WO2007051256A1

WO2007051256A1 - Method for monitoring protein digestion

Info

Publication number: WO2007051256A1
Application number: PCT/AU2006/001649
Authority: WO
Inventors: Peter Helmuth Karuso; Hung-Yoon Choi; Angela S. Crawford; Graham B. I. Scott
Original assignee: Fluorotechnics Pty Limited; Sigma-Aldrich Corporation
Priority date: 2005-11-04
Filing date: 2006-11-03
Publication date: 2007-05-10

Abstract

Methods of monitoring protein or peptide digestion by a proteolytic enzyme, comprising contacting a protein or peptide with a proteolytic enzyme in the presence of a fluorescent dye such as epicocconone or a related dye under conditions which allow digestion of the protein by the proteolytic enzyme and monitoring the fluorescence of the mixture over time. The protein may be a recombinant protein having a tag such as 6X HIS, 10X HIS, FLAG, CBP, HSV, S- tag or HA; or fusion proteins such as GST or MBP. Further, the protein or peptide may be present in a complex protein/peptide mixture, for example a biological sample such as serum or plasma or an entire proteome. The monitoring can be performed on the reaction mixture in real-time or via sampling. The temporal decrease in fluorescence signifies digestion of the protein or peptide by the proteolytic enzyme and the data can be used to extract kinetic rate constants for th digestion. The invention also relates to kits for carrying out the method.

Description

"METHOD FOR MONITORING PROTEIN DIGESTION"

TECHNICAL FIELD

The present invention relates to methods for monitoring proteolytic digestion of peptides and proteins and in particular to methods for monitoring protein and/or peptide digestion by proteases using a fluorescent dye.

BACKGROUND

Proteolytic digestion with a range of proteases is commonly used as a first and important step in techniques for protein identification in proteomics. One problem inherent to the use of proteases and peptidases in protein and peptide digestion is ensuring that digestion is as complete as possible. SDS-PAGE is often used to evaluate the totality of digestion, but this technique is laborious and semi-quantitative at best. On the other hand, over-digestion can also be problematic.

Consequently there is a high demand for quick and easy real-time assays for measuring protein, peptide and biological fluid (proteome) digestion in a way that allows the peptides generated to be analysed by mass spectrometry, CE or HPLC. Thus, there also exists a need for a new and versatile approach to monitoring the progress of protein, (including recombinant proteins with standard tags such as 6X-HIS, 1 OX-HIS, FLAG, CBP, HSV, S -tag or HA; or fusion proteins such as GST or MBP), peptide or biological fluid digestions, wherein the peptides generated are available for further down-stream analysis. Previously, there have not been any fluorescence-based methods to follow tryptic digestion of proteomic samples. However, Spencer et al. (Anal. Biochem. (1975) 64, 556-66) have reported a method to follow hydrolytic activity of some proteases on

intact protein substrates that contain hydrophobic pockets, such as BSA, α-casein,

urease and catalase, by the release of the fluorophore, l-anilino-8-naphthalenesulfonate from hydrophobic pockets.

It is therefore an object of the present invention to overcome or ameliorate at least one of the disadvantages of the prior art, or to provide a useful alternative.

SUMMARY OF THE INVENTION

Throughout this specification the term protein and proteins are to be taken to include, inter alia, recombinant protein(s). Such recombinant proteins may include, but are not limited to tags such as 6X HIS, 1OX HIS, FLAG, CBP, HSV, S-tag or HA; or fusion proteins such as GST or MBP. According to a first aspect, the invention provides a method of monitoring proteome, protein or peptide digestion by a proteolytic enzyme comprising: step 1 : contacting a proteome, protein or peptide with a proteolytic enzyme in the presence of a fluorescent dye selected from epicocconone and related dyes under conditions which allow digestion of the proteome, protein or peptide by the proteolytic enzyme; and step 2: monitoring fluorescence of the dye over time, wherein a change in fluorescence over time signifies digestion of the proteome, protein or peptide by the proteolytic enzyme.

The protein or peptides may be present in a complex protein/peptide mixture, for example a proteome or biological sample such as serum or plasma. The change in fluorescence may be an increase or a decrease in fluorescence.

According to a second aspect the invention provides a method of determining an end-point for proteome, protein or peptide digestion by a proteolytic enzyme comprising: step 1 : contacting a proteome, protein or a peptide with a proteolytic enzyme in the presence of a fluorescent dye selected from epicocconone and related dyes under conditions which allow digestion of the proteome, protein or peptide by the proteolytic enzyme, and step 2: monitoring a change in fluorescence of the dye over time, wherein the absence of a further change in fluorescence signifies the end- point for proteome, protein or peptide digestion.

According to a third aspect the invention provides a method of monitoring proteome, protein or peptide digestion by a proteolytic enzyme comprising: step 1 : contacting a proteome, protein or peptide with a proteolytic enzyme to form a reaction mixture; step 2: contacting a first sample of the reaction mixture with a fluorescent dye selected from epicocconone and related dyes, and determining fluorescence of the first sample; step 3: subjecting the reaction mixture of step 1 to conditions which allow digestion of the proteome, protein or peptide by the proteolytic enzyme; and step 4: at a desired time point during digestion of the proteome, protein or peptide, contacting a second sample of the reaction mixture with a fluorescent dye selected from epicocconone and related dyes; and step 5: determining fluorescence of the second sample, wherein a change in fluorescence of the second sample when compared to the first sample signifies the degree of digestion of the proteome, protein or peptide by the proteolytic enzyme. According to a fourth aspect the present invention provides a kit for use in the method according to any one of the preceding claims comprising: a fluorescent dye selected from epicocconone and related dyes; one or more proteolytic enzymes; and instructions on how to use the kit for monitoring proteome, protein or peptide digestion. Preferably the kit includes a standard protein or peptide substrate or any other biological standard. The standard protein substrate may be chosen from BSA, apo-

transferrin, α-casein, and carbonic anhydrase, and the peptide is preferably β-endorphin.

Preferably the kit includes standard buffers appropriate for the enzyme. The preferred buffer comprises one of the Good's buffers such as bicine, BES etc. An embodiment of the above method contemplates sampling the reaction mixture at regular intervals during digestion and after addition of a fluorescent dye selected from epicocconone and related dyes to each of the samples, measuring the decrease in fluorescence over time until no further decrease in fluorescence is observed. This variant of the method can be used to determine the end point of digestion of a protein (including recombinant proteins with tags such as 6X-HIS, 1 OX-HIS, FLAG,

CBP, HSV, S-tag or HA; or fusion proteins such as GST or MBP), a peptide, or complex protein/peptide mixture (e.g. a biological sample such as serum or plasma; i.e. a proteome). Advantageously, fluorescence is measured over time to provide data indicative of a reaction rate coefficient. Preferably the proteolytic enzyme is a protease or a peptidase capable of cleaving the protein or peptide in at least one position along the amino acid sequence of the protein or peptide. Non-limiting examples of proteases that can be used in the present invention are endopeptidases such as chymotrypsins and trypsins used in protein digestion for the purposes of protein identification. Examples of proteases provided herein include trypsin, Lys-C and chymotrypsin, merely as convenient systems to demonstrate the principles and working of the invention. Other proteases may be used for the purpose of cleaving tags from recombinant proteins. Non-limiting examples of such proteases that can be used in this aspect of the present invention include, but are not limited to, Factor Xa and Thrombin for 6X-HIS tags or GST₃ and Entrokinase for FLAG tags.

