SMART Digest and SMART Digest ImmunoAffinity (IA) Kit Technical Guide

Transcription

1 MART Digest and MART Digest ImmunoAffinity (IA) Kit Technical Guide marter protein preparation

2 MART Digest and MART Digest ImmunoAffinity (IA) Kits marter protein preparation The Thermo cientific MART Digest and the MART Digest ImmunoAffinity (IA) Kits are designed for biomarker and bio-therapeutic characterization and quantitation. The kits deliver sample preparation of proteins that is: Fast imple ighly reproducible ensitive Compatible with automation Challenges for biopharmaceutical laboratories Modern biopharmaceutical laboratories are tasked with providing high quality quantitative and qualitative analytical results, often in high-throughput, regulated environments. This is exacerbated by the increase in efficacy of biotherapeutics and the often low levels of biomarkers in complex biological matrices. ne of the key areas which affects these requirements is sample preparation. Current technologies employed are subject to high levels of irreproducibility, poor sensitivity, and protracted methodologies that are not amenable to automation and often require 24 hours to deliver results. 2

3 Addressing sample preparation challenges for biopharma The MART Digest Kit The kits deliver fast, simple and highly reproducible digestion of proteins for characterization and quantitation applications, achieved due to the heat-stable immobilized trypsin design. Immobilized, heat-stable trypsin (inactive at room temperature) Bead Biotherapeutic or Biomarker This design allows the digestion process to be significantly simplified compared to traditional in-solution digest protocols. 3

4 MART Digest Kit imple and easy to use workflow Add protein sample to MART Digest tube Incubate and shake MART Digest tube Thermo cientific LAµ PE or centrifuge/filter (optional) Analyze your sample tandard In-olution Digest Complex and laborious workflow Peptide sequence DTT 2 N 2 N I - Cys Cys Enzyme Enzyme Digested fragments Arginine: R Lysine: K Trypsin cleavage: Analyze your sample Quantitate protein concentration Reduce sample Alkylate sample Digest sample with trypsin Questions Answers Q: Why is the temperature set to 70 C? This has been shown to be the optimum temperature for enzyme activity and the unfolding of proteins. igher or lower temperatures may reduce the efficiency of the digestion. Q: What concentration of protein can I add to each MART Digest tube? You can add 200 pg to 3.5 mg of total protein to each MART Digest tube (protein/sample dependent). Q: Do I have to reduce and alkylate my protein? The MART Digest kits are thermally stable. When operated at high temperatures denaturation and digestion occur simultaneously. Therefore, for many quantitative workflows, there is no need to perform the additional steps of denaturation, reduction and alkylation. owever, during this process many disulfide bonds will remain intact. As a result, for characterization workflows where maximum sequence coverage is required, it is recommended that you perform reduction and alkylation after digestion. 4

5 MART Digest Kit Fast digestion of proteins Using the MART Digest kit simplifies the process and reduces the time needed for sample preparation. This not only speeds up your workflows, but also significantly reduces method development time. In the example below it can be seen that for carbonic anhydrase full digestion is achieved in 5 minutes. Carbonic Anhydrase, 29 KDa Time course experiment for digestion optimization digest time 0 min 5 min 10 min 20 min 30 min 60 min min Typical Digestion Times Protein Insulin 4 BA < 5 Carbonic anhydrase < 5 Lysozyme < 5 Apo-B 30 IgG 45 IgG in 50 µl plasma* 75 Ribonuclease A 150 Thyroglobulin 240 C-reactive protein 240 Digest Time (min) 200 μl protein solution (100 μg/ml) at 70 C *IgG in plasma (17.5 mg/ml total protein) at 70 C Q: My protein is very difficult to unfold so I need to use urea. What concentration of urea can I use? The MART Digest kit uses heat to unfold the protein. If urea is required then it is recommended to dilute the sample to 0.5 M or less of urea prior to transferring to the MART Digest tube. Q: What if I have less than 50 µl of sample? Q: Does digestion using the MART Digest kit at high temperatures result in an increase in post-translational modifications? In comparison to in-solution digests a comparable number of PTMs have been observed when screening for deamidation, amidation, methylation and oxidation. No modifications to existing PTMs, such as phosphorylated sites, have been observed. If your sample is less than 50 µl adjust to 50 µl with ultrapure water. 5

