI. Unless otherwise stated, all material and equipment were obtained from GE Healthcare. Simulation of Cy5-NHS-ester reactions The NHS-ester activated dye primarily reacts with amines but it can also undergo hydrolysis. Fig. 1 illustrates the major reaction paths that may occur in the labeling buffer and parameters used in simulation. If we know the total (active) dye concentration, total buffer concentration, total protein concentration, identity of the N-terminus and the number of lysines, we can calculate the concentration of free (nonprotonated) amines with the pH and pKa values (Equation 1) [reactive amine] = [total amine] × 10-pKa / (10-pKa + 10-pH)

(Equation 1) Using the measured half-life of the NHS ester dye (Fig. 2a), D/P ratios (Table 1), and the observation that the Cy5 signal from labeled transferrin was largely independent of Tris concentrations from 8 to 480 mM (data not shown), reaction rate constants for the reactions shown in Fig. 1 were estimated. The consumption of NHS-esters and the formation of dye-complexes were calculated using reaction rate constants, ka for the formation and kd for the hydrolysis of complexes. Based on the assumed rate constants, the progress of the reactions was calculated in Microsoft™ Excel™ (version 14.0.6129.5000, 32 bit) using numerical integration with a defined time step (∆t). In the Excel 9

calculations, each row (with index j) represented one time point and the concentration of the complex formed was calculated using Equation 2

[complex]j = [complex]j-1 + ka × [reactive amine] × [active dye] × ∆t – kd × [complex]j-1 × ∆t

(Equation 2) Reactive concentrations of dye and amines were corrected for their consumption whereas the concentration of hydroxyl ions (pH) was assumed to be constant. The results from the simulations include time plots of the amounts (expressed as percentages) of: remaining of NHS ester dye, dye in complex with Tris, labeled N-terminus amines, labeled lysines, and total protein labeled. The major difference between these simulations and those presented elsewhere [7] are first, different rate constants were assigned to alpha and epsilon amines in this study to account for differences in reactivity; and second, reactive amine concentrations were calculated using total protein concentrations, pH, and pKa values.

Results and Discussion Reaction kinetics In the absence of proteins, the Cy5 NHS ester dye will hydrolyze in an aqueous buffer at basic pH. In the case of a Tris-based labeling buffer, the dyes will also react with Tris, which is a primary amine. Fig. 2a shows the decline in the activity of the dye at a pH of 8.8 (at 23 °C). The reaction follows an exponential decay curve with a half-life of 11 min. The results from the simulations (Fig. 2b) were consistent with this observation. With the assumption that rate constants are independent of pH the simulations suggested that the half-life is extended to 1 h at pH 8.0 but reduced to less than 1 min at pH 10. The simulations also showed that Tris reacted relatively slowly with the NHS esters. A typical curve of the labeling kinetics is shown in Fig. 3a. The reaction reached a labeling plateau at about 30 min at room temperature. In the presence of protein, the reaction with hydroxyls may limit the extent of protein labeling and this idea was further explored by complementing the 10

experimental data with simulated studies (Fig. 3b). The simulations showed that the kinetics of protein labeling indeed is much affected by the pH of the labeling buffer. Dye concentration in the labeling reaction Fig. 4 shows the data from varying dye concentration while maintaining the protein concentration in order to investigate the effect on D/P ratio. We found that the D/P ratio after labeling was proportional to the dye concentration in the labeling reaction over a broad range of dye concentrationsover three orders of magnitude. At relatively high dye concentrations (i.e., D/P ratios above 0.2), we observed deviations from linearity and broadening of the α-lactalbumin bands. This result shows that the D/P ratio, and the Cy5 signal intensity per gram of protein, can be easily controlled by adjusting the dye concentration in the labeling reaction. Protein quantitation To investigate if pre-labeling can be used for protein quantitation we varied the protein concentration and kept the dye concentration constant. The results in Fig. 5 show that the signal was almost linear over a wide range of protein concentrations. This suggests that there were minimal changes in the D/P ratio even though the concentration ratio [dye]/[protein] varied greatly and the concentration of dye exceeded that of protein in the labeling reaction. In this experiment, the Cy5 signals from the protein ranged from 0.04 % to 10 % of the total Cy5 signal including the dye front, which shows that the majority of dye did not react with the protein and instead migrated in the front on the gel. This observation was confirmed in simulations and can be explained by competing hydrolysis reactions at the relatively high pH of 8.7. This result shows that it is possible to use pre-labeling with a fixed amount of dye for assays, e.g. to determine protein concentrations using standard curves. It would be possible to use the Cy5 signal response curve to determine a hitherto unknown concentration of a protein so long as the reference samples are labeled under identical conditions. Deviations from linearity in the response curve can be taken into account using non-linear functions.