In the context of the present invention the term "epicocconone and related dyes" is intended to encompass epicocconone itself as well as related fluorescent dyes as specifically disclosed in WO 2004/085546 incorporated in its entirety herein by reference.

Preferably, the fluorescent dye is of the formula (Ia), or isomer thereof:

(Ia) Preferably, X is O, NR⁴ or C. Preferably, R¹ is a straight or branched chain Ci_-20 conjugated alkenyl group optionally substituted 1-6 groups independently selected from hydroxy or oxo groups. Preferably, R² is a straight or branched chain C_1-20 alkyl group. Preferably, R³ is a straight or branched chain C_1-20 alkyl group, optionally substituted with a hydroxyl group. Preferably, R⁴ is N, O, straight or branched chain C_1-20 alkyl and/or aryl group, optionally substituted with a hydroxyl, halide, amine, carboxyl, carboxyl related or heteroaryl group or groups.

Preferably, the dye is of formula (Ib), including isomers thereof:

(Ib)

The compound of formula (Ib) is 5,6-dihydro-3- [(IZ, 4E, 6E, 8E)-l-hydroxy-3- oxo-1, 4, 6, 8-decatetraenyl]-6-hydroxymethyl-9a-methyl-2H-furo [3, 2g] [2] benzopyran-2-9-(9aH)-dione. However, this compound will be hereinafter referred to by its trivial name, which is "epicocconone".

In the context of the present invention, isomers of compounds of formulae Ia and Ib include tautomers and stereoisomers among other isomers.

BRIEF DESCRIPTION OF THE FIGURES

Preferred embodiments of the invention will now be described, by way of example only, with reference to the accompanying drawings in which:

Figure 1 shows real-time fluorescence monitoring of bicine buffer (♦), trypsin (A), Bovine Serum Albumin (BSA; ■) and a tryptic digest of BSA (X). Figure 2 shows gel electrophoresis scans of sub-sampled BSA tryptic digest

(containing epicocconone) from 0-128 minutes, quenched in gel-loading buffer at 85 °C (Panel "A"). A control gel, containing all components except trypsin (Panel "B"), shows no change over 128 minutes. The first lane showed a low molecular weight (LMW) marker and the last lane overnight digestion. Figure 3. Total fluorescent intensity measured from gated regions of the sub- samples shown in Figure 2. Figure 4. Kinetic analysis of the raw data from Figures 1 and 3. Panel "A" depicts the analysis of the reaction of epicocconone in bicine buffer (pH 8.4) showing development of the stain and subsequent decomposition. Apparent first order rate constants are indicated. The rate of association/dissociation calculated from part A was used in calculating the apparent first order rate constant for the tryptic digest (Panel "B"). A similar result was obtained by analysis of the data from protein gels (Panel "C") from Figure 3.

Figure 5. Visualisation of trypsin digests by SDS-PAGE. Lanes 1: Marker; 2: trypsin only (T = 0); 3: T = 0 BSA (no trypsin); 4: T = 0 BSA + trypsin; 5: BSA (0.25 h); 6: BSA (0.5 h); 7: BSA (1 h); 8: BSA (6 h); 9: BSA (18 h); 10: trypsin only (T = 18, no BSA)

Figure 6. Real-time monitoring of chymotrypsin digestion (30 ⁰C, pH 7.8) of BSA

followed by epicocconone. Bicine buffer (♦), chymotrypsin (A), Bovine Serum Albumin (■) and BSA+chymotrypsin (X).

Figure 7. Kinetic analysis of the data shown in Figure 6. Figure 7 A shows the analysis of the reaction of epicocconone with BSA in bicine buffer (pH 8.4) showing development of the stain and subsequent decomposition. Apparent first order rate constants are indicated. The rate of association/dissociation calculated from Figure 7A were used in calculating the apparent first order rate constant for the chymotryptic digestion (Figure 7B). Figure 8. Raw data on real-time monitoring of tryptic digestions of different proteins and validation of the digests by SDS-PAGE. Panels "A", "B", "C" and "D"

represent the real-time monitoring profiles of tryptic digestion of BSA, α-casein, apo-

transferrin and carbonic anhydrase (bovine), respectively; undigested samples (o), protein + Part A; digested (D), protein + Part A + trypsin. Panel "E" represents independent validation by SDS-PAGE of the sub-samples collected at the 100^th minute: Lane 1 represents LMW marker (97, 66, 45, 30, 20.1, and 14.4 KDa); 2, BSA; 3,

BSA/trypsin; 4, α-casein; 5, α-casein/trypsin; 6, apo-transferrin; 7, apo-

transferrin/trypsin; 8, carbonic anhydrase; 9, carbonic anhydrase/trypsin; 10, trypsin only. Figure 9. Kinetics of real-time monitoring of chymotrypsin digestion of different proteins using epicocconone, and validation of the digests by SDS-PAGE. Panel "A" depicts kinetics of chymotrypsin digestion of protein samples tested. Panel "B" depicts SDS-PAGE validation of chymotrypsin digests: Lane 1 represents LMW

marker; 2, BSA; 3, BSA/chymotrypsin; 4, α-casein; 5, α-casein/chymotrypsin; 6, apo-

transferrin; 7, apo-t/chymotrypsin; 8, carbonic anhydrase (bovine); 9, carbonic anhydrase /chymotrypsin; 10, chymotrypsin only.

Figure 10. Kinetics of real-time monitoring of Lys-C digestion of different proteins using epicocconone, and validation of the digests by SDS-PAGE. Panel "A" depicts kinetics of Lys-C digestion of protein samples tested. Panel "B" depicts represents SDS-PAGE validation of Lys-C digests: Lane 1 represents LMW marker; 2,

BSA; 3, BSA/Lys-C; 4, α-casein; 5, α-casein/ Lys-C; 6, apo-transferrin; 7, apo-

transferrin/ Lys-C; 8, carbonic anhydrase (bovine); 9, carbonic anhydrase / Lys-C; 10, Lys-C only.

Figure 11. Real-time fluorescence monitoring of tryptic digestion of the peptide

β-endorphin without added trypsin (O) and with trypsin added (P).

Figure 12. Sub-sampled tryptic digests of BSA (o) and bovine carbonic

anhydrase (V). In Panel "A", trypsin activity was inhibited by 100 μM leupeptin (a

small molecule inhibitor). The inset shows the apparent first order rate constant of digestion; In Panel "B", trypsin activity in a 40 μL sub-sample containing 0.6 μg of trypsin was inhibited by treating the samples with 2.4 μg of soybean trypsin inhibitor type H-S (a protein inhibitor).

Figure 13. Kinetics of real-time monitoring of trypsin digestion of a yeast proteome (at a ratio of 1 :20) using epicocconone, and validation of the digestions by SDS-PAGE. Panel "A" depicts kinetics of trypsin digestion of the yeast proteome sample tested. Panel "B" depicts SDS-PAGE validation of trypsin digests: Lane 1 represents the yeast protein; 2, yeast protein/trypsin; 3, trypsin only; 4, LMW marker (LMW marker 97, 66, 45, 30, 20.1, and 14.4 kDa).

