E+S ES E+P
Flash DigestTM Kit Next Generation Protein Digestion Flash Digest is a very active, highly stable immobilized trypsin reactor that is combined with optimized buffers and state of the art heating technology for fast, reproducible digestions. With this product there is no need to denature, reduce or alkylate samples prior to digestion. Even highly cross-linked proteins can be digested in as little as 5 minutes and complex matrices can be digested in as little as 75 minutes. Flash Digest is… Easy to Use Reproducible Fast Standardized Historically, the digestion of proteins has been the biggest bottleneck and one of the largest sources of variation in the sample preparation process. Fundamentally, this is because complete digestion is kinetically unfavorable. The drop in substrate concentration as a digestion nears completion makes it very difficult to obtain complete conversion to product. The process is further complicated by autolysis, a phenomenon that deactivates trypsin and changes its specificity over the course of the reaction. . In short, a typical enzyme produces product at rate that is approximately linear for a short period after the start of the reaction. As the reaction proceeds and substrate is consumed the rate continuously slows
Figure 1: Schematic representation of enzyme kinetics
K1
E+S
K2
ES
E+P
K-1
Substrate Binding
Catalytic Step
Figure 2: The amino acid sequence of human insulin Using the digestion of a simple protein (e.g. insulin) as an example it is easy to replicate this phenomenon in-situ. The digestion of insulin creates a maximum of 3 peaks, undigested protein, a large digested peptide, and the peptide product GFFYTPK.
FVNQHLCGSHLVEALYLVCGERGFFYTPK T S
S
S
S
GIVEQCCTSICSLYQLENYCN S S
With a typical digestion protocol (50:1 protein: enzyme ratio, 37°C) the conversion of insulin to products is, in its initial stages, approximately linear. However, as predicted, the reaction rate slows as a function of decreasing substrate concentration and autolytic loss of enzyme activity. Figure 3: Chromatograms of insulin digested for various times using typical reaction conditions
Solution Digestion 100
Conversion to Products (%)
80 60 40 20 0 0
6 12 18 24 Digest Time (Hours)
Immobilization eliminates autolysis. This preserves enzyme activity and specificity, while enabling the productive use of very high concentrations of enzyme. Perfinity has optimized the synthesis of the immobilized enzyme materials such that a complete digestion of insulin occurs in less than 8 minutes and achieves results similar to 24 hours in solution after only 4 minutes. Figure 4: Insulin digested for various times using Perfinity immobilized trypsin
Immobilized Trypsin Digestion Conversion to Products (%)
100 80 60 40 20 0 0
2 4 6 Digestion Time (min.)
8
As digest times were reduced through the use of the Perfinity immobilized trypsin, reduction and alkylation became the new bottleneck. For the Flash Digest product Perfinity engineered the enzyme such that it can withstand protein denaturing conditions. This eliminates the need to perform pretreatment. Shown below is the digestion of native bovine serum albumin (BSA). BSA contains 17 disulfide bonds. These bonds play the role of preserving the native confirmation of BSA. However, BSA is readily converted to peptides in 5 minutes. Figure 5: Digestion and LC/MS of native bovine serum albumin
The Flash Digest materials are placed into PCR tubes which are ideal for the rapid and uniform transfer of heat. This format was essential for obtaining uniformity across the plate (attempts to perform high temperature digests in standard polypropylene plates resulted in significant inside well to outside well bias). Figure 6: Flash DigestTM Kit Each Kit Contains - 12 Flash Digest Strips of 8 - Perfinity Flash Digest Buffer - 96 Well-Filter Plate (Optional) - 96 Well Collection Plate
A heating unit is required but sold separately. The recommended heating unit is a ThermoMixer C outfitted with a PCR Block and heated lid. The part numbers for these items are listed below. Please contact either Perfinity or your local Eppendorf sales representative for additional information. Description Part Number ThermoMixer C w/o thermoblock 5382000023 Eppendorf SmartBlock PCR 96 for PCR plates including lid 5360000006 ThermoTop with condensation protection technology 5308000003
Operation In order to perform a digestion add your samples to the tubes, place the tubes in the heating apparatus then following incubation either filter the samples or otherwise perform centrifugation followed by decanting… 1. 2. 3. 4.