11

Protein quantitation, sensitivity, and dynamic range We observed that the addition of 1 µl of 50-250 pmol Cy5 dye in DMSO in a reaction volume of 20 µl produced relatively low D/P ratios which were suitable for SDS-PAGE and still exhibited excellent sensitivity. The sensitivity and dynamic range of protein quantitation was measured for a labeling reaction with Cy5 at a concentration of 2.5 µM in the reaction mixture (Fig. 6). The deviations from linearity (Fig. 6) show that the Cy5 signal intensities were higher than those of the reference samples at high protein concentrations and lower at low protein concentrations. This indicates that for low protein concentrations, less than 1 ng/µl, side reactions compete for the available dye. The data shows that a wide range of protein concentrations, approximately 3 orders of magnitude, could be used for protein quantitation with standard curves. The sensitivity was in the subnanogram range, e.g., a limit of detection of 200 pg was measured for carbonic anhydrase. It is possible to improve the limit of detection using a higher dye concentration and thus increase the Cy5 signal per gram protein. However, increasing the dye concentration will increase D/P ratios (see Fig. 4) which may cause band broadening. We repeated the experiment using a more complex sample, a CHO cell lysate. The results in Fig. 7 show that the results also hold for lysate protein bands. Bands can be easily detected over a broad dynamic range. The bands are well defined for the entire dilution series indicating a low degree of conjugation for all labeled samples. We investigated the range of linearity and found typical CVs in the range 10-15% for identical bands when compared over a 100-fold dynamic range. The CVs increase as the dynamic range is broadened. Deviations from linearity are evident for samples with the highest protein concentration in Fig. 7, and to use such data for quantitation it is necessary to fit data with non-linear functions. Dye-to-protein ratios We used two different but complementary techniques to determine the D/P ratios. These were (i) absorbance solution measurements at 280 nm and 650 nm to determine the amount of protein and Cy5 after removal of excess dye; and (ii) in-gel fluorescence measurements of labeled proteins and the dye front. The data in Table 1 shows that both techniques produced similar results. The fluorescence 12

method is based on the assumption of a linear relationship between the signal and the amount of dye present. This assumption is reasonable in the absence of a quenching effect and the method has proven to be useful for measurements of D/P ratios less than 1. When D/P ratios are close to a value of 1, relatively large proteins exhibit single and distinct bands at the correct position on a gel (data not shown). In general, the bands of small proteins are most affected if D/P ratios are high. Protein-to-protein variation The data in Table 1 also reveals that Cy5 pre-labeling depends on the unique characteristics of the target protein. α-lactalbumin contains 12 lysine residues and a glutamic acid at its N-terminus whereas lysozyme contains 6 lysines and one lysine at its N-terminus: myoglobin has 18 lysine residues and an N-terminal glycine, and transferrin has 57 lysines and a valine residue at the Nterminus. The comparative empirical data in Table 1 showed that α-lactalbumin was labeled more efficiently than the other proteins under investigation. For the other proteins, the Cy5 signal per gram of protein was almost constant, even though their Mr ranged from 14 000 to 150 000. As shown in Table 1, when the same amount of protein in gram is labeled with a constant amount of Cy5, the proteins with high molecular mass also have higher D/P ratios. This is most likely a result of the fact that proteins with high Mr have more reactive lysine groups per protein molecule. The simulated D/P ratios for lysozyme, myoglobin and transferrin were identical to the empirical data. The predicted D/P ratio for α-lactalbumin was slightly lower (0.10 compared to 0.14). The simulation indicates that differences in dye labeling may be due to differences in active amine concentrations and also that the rate constants for forming dye complexes differ for N-termini and epsilon lysines. All the simulations were made with kaNterminus = 15 M-1 s-1, kalysine = 100 M-1s-1, disregarding any differences in local reactivity and assuming negligible hydrolysis of protein-dye complexes. In the absence of detailed sequence information, the amount of protein in gram may be a relevant factor for a first estimate of the Cy5 signal after labeling. We finally examined the versatility of Cy5 pre-labeling with a cell lysate. The proteins were separated on a 2-D gel with a broad pH range (pH 3-11). Two types of Cy5 dyes were also compared:

13

the sulphonated Amersham WB Cy5 and the non-sulphonated DIGE Cy5. Reference Coomassie gels were also made for comparison. The sulphonated Cy5 shifts the pI of the proteins and direct matching of all detected proteins spots, with different Cy5 dyes, was not possible. However, we compared the number of detected proteins and the spot distribution for the different dyes. The results in Figs. 8 and 9 suggest that there was no preferential labeling of proteins with respect to pI or Mr.

Conclusions A fixed amount of Cy5 dye can be used in the labeling reaction to label proteins over a broad concentration range for 1-D SDS-PAGE analysis. Within this broad range of sample concentrations the linearity in dose-response allows for quantitative comparisons of a protein in different samples. For best accuracy it is possible to perform assay-like protein quantitation with high sensitivity using standard curves. The observed reaction kinetics and degree of labeling were reproduced in simulations. The simulations show that the pH of the labeling buffer is a critical factor. For routine SDS-PAGE analysis we found the optimal range of Cy5 concentrations for effective pre-labeling reactions to be 2.5-12.5 µM (i.e., 50-250 pmol in a 20 µ l reaction volume) at pH 8.7. At this pH we observed that the degree of modification is low, and almost constant, even in the presence of excess dye. If the labeling results in broad or multiple bands the dye can simply be diluted to lower the D/P ratios. The pre-labeling method is applicable to a wide range of proteins and provides significant advantages compared to traditional post-staining techniques.

References 1. A. T. Andrews, Electrophoresis – Theory, Techniques, and Biochemical and Clinical Applications, Clarendon Press, Oxford, 1986. 2. P. Z. O’Farrell, H. M. Goodman, P. H. O’Farrell, High resolution two-dimensional electrophoresis of basic as well as acidic proteins, Cell 12 (1977) 1133-1142. 3. V. E. Urwin, P. Jackson, Two-dimensional polyacrylamide gel electrophoresis of proteins labeled with the fluorophore monobromobimane prior to first-dimensional isoelectric focusing: imaging of

14

the fluorescent protein spot patterns using a cooled charge-coupled device, Anal. Biochem. 209 (1993) 57-62. 4. M. Ünlü, M. E. Morgan, J. S. Minden, Difference gel electrophoresis. A single gel method for detecting changes in protein extracts, Electrophoresis 18 (1997) 2071-2077. 5. R. Westermeier and T. Naven, Proteomics in Practice, Wiley-VCH, Weinheim, 2002. 6. Imaging, Principles and Methods (29-0203-01), GE Healthcare, 2011. 7. G. P. Smith, Kinetics of amine modification of proteins, Bioconjug. Chem., 17 (2006) 501-506. 8. H. J. Gruber et al., Anomalous fluorescence enhancement of Cy3 and Cy3.5 versus anomalous fluorescence loss of Cy5 and Cy7 upon covalent linking to IgG and noncovalent binding to avidin, Bioconjug. Chem. 11(5) (2000) 696-704. 9. G. W. Cline and S. B. Hanna, Kinetics and mechanisms of the aminolysis of Nhydroxysuccinimide esters in aqueous buffers, J. Org. Chem. 53 (1988) 3583-3586. 10. G. W. Anderson, J. E. Zimmerman, F. M. Callahan, N-hydroxysuccinimide esters in peptide synthesis, J. Am. Chem. Soc. 85 (19) (1963) 3039. 11. J. M. Becker and M. Wilchek, Inactivation by avidin of biotin-modified bacteriophage, Biochim. Biophys. Acta, 264 (1) (1972)165-170.

Acknowledgments We would like to thank Oki K. Dzivenu, D.Phil., ELS, of GE Healthcare for editorial assistance.

15

Figure Legends

Fig. 1. Schematic illustration of the different reaction pathways of the NHS ester and the parameters used to simulate the labeling reaction.