DESCRIPTION OF THE PREFERRED EMBODIMENT

The present invention is based on a surprising finding that the fluorescence of a fluorescent dye epicocconone, when used in a tryptic digest reaction comprising a protein and a proteolytic enzyme (e.g. trypsin or the like), decreases as the protein digestion progresses to completion. Epicocconone, related dyes and their uses have been described in WO 01/81351 and WO 2004/085546 incorporated in its entirety herein by reference. Epicocconone and related dyes have been used successfully inter alia for detection and quantification of proteins and other biological macromolecules. These methods are based on enhancement in the fluorescence of a dye such as epicocconone with increasing concentration of protein. With respect to the studies described herein it was hypothesised that fluorescence intensity of the digested protein samples would increase over time proportionally to an increase in exposure of lysine residues as a consequence of protein digestion. Unexpectedly, however, inclusion of epicocconone in a tryptic digest of BSA (bovine serum albumin) showed a rapid decrease in fluorescence, which effectively followed the digestion process and reached its lowest level at the completion of protein digestion, as confirmed by SDS-PAGE, HPLC and LC mass spectrometry.

Specifically, it is observed that fluorescence drops exponentially during the proteolysis reaction until it reaches a low baseline steady state level, indicating that the reaction has been driven to completion. With suitable controls, it is also possible to extract kinetic data from the fluorescence progress curves, making this a novel method for following protease kinetics on real substrates. Additionally, it was demonstrated that epicocconone does not create adducts that interfere with the downstream mass spectrometric or HPLC analyses. It should be clear that the observed reduction in fluorescence of epicocconone would also apply to digestion of proteins and peptides by a range of proteases useful for protein identification, as discussed above or for the cleavage of fusion proteins or tags from the protein of interest.

Thus, the invention described herein relates to methods of monitoring, either in real time or sequential sampling mode, the proteolytic digestion of one or more proteins in a reaction comprising a proteolytic enzyme, a protein (including recombinant proteins with tags), or a peptide, or complex protein/peptide mixture (proteome) and the fluorescent dye epicocconone or a related dye as defined above. In certain embodiments of the invention the dye can be omitted from the reaction mixture comprising the proteolytic enzyme and the protein or biological sample substrate. Ih such a method, samples of the reaction mixture can be taken at the start of the digestion reaction and at intervals throughout the reaction, and epicocconone added to each sample, to determine its fluorescence and thus progression of digestion to its end-point.

The invention will now be described in more detail, with reference to specific non-limiting examples. EXAMPLES Example 1: Real-time monitoring of trypsin digestion using epicocconone

1.1 Materials

• Bicine (50 mM₅ pH 8.4, Sigma-Aldrich B3876)

• BSA (10 mg/mL in 50 niM Bicine, Sigma-Aldrich A3059)

• Trypsin (20 μg/20 μl mM HCl, Sigma-Aldrich T6567)

• Iodoacetamide (1 M in 100 mM bicine, Sigma-Aldrich 16125)

• DTT (200 mM in 100 mM bicine, BioRad 161-0611)

• 96-well plate with clear bottom (Greiner bio-one, 655096)

• Epicocconone (24 mM in DMSO, FLUOROtechnics)

• Deep Purple total protein gel stain (GE Healthcare)

• NuPAGE Novex 12 % Bis-Tris Gels (Invitrogen, NP0341 )

• LMW Marker (Amersham Biosciences, 17-0446-01)

1.2 Equipment

• Typhoon 9200 (Amersham Biosciences)

• FluoStar (BMG)

• Electrophoresis system (XCeIl SureLock, Invitrogen)

1.3 Methods

1.3.1 Preparation of BSA for digestion

1 Trypsin digestion was carried out in bicine buffer (pH 8.4).

2 BSA was prepared in 10 mg/mL in 50 mM bicine buffer.

3 One hundred microlitres of the BSA sample was used for trypsin digestion. 1.3.2 Reduction and alkylation

1 The 100 μL of BSA sample was reduced by adding 5 μL of DTT stock for 10

min at 80 °C.

2 The sample was alkylated by adding 4 μL of the iodoacetamide stock at room

temperature for 45 min to 1 hr.

3 The remaining iodoacetamide in the sample was neutralised by adding 20 μL of

the DTT at room temp for 45 min to 1 hr.

1.3.3 Real-time monitoring of trypsin digestion using epicocconone (FluoStar assay)

1 The reduced and denatured BSA sample from 1.3.2 above was diluted 10-fold in

50 mM bicine buffer (25 μL + 225 μL bicine buffer). BSA molar concentration was

calculated to be approx. 4 μM.

2 One hundred microlitres of the sample (step 1) was prepared in duplicate and added to a microtitre plate. Controls included a bicine-based digestion buffer, a trypsin sample only and an undigested BSA sample (no trypsin).

3 Epicocconone stock solution was diluted 100-fold in 50 mM bicine. One hundred microlitres of diluted epicocconone solution was added to each well, making the final

concentration 12 μM. The FluoStar required approximately 10 min to obtain

appropriate setting conditions. 4 Trypsin (Sigma- Aldrich T6567), reconstituted in 1 mM HCl, was added at a ratio of 1:40.

5 Fluorescence development was monitored in real time every 2 minutes up to 6

hours using FluoStar (Ex/Em = 540/630±12 nm). FluoStar settings were as follows:

temperature, 37 ⁰C; 10 flashes/cycle to 180 cycles. 6 The raw data was plotted - see Figure 1.

1.3.4 Visualisation of trypsin digests in SDS-PAGE

1 The reduced and denatured BSA sample from 1.3.2 above was diluted 10-fold in

calculated to be approx. 4 μM.

2 One hundred microlitres of the sample (step 1) was added to a 1.5 mL microtube. Controls included a bicine-based digestion buffer and an undigested BSA sample (no trypsin).

3 Epicocconone stock solution was diluted 100-fold in 50 mM bicine. One hundred microlitres of diluted epicocconone solution was added to each corresponding well,

making the final concentration 12 μM. At time = 0, 15 μL of the samples were taken

out and mixed with 15 μL of a protein gel loading buffer (2 x), then denatured for 5 min at 85 ⁰C.

4 Trypsin, reconstituted in 1 mM HCl, was added at a ratio of 1 :40. The sample tubes were then incubated at 37 ⁰C.

5 Sub-samples were collected at 2, 4, 8, 16, 32, 64, 128 min, and overnight (18

hours). At each sampling point, 15 μL of the samples were taken out, immediately

mixed with 15 μL of a protein gel loading buffer (2 x), denatured for 5 min at 85 °C, and

stored at -80 °C. 6 The sub-samples collected as described above were run by SDS-PAGE

(NuPAGE, 12% BT gel), and gels were stained with Deep Purple for visualisation.

7 The Deep Purple-stained gels were imaged by Typhoon scanner (Ex:Em=532:560 run LP; 440 PMT). 8 All gel lanes showing digested (Figure 2A) and undigested (Figure 2B) protein samples were equally gated, and the fluorescent intensity was measured by ImageQuant (v 5.2) software.