Add your undigested sample Incubate Filter (or Centrifuge/Decant) Analyze
Figure 7: The Flash Digest workflow
It is important to note that the buffer used for Flash Digestion contains various salts (e.g., Tris, CaCl2). Results have shown that with trypsin these salts are needed to maintain specificity and activity. As such, it is important to employ a divert valve as part of the LC/MS method. Alternatively, an offline desalting procedure (e.g. SPE) can be used. Applications In order to demonstrate the utility of the Flash Digest technology an assay for the quantification of IgG was developed. Initial results were obtained in bovine serum albumin (0.05% BSA). The peptide being monitored is TTPPVLDSDGSFFLYSK. This peptide is unique to humanized monoclonal antibodies and is frequently monitored in support of preclinical bioanalysis. Monitoring this peptide, the optimal digestion time for IgG was determined by dispensing 200uL of 1ug/mL IgG1, 0.05% BSA in each of 8 FD wells. The sample strip was vortexed then placed into the heated block. Aliquots were removed at 15 minute intervals, filtered, collected and analyzed by LC/MS. It is clear that the digestion of IgG reaches completion after 60 minutes. Following the determination of digestion time linearity was established, monitoring concentrations ranging from 50ng/mL to 50ug/mL IgG1.
Figure 8: Determining the optimal digestion time for IgG
Figure 9: Validation of IgG quantitation in proxy matrix Reproducibility was determined at 0.05, 0.5, 5, 50ug/mL concentrations respectively.
Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Sample 6 Sample 7 Average StDev CV(%)
Concentration in ng/mL 50 500 5000 153 1533 19159 177 1545 18269 162 1696 17823 181 1511 17548 162 1584 17504 158 1562 17890 151 1717 18012 163 1593 18029 11 81 563 7.0 5.1 3.1
50000 213779 203635 199707 205497 205397 209014 208844 206553 4497 2.2
Evaluation of digestion time for IgG in monkey, mouse and beagle plasma was determined to be 75 minutes. Without internal standard or immunocapture, the CV values for IgG in beagle plasma ranged from 8.3% at the LLOQ to 5% at the ULOQ. Front and back five point calibration curves of IgG in plasma yielded linear fits with R2 values of 0.999 and 0.998 respectively. Figure 10: Validation of IgG quantitation in plasma Reproducibility was determined at 0.1, 1, and 100ug/mL concentrations respectively.
Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Sample 6 Sample 7 Sample 8 Average StDev CV(%)
IgG in Beagle Plasma (ng/mL) (ng/mL) (ng/mL) 100 1000 10000 3938 35256 391028 3843 30713 400077 3725 31881 368354 4173 34346 362317 3449 27114 381921 3286 29304 424200 4033 30125 400099 4112 34953 388384 3820 31712 389548 317 2940 19550 8.3 9.3 5.0
Peptide Mapping A comparison of the Flash Digest protocol to existing overnight methods was performed by digesting a commercially available monoclonal antibody at 70°C using the temperature stable immobilized enzyme. For this antibody it was determined that the reaction was complete after 30 minutes. These results where compared to an in-solution digestion of the same molecule performed over 24 hours at 37°C using a 10:1 protein:enzyme ratio. LC/UV Analysis of the resulting peptides showed that a large percentage of the same peptides were generated in each case. However, the peak intensity was generally larger from the Flash Digest protocol. It should be noted that a small amount (0.1%) of octyl-beta glucoside was added to the Perfinity Flash Digest Buffer when performing this peptide mapping protocol. Figure 11: A compatison of mAb peptide maps Top: mAb digested using Flash Digest Bottom: mAb digested using a solution digestion method with a 10:1, protein: enzyme ratio
Standardization/Quality Control To ensure lot-to-lot reproducibility of the Flash Digest materials each lot is tested and standardized for activity. Each strip of 8 tubes is checked for accuracy and each well is checked for precision. The QC test is a 1 minute digest of native human insulin. It was purposefully designed for partial digestion to maximize our ability to differentiate batch to batch variances. This procedure validates that each lot, each strip, and each well will perform the same. Figure 12: Example of a certificate of analysis Consistent activity is analyzed by creating an external test strip during plate preparation, each plate is tested for uniformity by weight; representative results
Discussion There has long been a need for a robust, standardized preparation technique that enables quantification of proteins in various matrices and rapid qualification of purified proteins. Flash Digest enables a dramatic simplification of the digestion process. Complete digestions ensure maximum sensitivity, method development can be performed in hours instead of days and the final workflow is reproducible, simple and fast. Ordering Information http://www.perfinity.com/product-category/flash-digest/ Description Part Number Flash Digest With Filter Plate FD-KIT-WP Flash Digest Without Filter Plate FD-KIT-WOP www.perfinity.com
Toll free phone: 888.775.1026
Email: [email protected]
Figure 1: Schematic representation of enzyme kinetics
K1
E+S
K2
ES
E+P
K-1
Substrate Binding
Catalytic Step
Figure 2: The amino acid sequence of human insulin Using the digestion of a simple protein (e.g. insulin) as an example it is easy to replicate this phenomenon in-situ. The digestion of insulin creates a maximum of 3 peaks, undigested protein, a large digested peptide, and the peptide product GFFYTPK.