Fig. 2a. NHS ester activity as a function of time in Tris-buffer (pH 8.8 at 23 °C). The Cy5 assay signal was proportional to the concentration of reactive dye present in the labeling buffer. The decay of the NHS ester activity was exponential with a half-time of 11 min. Error bars are +/- two pooled standard deviations.

Fig. 2b. Simulated Cy5 NHS ester activity. The parameters used for simulation were pH, the total dye concentration set to 12.5 µM, and the ka for reaction with hydroxyls set to 180 M-1 s-1. The calculated half-lives were 1 hour at pH 8.0, 12 min at pH 8.7, and less than 1 min at pH 10.

Fig. 3a. Labeling reaction kinetics with 0.1 mg/ml of total protein concentration and a dye concentration of 12.5 µ M in the labeling reaction (pH 8.7 at 25 °C). Error bars are +/- two pooled standard deviation.

Fig. 3b. Simulated labeling kinetics for the labeling of 20 µ g of lysozyme at different pH. At pH 8, the reaction was relatively slow and the amount of labeled protein leveled off after 2 h, at pH 10 the dye was rapidly consumed by hydrolysis and the amount of labeled protein was less than 7 %. At pH 8.7, the reaction proceeded at an intermediate rate and about 9 % of the protein was labeled. For rate constants used in simulations see footnote Table 1.

16

Fig. 4. The amount of dye in the labeling reaction determines the extent of protein conjugation. A) Log-log plot of D/P ratios after labeling with variable dye concentrations. The D/P ratios were measured using fluorescence in-gel measurements of labeled α-lactalbumin and dye front. Increasing the amount of dye in the labeling reaction while keeping the protein concentration fixed (at 1 mg/ml) resulted in a linear increase in Cy5 signal intensity and D/P ratio. B) A D/P ratio in the range 0.1-0.2 produced a high signal intensity without band broadening, which corresponds to 7-13 µM Cy5 in the labeling reaction. Amersham WB molecular weight marker was loaded in the outer right lane. C) A high dye concentration (130 µM) in the labeling reaction gave a D/P ratio > 1 and extra bands occur above the α-lactalbumin band. The relationship between dye concentration and D/P-ratio was nonlinear with high dye concentrations.

Fig 5. A) With a constant Cy5 dye concentration in the labeling reaction (10 µM) a dilution series of myoglobin produced an almost linear response in Cy5 signal on an SDS-PAGE gel, even in the presence of excess dye (see inset gel image). B) The average D/P ratio estimated from in-gel fluorescence measurements was 0.2. Although the [Cy5]/[protein] ratios in the reaction ranged from 2 to 440, the measured D/P ratios were within +/- 20 % of the average. Error bars are +/- one pooled standard deviation.

17

Fig. 6. The dynamic range and sensitivity of Cy5 pre-labeling were measured using a constant amount of dye (2.5 µM) and variable amounts of protein in the labeling reaction (filled circles). The protein bands of carbonic anhydrase were analyzed (indicated by arrow in inset image). Reference samples were prepared in which one labeled sample was serially diluted after labeling (crosses).

Fig. 7. The dynamic range of Cy5 pre-labeling for SDS-PAGE was measured using a constant amount of dye (10 µ M) and variable amounts of CHO cell lysate. A broad dynamic range was observed and different contrast settings are required to view the labeled proteins in all lanes (see zoom image). Four protein bands (A-D) were analyzed and the calculated band volumes were plotted versus the amount of protein (log-log scale).

Fig. 8. A 2-D electrophoresis experiment with IPG DryStrip pH 3-11 NL (nonlinear gradient) strips was performed to determine the protein coverage of the labeling reaction with Cy5. A CHO cell lysate was pre-labeled with either sulphonated Cy5 (Amersham WB Cy5) or the DIGE Cy5. There was no significant difference in the number of detected spots using the two different Cy5 dyes; 1601 for the DIGE Cy5 gel and 1587 for the Amersham WB Cy5 gel (figure). The gel image indicates efficient labeling across the gel with respect to both Mr and pI.