1.4 Data Analysis and Results

The results of this study are summarised in Figures 1 to 4. The raw data obtained from the reaction of BSA and BSA+trypsin with epicocconone was measured as described and fitted to simple first order kinetic models using Prism (Version 4.0.3, GraphPad Software, San Diego, USA). For the reaction of BSA with epicocconone in bicine buffer it is clear that there are at least two reactions, one is a time dependant association of epicocconone with BSA and then a slower decomposition, and/or photobleaching of this fluorescent conjugate. This process was modelled with a simple

association/dissociation model Y = spanl(l - e^~klX) + span! x e^~klX + bottom where the

first term refers to the exponential increase in signal and the second the exponential decrease. The values from Figure 1 were subtracted from background fluorescence (bicine + trypsin) and adjusted for the actual start time of the experiment, estimated as 10 minutes from the first reading. An r² value of 0.9992 was obtained for this model. As the degradation of signal with time is a characteristic of the interaction of epicocconone with protein (and presumably peptides) the value for k₂ was used in the analysis of the trypsin kinetics. A two phase exponential decay

(Y = span\(e^~k{X) + span2(e^~k2X) + plateau) was used where k₂ was set to the value found

for BSA alone. Thus the estimated apparent first order rate constant for trypsin under the experimental conditions was found to be 0.109 mm^"1 (r² = 0.9871). Clearly the actual kinetics is more complex, but this is a good approximation that could be only slightly improved on by allowing k₂ to vary independently. The results firom the sub-sampled reaction were similarly analysed but without the term associated with degradation of the signal with time, as this is not relevant in gels. Thus the apparent first order rate constant was found to be 0.3136 min^"1 (r² = 0.9757). However, with so few points, a large 95% confidence interval was found (0.1976 to 0.4296 min^"1). Coupled with the relatively imprecise mode of measurement suggests that in situ monitoring of tryptic digestion with epicocconone is comparable to the laborious process of sub-sampling and that the presence of the dye does not significantly affect the enzyme kinetics.

The experiment described above was also conducted in a carbonate buffer (NH₄HCθ₃, 10OmM or 5OmM, pH 8.2) and results compared with data obtained using bicine buffer. The comparative results are shown in Figure 5. Sigma- Aldrich Proteomics Grade trypsin (T 6567) worked well in both digestion buffers. The digestion appeared to reach completion within 0.5-1 hr. The data presented indicate that reduced and alkylated BSA is rapidly digested with trypsin and that this process can be followed in situ with epicocconone.

Example 2 - Kinetics of chymotrypsin in BSA-digestion using epicocconone

2.1 Materials and Equipment

• Materials and Equipment as per 1.1 and 1.2 above, and:

• Chymotrypsin (Sigma-Aldrich C4129, 1 mg/mL of 1 mM HCl) • CaCl₂ (IM in RO water)

2.2 Methods

2.2.1 Preparation of BSA for digestion

1. Chymotrypsin digestion was carried out in bicine buffer. 2. BSA was prepared in 10 mg/mL in 50 mM bicine buffer.

3. One hundred microlitres of the BSA sample was used for chymotrypsin digestion.

2.2.2 Reduction, AIkylation and Neutralisation

1. The BSA sample (100 μL) was reduced by adding 5 μL of DTT stock and

heating to 80⁰C for 10 min.

2. The sample was alkylated by adding the iodoacetamide (4 μL) stock at room temperature for 45 min to 1 hr.

3. The remaining iodoacetamide in the sample was neutralised by adding DTT (20

μL) and incubating at room temp for 45 min to 1 hr.

2.2.3 Real-time monitoring of chymotrypsin digestion using epicocconone (FluoStar assay)

1. The reduced and denatured BSA sample from 2.2.2 was diluted 10-fold in 50

mM bicine buffer (25 μL + 225 μL bicine buffer). BSA molar concentration was

calculated to be approx. 6 μM. 2. One hundred microlitres of the sample (step 1) was prepared in duplicate and added to a microtitre plate. Controls included a bicine-based digestion buffer, a chymotrypsin sample only and an undigested BSA sample (no chymotrypsin).

3. Epicocconone stock solution was diluted 100-fold in 50 mM bicine and 100 μL

added to each well making the final concentration of epicocconone 12 μM. The

FluoStar required approximately 10 min to obtain appropriate setting conditions.

4. 2 μL of CaCl₂ was added

5. Chymotrypsin (C4129), reconstituted in 1 mM HCl, was added at a ratio of 1 :30. 6. Fluorescence development was monitored in real time every 2 minutes up to 6

hours using FluoStar (Ex/Em = 540/630+12 run). FluoStar settings were as

follows: temperature = 30 ⁰C and 10 flashes/cycle to 180 cycles.

7. The raw data was plotted - see Figure 6.

2.3 Data Analysis and Results

Results of these examples are shown in Figures 6 and 7. Figure 6 shows real-time monitoring of chymotrypsin digestion of BSA. Chymotrypsin displays similar kinetics to that of trypsin (Figure 1) whereby fluorescence exponentially increased in the undigested BSA and exponentially decayed in the digested BSA (with added chymotrypsin). Similar to Figure 4, it is possible to extract the kinetic constants for chymotryptic digestion (see Figure 7) through non-linear least squares fitting of the progress curves.

Example 3. Real-time monitoring of tryptic digestion of a range of different proteins using epicocconone

3.1 Materials and Equipment

• Materials and Equipment as per 1.1 and 1.2 above, and:

• Part A, epicocconone (1 mg/mL DMSO₅ FLUOROtechnics)

• Part B, Bicine buffer (100 niM, pH 8.4-5; B3876, Sigma-Aldrich)

• Protein samples for the real-time monitoring of trypsin digestion: bovine serum

albumin (A-3059, Lot 083K1291), apo-transferrin (T-2036, Lot 043K0911), α-

casein (C-6780, Lot 041K7420), and carbonic anhydrase (C-7025, Lot 093K9310)

• Sodium dodecyl sulfate (442444H₅ BDH) • NuPAGE LDS sample buffer (4χ, Invitrogen, NP0007)

3.2 Methods:

3.2.1 Preparation of protein samples 1. Prepared 200 mM dithiothreitol (DTT) and 1 M iodoacetamide in Part B.

2. Prepared protein samples at a concentration of 10 mg/mL in Part B.

3. Placed 100 μL of the protein sample in a 1.5 mL microtube.

4. Reduced the sample by adding 1 μL of 10% SDS, 5 μL of DTT stock and

incubated at 80 ⁰C for 10 min.

5. Alkylated the sample by adding 4 μL of the iodoacetamide stock and incubated at room temperature for 45 min to 1 hr.

6. Neutralised the remaining iodoacetamide by adding 20 μL of DTT stock and incubated at room temp for 45 min to 1 hr.

7. The samples were then diluted 1 : 10 in Part B. 3.2.2 Real-time monitoring assay (FluoStar assay)

1. One hundred microlitres of the sample was added to two wells.

2. One hundred microlitres of the control sample containing SDS was added to another two wells.

3. Epicocconone stock solution was diluted 1 :100 in the bicine buffer. One hundred microlitres of the diluted epiccconone was added to all four wells. The

FluoStar required approximately 2 min to obtain appropriate setting conditions.