FVNQHLCGSHLVEALYLVCGERGFFYTPK T S
S
S
S
GIVEQCCTSICSLYQLENYCN S S
With a typical digestion protocol (50:1 protein: enzyme ratio, 37°C) the conversion of insulin to products is, in its initial stages, approximately linear. However, as predicted, the reaction rate slows as a function of decreasing substrate concentration and autolytic loss of enzyme activity. Figure 3: Chromatograms of insulin digested for various times using typical reaction conditions
Solution Digestion 100
Conversion to Products (%)
80 60 40 20 0 0
6 12 18 24 Digest Time (Hours)
Immobilization eliminates autolysis. This preserves enzyme activity and specificity, while enabling the productive use of very high concentrations of enzyme. Perfinity has optimized the synthesis of the immobilized enzyme materials such that a complete digestion of insulin occurs in less than 8 minutes and achieves results similar to 24 hours in solution after only 4 minutes. Figure 4: Insulin digested for various times using Perfinity immobilized trypsin
Immobilized Trypsin Digestion Conversion to Products (%)
100 80 60 40 20 0 0
2 4 6 Digestion Time (min.)
8
As digest times were reduced through the use of the Perfinity immobilized trypsin, reduction and alkylation became the new bottleneck. For the Flash Digest product Perfinity engineered the enzyme such that it can withstand protein denaturing conditions. This eliminates the need to perform pretreatment. Shown below is the digestion of native bovine serum albumin (BSA). BSA contains 17 disulfide bonds. These bonds play the role of preserving the native confirmation of BSA. However, BSA is readily converted to peptides in 5 minutes. Figure 5: Digestion and LC/MS of native bovine serum albumin
The Flash Digest materials are placed into PCR tubes which are ideal for the rapid and uniform transfer of heat. This format was essential for obtaining uniformity across the plate (attempts to perform high temperature digests in standard polypropylene plates resulted in significant inside well to outside well bias). Figure 6: Flash DigestTM Kit Each Kit Contains - 12 Flash Digest Strips of 8 - Perfinity Flash Digest Buffer - 96 Well-Filter Plate (Optional) - 96 Well Collection Plate
A heating unit is required but sold separately. The recommended heating unit is a ThermoMixer C outfitted with a PCR Block and heated lid. The part numbers for these items are listed below. Please contact either Perfinity or your local Eppendorf sales representative for additional information. Description Part Number ThermoMixer C w/o thermoblock 5382000023 Eppendorf SmartBlock PCR 96 for PCR plates including lid 5360000006 ThermoTop with condensation protection technology 5308000003
Operation In order to perform a digestion add your samples to the tubes, place the tubes in the heating apparatus then following incubation either filter the samples or otherwise perform centrifugation followed by decanting… 1. 2. 3. 4.