Fig 9. Protein coverage on 2D gels was compared using Coomassie post-staining and Cy5 prelabeling. Plots of cumulative sums of spot volumes versus pixel in x- and y-directions in the gel image show the distribution of spot volumes on the gel with respect to pI and Mr. A comparison of the pixel distribution of Cy5 (Amersham WB Cy5) spot volumes to that of Coomassie stained spots shows that the two techniques produced similar curves, which suggests that there is no preferential labeling and Cy5 pre-labeling is applicable to a wide diversity of proteins.

18

Table 1. Comparison of measured and simulated D/P ratios for different proteins.

Table 1 footnote:

The pooled D/P ratio standard deviations were 0.01 (fluorescence) and 0.02 (absorbance). We used the following parameters for the simulation: pH 8.7, pKa values for respective N-termini, pKa 10.53 for epsilon lysine, a dye concentration -1 -1

-1 -1

-1

of 12.5 µM, a Tris concentration of 120 mM, and rate constants kaOH 180 M s , kaTris 0.0001 M s , kdTris 0.0001 s , -1 -1

-1

-1 -1

-1

kaNterminus 15 M s , kdNterminus 0.00001 s , kalysine 100 M s , kdLysine 0.00001 s .

19

Table 1

Protein

[Cy5]/[protein] in labeling reaction

D/P ratio absorbance measurement

D/P ratio fluorescence measurement

D/P ratio simulation

Fluorescence per gram protein (rel. units)

α-Lactalbumin (Mr = 14.1 × 103)

0.18

0.15

0.14

0.10

1.53

Lysozyme (Mr = 14.3 × 103)

0.19

0.08

0.08

0.09

0.89

Myoglobin (Mr = 16.7 × 103)

0.22

0.13

0.10

0.13

0.98

Transferrin (Mr = 80 × 103)

1.04

0.51

0.46

0.51

0.87

Anti-Transferrin (Mr = 150 × 103)

2.29

0.82

0.84

-

0.87

20

Figure 1

Cy5 signal relative to Cy5 signal at t = 0 min

Figure 2a

y = e-0.064x R² = 0.999

1 0.8 0.6 0.4 0.2 0 0

10

20 Time (min)

30

Figure 2b

Cy5 signal relative to Cy5 signal at t=60 min

Figure 3a

1

0.8

0.6

0.4

0.2

0 0 10 20 Time (min) 30

Figure 3b

Figure 4

A) D/P ratio

0.1 0.01

0.001 0.0001 0.0001

0.001 0.01 0.1 [Cy5]/[protein]

B)

C)

[Cy5]/[protein]:

[Cy5]/[protein]:

0.003 0.012 0.05

0.006 0.024

0.2

3.7

0.1

[protein]=constant

B)

C)

Figure 5

A)

Cy5 intensity myoglobin band

1.0E+07

1.0E+06

1.0E+05

1.0E+04 0.001

0.01

0.1

1

Amount of myoglobin in band (µg)

9.0E+06 8.0E+06 7.0E+06 6.0E+06 5.0E+06 4.0E+06 3.0E+06 2.0E+06 1.0E+06 0.0E+00

0.5 0.45 0.4 0.35 0.3 0.25 0.2 0.15 0.1 0.05 0

Cy5 intensity / amount of myoglobin D/P ratio

-2 -1 0 log (µM myoglobin in labeling reaction)

1

D/P ratio

Cy5 intensity / amount myoglobin

B)

Figure 6

Figure 7 Contrast 1

Zoom - Contrast 2

A B C D

Band A

log (Cy5 intensity)

7

Band B Band C

6

Band D 5

4 -3.5

-2.5

-1.5

-0.5

log (µg total protein)

0.5

Figure 8

Figure 9 High Mr

Cumulative sum of Cy5 spot volumes

4.0E+06

Low Mr

3.0E+06 2.0E+06 1.0E+06 0.0E+00 0

500

1000

1500

Pixel High Mr

Cumulative sum of Coomassie spot volumes

3.0E+04

Low Mr

2.0E+04 1.0E+04 0.0E+00 0

500

1000

1500

Pixel Low pI

Cumulative sum of Cy5 spot volumes

4.0E+06

High pI

3.0E+06 2.0E+06 1.0E+06 0.0E+00 0

500

1000

1500

Pixel Low pI

Cumulative sum of Coomassie spot volumes

3.0E+04

High pI

2.0E+04 1.0E+04 0.0E+00 0

500

1000

1500

Pixel