4. Trypsin (1.93 μg/1.93 μL in 1 mM HCl) was added to one protein and one control sample at a ratio of 1 :40, and 1.93 μL of 1 mM HCl was added to the remaining protein and control samples. 5. Fluorescence development was monitored in real time every 2 minutes up to 400

minutes using FluoStar (Ex/Em = 540+10/630+10 nm) with 10 flashes.

The data were plotted by subtracting CTL from undigested protein samples and by subtracting CTL + trypsin from digested protein samples. 3.2.3 Validation of tryptic digests by SDS-PAGE

1. Sub-samples (6.5 μL) were collected at the 100th minute during the assay.

2. The sub-samples were then immediately mixed with 1 μL of 500 mM DTT and

2.5 μL of NuPAGE sample buffer (4x), and denatured for 10 min at 70 °C.

3. The sub-samples described above were run by SDS-PAGE (NuPAGE, 12% BT gel), and the gels stained with Deep Purple for visualisation.

4. The Deep Purple-stained gels were imaged by Typhoon scanner (Ex/Em = 532/560 nm LP; 500 PMT).

3.3 Data Analysis and Results

By referring to Figure 8, it can be seen that when the fluorescence level of the digested sample (o) reaches 1 to 2.5% of the initial fluorescence, the digestion is completed (typically 30-600 minutes). The real-time monitoring data can be used to precisely predict the status of the tryptic digestion.

The exponential decay in the fluorescence of BSA, α-casein and apo-transferrin

reactions to 1 to 2.5% of the initial fluorescence was observed within 120 minutes by real-time assays (Figure 8-A to 8-C). This was validated by an independent analysis, e.g.

SDS-PAGE (lane 3, BSA/trypsin; lane 5, α-casein/trypsin; lane 7, apo-

transferrin/trypsin, Figure 8-E) or by subsampling (see Example 7).

However, as shown in Figure 8-D, the exponential decay in the fluorescence of carbonic anhydrase (bovine) tested for tryptic digestion did not reached 2.5% of the initial fluorescence, indicting the protein was not completely digested by trypsin. This was validated by SDS-PAGE, showing an incomplete digestion of the protein by trypsin (lane 9 in Figure 8-E). The carbonic anhydrase (from bovine erythrocytes) was shown to be resistant to tryptic digestion.

Using a range of different proteins, it can be seen that the real-time monitoring assay using epicocconone distinguishes complete or incomplete digestion of protein samples. This demonstrates that a kit would provide a useful fluorescent assay for realtime monitoring of in-solution digestion of different proteins with trypsin or other protease(s). Further, the unique protein staining mechanism of epicocconone (Coghlan et al, Org. Lett. (2005) 7, 2401) lends itself to the detection of proteins in solution without interfering with protein digestion by trypsin. Real-time monitoring of protein digestion using epicocconone does not appear to rely on hydrophobic pockets and this method has application in proteomics where the end point of tryptic digestion is required.

Example 4: Real-time Monitoring of Protein Digestion of a range of different proteins with chymotrypsin using epicocconone 4.1 Materials and Methods

Protein samples for the real-time monitoring of trypsin digestion included bovine serum albumin (A-3059, Lot 083K1291, Sigma-Aldrich), apo-transferrin (T-2036, Lot

043K0911, Sigma-Aldrich), α-casein (C-6780, Lot 041K7420, Sigma-Aldrich), and

carbonic anhydrase (C-7025, Lot 093K9310, Sigma-Aldrich). The samples were treated in the same manner for reduction and alkylation, as previously described in Example 3.

Chymotrypsin (C-4129, Lot 023K7660, Sigma-Aldrich) (1 mg/mL in 1 mM HCl) mediated digestion of different protein samples was carried out in 100 mM bicine buffer (pH 7.7) containing 10 mM CaCl₂. The ratio of enzyme to protein sample was 1 :40. Materials and methods were previously described in Example 3. Methods for the real-time monitoring assay using epicocconone and its SDS- PAGE validation were previously described in Example 3. 4.2 Data Analysis and Results

Figure 9 A shows the kinetics of real-time monitoring of digestion of four different proteins with chymotrypsin., and SDS-PAGE validation of the digests can be seen in Figure 9B. Compared to tryptic digestion of the same proteins, a similar trend of

exponential decays in the fluorescence of BSA, α-casein and apo-transferrin to 1 - 2.5%

of the initial fluorescence were observed within ~100 minutes. This was validated by

SDS-PAGE (lane 3, BSA/chymotrypsin; lane 5, α-casein/chymotrypsin; lane 7, apo- transferrin/chymotrypsin, Figure 9-B).

Carbonic anhydrase (bovine), tested in the real-time assay, was not completely digested by the enzyme in 100 minutes, as validated in SDS-PAGE (lane 9, Figure 9-B).

Real-time monitoring of protein digestion with chymotrypsin using epicocconone does not rely on hydrophobic pockets or on the production of peptides terminating in lysine or arginine (as with trypsin). Therefore, this method has application in proteomics where the end point of tryptic digestion is required before further downstream analysis such as peptide mass fingerprinting (PMF), peptide mapping or HPLC can be performed.

Example 5: Real-time Monitoring of Protein Digestion of a range of different proteins with Lys-C using epicocconone 5.1 Materials and Methods

043K0911, Sigma-Aldrich), α-casein (C-6780, Lot 041K7420, Sigma-Aldrich), and carbonic anhydrase (C-7025, Lot 093K9310, Sigma- Aldrich). The sanipres were treated in the same manner for reduction and alkylation, as previously described. Lys-C (P- 3428, Lot 102Kl 129, Sigma-Aldrich) (50 μg/50 μL water) was used for digestion of protein samples in 100 mM bicine buffer (pH 8.5) at the ratio of 1 : 20 for the real-time assay.

Methods for the real-time monitoring assay using epicocconone and its SDS- PAGE validation were previously described. 5.2 Data Analysis and Results

Figure 10 shows real-time monitoring of digestion of four different proteins with Lys-C (Figure 10A), and SDS-PAGE validation of the digests (Figure 10B). The major

molecular bands of BSA, α-casein and apo-transferrin would be completely cleaved by

the 600th minutes with enzyme at a ratio of 1 :20. This was validated by SDS-PAGE

(lane 3, BSA/Lys-C; lane 5, α-casein/Lys-C; lane 7, apo-transferrin/Lys-C). However,

the Lys-C-derived end point fluorescence decays to completed digestion (< 1-2.5%)

were slightly higher than for trypsin or chymotrypsin, e.g. BSA (3%) and casein (4.9%), and equal to that of apo-transferrin (2.5%). The slightly higher baselines after digestion observed with BSA and casein is probably due to the nature of Lys-C, having a more specific cleavage at the carboxyl side of Lys-residues. This would result in relatively larger peptides as opposed to the smaller peptides generated by trypsin or chymotrypsin. Carbonic anhydrase was not completely digested by Lys-C within the time-frame of the experiment. This shows the utility of the present invention to assess complete digestion in real-time. The fluorescence decay was recorded to be over 8% at the 400th minute, indicating the protein was somewhat resistant to Lys-C digestion. The incomplete digestion was validated by SDS-PAGE (lane 9 for CA/Lys-C in Figure 10- B). Real-time monitoring of protein digestion with the proteomics protease Lys-C does not rely on hydrophobic pockets or on the production of very short peptides (as with trypsin) and this method has application in proteomics where the end point of tryptic digestion is required before further downstream analysis such as peptide mass fingerprinting (PMF), peptide mapping or HPLC is performed.