Add your undigested sample Incubate Filter (or Centrifuge/Decant) Analyze
Figure 7: The Flash Digest workflow
It is important to note that the buffer used for Flash Digestion contains various salts (e.g., Tris, CaCl2). Results have shown that with trypsin these salts are needed to maintain specificity and activity. As such, it is important to employ a divert valve as part of the LC/MS method. Alternatively, an offline desalting procedure (e.g. SPE) can be used. Applications In order to demonstrate the utility of the Flash Digest technology an assay for the quantification of IgG was developed. Initial results were obtained in bovine serum albumin (0.05% BSA). The peptide being monitored is TTPPVLDSDGSFFLYSK. This peptide is unique to humanized monoclonal antibodies and is frequently monitored in support of preclinical bioanalysis. Monitoring this peptide, the optimal digestion time for IgG was determined by dispensing 200uL of 1ug/mL IgG1, 0.05% BSA in each of 8 FD wells. The sample strip was vortexed then placed into the heated block. Aliquots were removed at 15 minute intervals, filtered, collected and analyzed by LC/MS. It is clear that the digestion of IgG reaches completion after 60 minutes. Following the determination of digestion time linearity was established, monitoring concentrations ranging from 50ng/mL to 50ug/mL IgG1.
Figure 8: Determining the optimal digestion time for IgG
Figure 9: Validation of IgG quantitation in proxy matrix Reproducibility was determined at 0.05, 0.5, 5, 50ug/mL concentrations respectively.
Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Sample 6 Sample 7 Average StDev CV(%)
Concentration in ng/mL 50 500 5000 153 1533 19159 177 1545 18269 162 1696 17823 181 1511 17548 162 1584 17504 158 1562 17890 151 1717 18012 163 1593 18029 11 81 563 7.0 5.1 3.1
50000 213779 203635 199707 205497 205397 209014 208844 206553 4497 2.2
Evaluation of digestion time for IgG in monkey, mouse and beagle plasma was determined to be 75 minutes. Without internal standard or immunocapture, the CV values for IgG in beagle plasma ranged from 8.3% at the LLOQ to 5% at the ULOQ. Front and back five point calibration curves of IgG in plasma yielded linear fits with R2 values of 0.999 and 0.998 respectively. Figure 10: Validation of IgG quantitation in plasma Reproducibility was determined at 0.1, 1, and 100ug/mL concentrations respectively.
Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Sample 6 Sample 7 Sample 8 Average StDev CV(%)
IgG in Beagle Plasma (ng/mL) (ng/mL) (ng/mL) 100 1000 10000 3938 35256 391028 3843 30713 400077 3725 31881 368354 4173 34346 362317 3449 27114 381921 3286 29304 424200 4033 30125 400099 4112 34953 388384 3820 31712 389548 317 2940 19550 8.3 9.3 5.0
Peptide Mapping A comparison of the Flash Digest protocol to existing overnight methods was performed by digesting a commercially available monoclonal antibody at 70°C using the temperature stable immobilized enzyme. For this antibody it was determined that the reaction was complete after 30 minutes. These results where compared to an in-solution digestion of the same molecule performed over 24 hours at 37°C using a 10:1 protein:enzyme ratio. LC/UV Analysis of the resulting peptides showed that a large percentage of the same peptides were generated in each case. However, the peak intensity was generally larger from the Flash Digest protocol. It should be noted that a small amount (0.1%) of octyl-beta glucoside was added to the Perfinity Flash Digest Buffer when performing this peptide mapping protocol. Figure 11: A compatison of mAb peptide maps Top: mAb digested using Flash Digest Bottom: mAb digested using a solution digestion method with a 10:1, protein: enzyme ratio
Standardization/Quality Control To ensure lot-to-lot reproducibility of the Flash Digest materials each lot is tested and standardized for activity. Each strip of 8 tubes is checked for accuracy and each well is checked for precision. The QC test is a 1 minute digest of native human insulin. It was purposefully designed for partial digestion to maximize our ability to differentiate batch to batch variances. This procedure validates that each lot, each strip, and each well will perform the same. Figure 12: Example of a certificate of analysis Consistent activity is analyzed by creating an external test strip during plate preparation, each plate is tested for uniformity by weight; representative results
Discussion There has long been a need for a robust, standardized preparation technique that enables quantification of proteins in various matrices and rapid qualification of purified proteins. Flash Digest enables a dramatic simplification of the digestion process. Complete digestions ensure maximum sensitivity, method development can be performed in hours instead of days and the final workflow is reproducible, simple and fast. Ordering Information http://www.perfinity.com/product-category/flash-digest/ Description Part Number Flash Digest With Filter Plate FD-KIT-WP Flash Digest Without Filter Plate FD-KIT-WOP www.perfinity.com
Toll free phone: 888.775.1026
Email: [email protected]
