Flash Digest

Next Generation Protein Digestion

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. This product eliminates the need to denature, reduce or alkylate samples prior to digestion. Even highly cross-linked proteins can be digested in as little as 1 minute and complex matrices can be digested in as little as 75 minutes. Flash Digest is…

Easy to Use

Reproducible

Fast

Standardized

The digestion of proteins has been the biggest bottleneck and one of the largest sources of variation in sample preparation. 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. The process is further hindered 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.

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.

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. 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 1: Schematic representation of enzyme kinetics

Figure 2: The amino acid sequence of human insulin

FVNQHLCGSHLVEALYLVCGERGFFYTPK T

GIVEQCCTSICSLYQLENYCN

S

S

S

S

S S

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. Take, for instance, 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 3: A,B–insulin digested for various times using typical reaction conditions; C,D–Insulin digested for various times using Perfinity immobilized trypsin

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Digestion Time (min.)

Immobilized Trypsin Digestion

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Digest Time (Hours)

Solution Digestion A B

C D

The Flash Digest materials are sold in PCR tubes which are ideal for the rapid and uniform transfer of heat. This format is essential for obtaining uniformity across the plate. Figure 5: Flash DigestTM Kit 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 5306000006

ThermoTop with condensation protection technology 5308000003

Operation

Figure 6: The Flash Digest workflow 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. Add your undigested sample 2. Incubate 3. Filter (or Centrifuge/Decant) 4. Analyze

Each Kit Contains

- 12 Flash Digest Strips of 8 - Perfinity Flash Digest Buffer - 96 Well Filter Plate (Optional) - 96 Well Collection Plate

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: Validation of IgG quantitation in proxy matrix Reproducibility was determined at 0.05, 0.5, 5, 50ug/mL concentrations respectively.

50 500 5000 50000

Sample 1 153 1533 19159 213779

Sample 2 177 1545 18269 203635

Sample 3 162 1696 17823 199707

Sample 4 181 1511 17548 205497

Sample 5 162 1584 17504 205397

Sample 6 158 1562 17890 209014

Sample 7 151 1717 18012 208844

Average 163 1593 18029 206553

StDev 11 81 563 4497

CV(%) 7.0 5.1 3.1 2.2

Concentration in ng/mL

Figure 7: Determining the optimal digestion time for

IgG

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 9: Validation of IgG quantitation in plasma Reproducibility was determined at 0.1, 1, and 100ug/mL concentrations respectively.

(ng/mL) (ng/mL) (ng/mL)

100 1000 10000

Sample 1 3938 35256 391028

Sample 2 3843 30713 400077

Sample 3 3725 31881 368354

Sample 4 4173 34346 362317

Sample 5 3449 27114 381921

Sample 6 3286 29304 424200

Sample 7 4033 30125 400099

Sample 8 4112 34953 388384

Average 3820 31712 389548

StDev 317 2940 19550

CV(%) 8.3 9.3 5.0

IgG in Beagle Plasma

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 10: A comparison 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 11: 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/

www.perfinity.com Toll free phone: 888.775.1026 Email: info@perfinity.com