Example 6: Real-time monitoring of tryptic peptide digestion using epicocconone

6.1 Materials and equipment

• Materials and Equipment as per 3.1 above, and:

• β-endorphin (Molecular Mass, 3438.01; H2705, Auspep), H-Try-Gly-Gly-Phe-

Met-Thr- Ser-Glu-Lys-Ser-Gln-Thr-Pro-Leu-Val-Thr-Leu-Phe-Lys-Asn-Ala-Ile- Ile-Lys-Asn-Ala-His-Lys-Lys-Gly-Gln-OH

6.2 Methods

6.2.1 Preparation of a peptide sample 1. β-endorphin peptide was prepared in 100 mM bicine buffer (pH 8.4-5) at a

concentration of 2 mg/mL.

2. The peptide sample was diluted 1 :4 in 100 mM bicine buffer to give a final concentration of 0.5 mg/mL.

3. To 99 μL of the diluted peptide sample (49.5 μg) was added 1 μL of 1% SDS stock solution.

4. Controls (no peptide) were prepared in the bicine/SDS buffer.

6.2.2 Real-time monitoring assay

1. One hundred microlitres of the peptide sample was added to two wells.

2. One hundred microlitres of the bicine/SDS control was added to another two wells. 3. Epicocconone stock solution was diluted 1 : 100 in the bicine buffer. One hundred microlitres of the diluted epiccconone was added to all four wells. The FluoStar required approximately 50 sec to obtain appropriate setting conditions. 4. Trypsin (1 μg/μL in 1 mM HCl) was added to one peptide and one control sample, and 1 μL of 1 mM HCl was added to the remaining peptide and control samples. 5. Fluorescence development was monitored in real time every 3 minutes for 3 hours using FluoStar (Ex/Em = 540-10/630-10 nm) with 10 flashes. 6. The data were plotted in Prism graphic software by subtracting fluorescence background (obtained from the appropriate controls) from peptide and peptide + trypsin. 6.3 Data Analysis and Results

Figure 11 shows the real-time monitoring of tryptic digestion of the β -endorphin

peptide using epicocconone. An exponential decay in fluorescence was observed when trypsin was added, and the pseudo-first order rate constant for digestion can be obtained by non-linear regression analysis of the data.

The β-endorphin peptide has 5 lysine residues (molecular mass of approx. 3500)

to which epicocconone could bind to as well as the iV-terminal amine. The exponential decrease in fluorescence indicates that epicocconone does not interfere with the digestion of peptides by proteases.

Example 7. An alternative method to monitor tryptic digestion using epicocconone by sub-sampling 7.1 Materials and equipment • As per previous examples. 7.2 Methods

1. The trypsin inhibitors, leupeptin and trypsin-inhibitor (soybean) were prepared at a concentration of 1 mM in Reverse Osmosis (RO) treated water and 0.48 mg/mL in RO water, respectively.

2. The protein samples were prepared as previously described in Example 3. The reduced and alkylated protein samples were diluted 1 : 10 in 100 mM bicine buffer (pH 8.4-5).

3. Trypsin (4.62 μg) was added to each protein sample (231 μg / 300 μL) at a ratio

of 1 :50 and the samples incubated at 37 °C.

4. Sub-samples were collected for inhibition of trypsin activity either by leupeptin or by trypsin inhibitor (soybean). a. Forty-five microlitres of the tryptic digests were sub-sampled at 0, 10, 20, 30, 60, 60, 90, and 120 minutes and immediately added to 1.5 mL tubes containing 5 μL of 1 mM leupeptin. The sub-samples treated with leupeptin inhibitor were left at room temperature until fluorescence was read. b. Forty-five microliters of the tryptic digests were sub-sampled at 0, 10, 20, 30, 60, 60, 90, and 120 minutes and immediately added to 1.5 mL tubes containing 5 μL of soybean trypsin inhibitor. The sub-samples treated with soybean trypsin inhibitor were left at room temperature until fluorescence was read.

5. Controls for leupeptin inhibitor included the bicine buffer only and the bicine buffer containing trypsin or trypsin + leupeptin. Controls for trypsin inhibitor (soybean) also included the bicine buffer only, the bicine buffer containing trypsin or trypsin + soybean trypsin inhibitor. 6. Epicocconone solution was prepared by diluting it 1 : 100 in 100 mM bicine (pH 8.4-8.5).

7. The sub-samples (40 μL), as prepared above, were added to a 96-well plate, to which an equal volume of epicocconone solution was added. The plate was inserted into

FluoStar and incubated for 50 min at 37 °C.

8. Fluorescence of the sub-samples was read at Ex/Em = 540-10/630-10 run with 10 flashes.

9. The fluorescence value was normalised by subtracting basal fluorescence of corresponding controls from the raw fluorescence of the sub-samples. 10. The normalised data was plotted and can be seen in Figure 12.

7.3 Data Analysis and Results

Figure 12 shows the fluorescence decay of tryptic digestion of BSA and CA that were sub-sampled at various time points. At each sub-sample, the trypsin activity was inhibited either by leupeptin (A) or by soybean trypsin inhibitor (B). This alternative method was tested for its applicability to monitoring complete Bovine Serum Albumin

(BSA) or incomplete Carbonic Anhydrase (CA) tryptic digestion, and produced similar fluorescence decay results compared to the results generated from the real-time assay

(Example 3).

The rates of digestion of both BSA and CA in the present method appeared to be slightly faster than those in the real-time assay. This could be due to the time required to effect complete inhibition of trypsin by the inhibitors, or that the tryptic digestion runs slightly faster in the absence of epicocconone and related dyes.

An alternative embodiment of the invention includes the running of proteolytic digestion without the presence of epicocconone (or related dyes). In this example, the tryptic digestion of two proteins, or protein mixtures, including biological samples and the like can be followed by sub-sampling and quenching the digestion with protease inhibitors. Considering that the inhibition of trypsin is time dependant and may not be complete, the measured pseudo-first order rate constant for digestion of BSA (0.3 mm^"1) was similar to that found by the method of example 3 (0.14 miiT¹).

Example 8: Mass Spectrometry compatibility of peptides generated from proteolytic digestion monitored by epicocconone.

8.1 Materials and Methods

8.1.1 Sample preparation for MS analysis The different protein samples used for tryptic digestion included bovine serum albumin (A-3059, Lot 083K1291), apo-transferrin (siderophilin; T-2036, Lot 043K0911),

α-casein (C-6780, Lot 041K7420), and carbonic anhydrase (C-7025, Lot 093K9310).

The proteins were reduced, alkylated and neutralised for trypsin digestion, as described previously in example 3. Each protein sample was diluted 1:10 in 100 mM bicine (pH

8.4-5. The final concentration of each protein was 770 μg/mL.

1. The protein solution (100 μL) in triplicate was mixed with 100 μL of

epicocconone solution diluted 1:100 in 100 mM bicine buffer (pH 8.4-5).

2. The control sample without Ix epicocconone solution was prepared in an

identical manner. 3. To the protein samples 2.0 μg of trypsin was added, and the digestion was carried out at 37 °C.

4. Sub-samples (18 μL) of tryptic digestion of each protein were collected at 0, 30

and 180 minutes.

5. The sub-samples were quenched in 0.1% TFA and immediately frozen at -80 °C prior to Peptide Mass Fingerprinting (PMF) analysis. 8.1.2 MS analysis of the samples added to epicocconone solution

The sub-sampled protein digests were analysed by the Australian Proteome Analysis Facility Ltd. (Sydney, Australia). The Samples were desalted and concentrated using an Eppendorf Cl 8 spin column, and an aliquot (1 μL) was spotted onto a MALDI

sample plate with α-cyano-4-hydroxycinnamic acid matrix (1 mL; 5 mg/mL in 70% v/v

can, 0.1% v/v TFA) and allowed to air dry. Matrix assisted laser desorption ionisation (MALDI) Time of Flight (TOF) mass spectrometry was performed with an Applied Biosystems 4700 Proteomics Analyser with TOF/TOF optics in MS mode. A pulsed Nd: YAG laser (355 run) was used to irradiate and volatilise the sample. The spectra were acquired in reflection mode in the mass range from 750 to 3500 Da. The eight strongest peptides from the MS scan were isolated and fragmented (by collision with a buffer gas). 8.2 Data Analysis and Results

The sub-samples (in triplicate) with and without the epicocconone solution included in the tryptic digestion were analysed via PMF and submitted to the database search program, Mascot (Matrix Science Ltd, London, UK). The peptides generated from tryptic digestion (with and without epicocconone) identified each protein to be

bovine albumin serum (P02769; Mr, 69248 Da; pi, 5.82), bovine α-casein (P02662; Mr,

24513 Da; pi, 4.98), human transferrin siderophilin (P02787; Mr, 77000 Da; pi, 6.81) and bovine carbonic anhydrase (P00921; Mr, 28963 Da; pi, 6.40), respectively. The number of peptides identified and sequence coverage are shown in Table 1. There were no significant differences of the sequence coverage between the sub-samples with and without epicocconone added to the tryptic digestion.

The sub-samples containing epicocconone solution are fully compatible with PMF analysis, demonstrating that the fluorophore does not permanently derivatise peptides, allowing both assessment of tryptic digestion and MS analysis of the same sample. Peptides would also be available for other purposes such as HPLC or other analytical applications / techniques.

Table 1. The number of peptides identified and sequence coverage of the parent protein

generated from tryptic digestion of bovine serum albumin, α-casein, apo-transferrin and

bovine carbonic anhydrase using peptide mass fingerprinting (PMF). The sub-samples (in triplicate) with and without epicocconone added in the tryptic digestion were analysed and submitted to the database search program, Mascot (Matrix Science Ltd, London, UK). Note: ' — ' indicated test failed.

Example 9: Real-time monitoring of proteolysis of a complex proteome using epicocconone

9.1 Materials and equipment

• As per previous examples.

• Compressed baker's yeast (Microbiogen Pty Ltd, Australia)

• NaOH (1 M, Sigma-Aldrich 480878)

• FluoroProfile (Sigma-Aldrich FPOO 10) 9.2 Methods

9.2.1 Yeast Preparation

1 A small pellet of compressed baker's yeast (100 mg) was used for trypsin digestion. 2 The pellet was suspended in 2 mL of 1 M NaOH. The sample was then

centrifuged at 2100 x g for 10 min.

3 An aliquot of the supernatant was diluted 5 -fold in RO water to reduce the TSf aOH concentration to 200 rnM.

4 The protein content was measured at 1.3 mg/mL by using FluorProfile Kit.

5 A small aliquot of the protein extract (15 μL) was mixed with 85 μL of bicine

buffer (50 rnM, pH 8.5). 6 One hundred microlitres of the final yeast sample was used for trypsin digestion.

9.2.2 Real-time monitoring of tryptic digestion of yeast proteome using epicocconone

1 One hundred microlitres of the final yeast sample was added to a microtitre plate well. Controls included a bicine-based digestion buffer, a trypsin sample only and an undigested yeast sample (no trypsin).

3 Epicocconone stock solution was diluted 100-fold in 50 rnM bicine. One hundred microlitres of diluted epicocconone solution was added to each well, making a 4- well assay. The FluoStar required approximately 30 seconds obtaining appropriate setting conditions.

4 Trypsin (Sigma- Aldrich T6567), reconstituted in 1 rnM HCl₅ was added at a ratio of 1:20. 5 Fluorescence development was monitored in real time every 2 minutes up to 400

minutes using FluoStar (Ex/Em = 540+10/630+10 nm). FluoStar settings were

as follows: temperature, 37 ⁰C; 10 flashes/cycle.

6 Progress curves were manipulated in Microsoft Excel by subtracting controls. bicne buffer was subtracted from the sample containing protein only and trypsin

+ buffer was subtracted from the protein/trypsin sample.

7 The normalised data was plotted using Prism (GraphPad v.4) - see Figure 13. The progress curve for the yeast proteome with epicocconone was fitted to a two phase exponential association/dissociation (Y=Ymax*(l-exp(- kl *X))+span*(exp(~k2*X))-bottom) to derive values for kl (association constant) and k2 (dissociation constant). These values were used to fit the tryptic digestion of the yeast proteome to a three phase exponential keeping kl and k2 fixed to the values found above into the equation Y=spanl*(l-exp(- kl*X))+span2*(exp(-k2*X))+ span3 exp(-k3*X)-bottom to derive a value for k3.

9.2.3 Visualization of trypsin digests in SDS-PAGE

1. Sub-samples were collected at the end of assay. The sub-sample (2 μL), e.g.

undigested and digested protein sample, and bicine/trypsin sample, was mixed with

8 μL of a sample loading buffer (2.5 μL of NuPAGE sample buffer/1 μL of 500

mM DTT/4.5 μL RO water) and incubated at 80 °C for 10 min.

2. Each sample was then loaded onto a 12% polyacrylamide gel (NuPAGE Bis-Tris, Invitrogen) and run (200V constant) for 50 min. until the blue loading buffer dye just ran off the gel.

3. The Deep Purple-stained gels were imaged by Typhoon scanner (Ex:Em=532:560 LP; 440 PMT). 9.3 Data Analysis and Results

This example shows the utility of the method for monitoring the proteolysis of a complex protein mixture, in the non-limiting example, a yeast proteome. Figure 13 A shows the real-time monitoring of tryptic digestion of a yeast proteome using the reaction of epicocconone with proteins to follow the digestion through a decrease in fluorescence. In contrast, the reaction of epiccconone with the yeast proteome results in an initial increase in fluorescence indicating the reaction of epicocconone with the proteins and then a slow exponential decrease due to photobleaching and/or decomposition of the fluorophore and/or fluorophore-protein adduct. This can be modelled to a two phase exponential association/dissociation to obtain the pseudo-first order rate constants for these processes. These values can then be used to determine the pseudo-first order rate constants for proteolysis by non-linear regression. This technique resulted in a rate of proteome digestion of 0.1886 min^~ in this example.

In summary, the present invention provides a generic method to follow the proteolytic digestion of any protein (including recombinant protein), peptide, and complex protein/peptide mixture by employing the unique chemical properties of epicocconone.

Although the invention has been described with reference to certain preferred embodiments, it will be understood that variations in keeping with the principles and spirit of the invention described herein are also within its scope.

Claims

1. A method of monitoring proteome, protein or peptide digestion by a proteolytic enzyme comprising: step 1 : contacting a proteome, protein or peptide with a proteolytic enzyme in the presence of a fluorescent dye selected from epicocconone and related dyes under conditions which allow digestion of the proteome, protein or peptide by the proteolytic enzyme; and step 2: monitoring fluorescence of the dye over time, wherein a change in fluorescence over time signifies digestion of the proteome, protein or peptide by the proteolytic enzyme.

2. A method of determining an end-point for proteome, protein or peptide digestion by a proteolytic enzyme comprising: step 1 : contacting a proteome, protein or peptide with a proteolytic enzyme in the presence of a fluorescent dye selected from epicocconone and related dyes under conditions which allow digestion of the proteome, protein or peptide by the proteolytic enzyme, and step 2: monitoring a change in fluorescence of the dye over time, wherein the absence of a further change in fluorescence signifies the end- point for proteome, protein or peptide digestion.

3. A method of monitoring proteome, protein or peptide digestion by a proteolytic enzyme comprising: step 1 : contacting a proteome, protein or peptide with a proteolytic enzyme to form a reaction mixture; step 2: contacting a first sample of the reaction mixture with a fluorescent dye selected from epicocconone and related dyes, and determining fluorescence of the first sample; step 3: subjecting the reaction mixture of step 1 to conditions which allow digestion of the proteome, protein or peptide by the proteolytic enzyme; and step 4: at a desired time point during digestion of the proteome, protein or peptide contacting a second sample of the reaction mixture with a fluorescent dye selected from epicocconone and related dyes; and step 5: determining fluorescence of the second sample, wherein a change in fluorescence of the second sample when compared to the first sample signifies the degree of digestion of the proteome, protein or peptide by the proteolytic enzyme.

4. A method according to claim 3 further including the steps of, where necessary: additionally sampling the reaction mixture at intervals during digestion and, after addition of a fluorescent dye selected from epicocconone and related dyes to each additional sample, determining fluorescence of the additional sample.

5. A method according to claim 4 including repeating sampling of the mixture, addition of the dye and determining the fluorescence until no further decrease in fluorescence is observed. 6. A method according to any one of the preceding claims wherein the dye is the fluorescent dye of the formula (Ia), or isomer thereof:

(Ia) wherein X is O, NR⁴ or C;

R¹ is a straight or branched chain C_1-20 conjugated alkenyl group optionally substituted 1-6 groups independently selected from hydroxy or oxo groups; R² is a straight or branched chain C_1-20 alkyl group; R³ is a straight or branched chain C_1-20 alkyl group, optionally substituted with a hydroxyl group; R⁴ is N, O, a straight or branched chain C_1-20 alkyl and/or aryl group, optionally substituted with a hydroxyl, halide, amine, carboxyl, carboxyl related or heteroaryl group or groups; and tautomers and stereoisomers of (Ia). A method according to claim 6 wherein the dye is epicocconone, (5,6-dihydro-3- [(1Z, 4E, 6E₅ 8E)-l-hydroxy-3- oxo-1, 4, 6,

8-decatetraenyl]-6-hydroxymethyl-9a- methyl-2H-furo [3, 2g] [2] benzopyran-2-9 (9aH)-dione) as shown in formula (Ib), including isomers thereof:

(Ib) tautomers and stereoisomers among other isomers.

A method according to any one of the preceding claims wherein the proteolytic enzyme is a protease or a peptidase capable of cleaving the proteome, protein or peptide in at least one position along the amino acid sequence of the protein or peptide.

9. A method according to claim 8 wherein the protease is an endopeptidase.

10. A method according to claim 9 wherein the endopeptidase is a chymotrypsin.

11. A method according to claim 9 wherein the endopeptidase is a trypsin.

12. A method according to claim 9 wherein the endopeptidase is Lys-C.

13. A method according to any one of the preceding claims wherein the digestion is carried out in the presence of a buffer.

14. A method according to claim 13 wherein the buffer is a Good's buffer.

15. A method according to claim 13 wherein the buffer is a bicine buffer.

16. A method according to claim 13 wherein the buffer is a carbonate buffer.

17. A method according to any one of the preceding claims wherein digestion of proteome, proteins or peptides is substantially unaffected by the fluorescent dye.

18. A method according to any one of the preceding claims wherein fluorescence is measured over time to provide data indicative of a reaction rate coefficient.

19. A method according to any one of the preceding claims wherein digestion is stopped when an end point is achieved.

20. A method according to claim 19 wherein further analysis of the reaction mixture takes place after digestion is stopped.

21. A method according to claim 20 wherein said further analysis is selected from the group consisting of peptide mass fingerprinting (PMF), peptide mapping and HPLC.

22. A method according to any one of the preceding claims further including the addition of a base and/or a detergent to the epicocconone or related dye.

23. A method according to claim 22 wherein the detergent is SDS.

24. A method according to any one of the preceding claims wherein said protein or peptide are derived from a biological sample or proteome.

25. A method according to claim 24 wherein said biological sample is cellular extract.

26. A method according to claim 24 wherein said biological sample is serum or plasma.

27. A method according to any one of the preceding claims wherein said protein includes a recombinant protein.

28. A method according to claim 27 wherein said recombinant protein includes a tag.

29. A method according to claim 28 wherein said tag is chosen from the group consisting of 6X-HIS, 1 OX-HIS, FLAG, CBP, HSV, S-tag and HA.

30. A method according to claim 29 wherein said protease is Factor Xa or Thrombin for proteins tagged with 6X-HIS or GST.

31. A method according to claim 29 wherein said protease is Entrokinase for proteins tagged with FLAG.

32. A method according to any one of claims 1-26 wherein said protein includes a fusion protein.

33. A method according to claim 32 wherein said fusion protein is GST or MBP.

34. A kit for use in the method according to any one of the preceding claims comprising: ... a fluorescent dye selected from epicocconone and related dyes; one or more proteolytic enzymes; and instructions on how to use the kit for monitoring proteome, protein or peptide digestion.

35. A kit according to claim 34 further including a standard protein or peptide substrate.

36. A kit according to claim 35 wherein the standard protein substrate is BSA, apo-

transferrin, α-casein, carbonic anhydrase and the peptide is β -endorphin.

37. A kit according to any one of claims 34 to 36 further including a buffer.

38. A kit according to claim 37 wherein said buffer is a Good's buffer.

39. A kit according to claim 37 wherein said buffer is a bicine buffer.

40. A kit according to claim 37 wherein said buffer is a carbonate buffer.