6 MART Digest Kit igh quality analytical results The MART Digest kit provides significant improvements in reproducibility over existing protocols, which results in fewer sample failures, higher throughput and the ability to more easily interrogate data. The following chromatogram shows 13 overlaid traces of mouse mab peptides, digested with the MART Digest kit. Excellent reproducibility of <0.082 %RD is achieved when using the Thermo cientific Vanquish UPLC system, as indicated in the table below Retention Time Repeatability peak # RT (min) RD (%) mau Min verlay of 13 consecutive chromatographic runs of a peptide sample separated on an analytical Thermo cientific Acclaim VANQUI C18 column (2.2 μm, mm) and prepared from a mab, digested with the MART Digest kit %C: 0.0 % 150 WVL:214 nm Reduced Non-reduced Reproducible peptide maps and excellent retention time repeatability enable confidence in direct comparisons. 75 Area Ratio %B: 10.0 % Flow: 400 μl/min Min 6 Monitoring of conditional differences

7 Characterization The MART Digest kit, in combination with the igh Resolution Accurate Mass (RAM) capabilities of the Thermo cientific Q Exactive mass spectrometers, and high resolution chromatographic separation with the Vanquish UPLC family provides us with high quality data sets that can be more effectively interrogated with Thermo cientific BioPharma Finder oftware. The BioPharma Finder software allows us to map our protein, in the case for Rituximab ~100% sequence coverage is achieved, as well as quantitation of modifications on both the light and heavy chains. Rituximab light chain Rituximab heavy chain This is of particular importance in understanding how the molecule will interact within a biological system. equence coverage ~100% Protein Modification Recovery Abundance Rituximab_LC Q1+N 3 loss Good 87.81% Rituximab_LC W90+xidation Good 2.06% Rituximab_C ~Q1+N 3 loss Good % Rituximab_C W281+xidation Good 4.98% Rituximab_C N301+A1G0F Fair 2.87% Rituximab_C N301+A1G1F Fair 1.22% Rituximab_C N301+A2G0 Fair 1.30% Rituximab_C N301+A2G0F Fair 37.69% Rituximab_C N301+A2G1F Fair 44.86% Rituximab_C N301+A2G2F Fair 10.77% Rituximab_C N301+M5 Fair 1.07% Rituximab_C N365+Deamidation Good 2.72% Rituximab_C W385+xidation Good 5.37% Rituximab_C G450+Lys Good % 7

8 Quantitation The MART Digest kit allows confident detection of biomarkers with high sensitivity within a wide dynamic range, as can be seen below with the example of Thyroglobulin. Greater sensitivity is achieved in 3.5 hours compared to an in-solution digest protocol taking 20 hours to complete Clarke (2012) MART Digest kit y = 76.26x R² = Peak Area Area Counts Precursor Ion (m/z) Concentration in plasma (μg/ml) Measurement of serum Thyroglobulin after tryptic digestion of serum samples. n MART Digest kit: 25% plasma, 3.5 h digestion n In-solution digest: 20% plasma, R/A, h digestion Clarke et al. (2012), J. Investigative Medicine, 60(8) Calibration curve for thyroglobulin signature peptide in murine plasma ( μg/ml) m/z m/z m/z 3000 Peak Area Digestion Time (min) Digestion time curve 70 C. ptimum digestion time of 3.5 hours for Thyroglobulin was determined by monitoring the peak area response for the three signature peptides. 8

9 The MART Digest IA Kit The MART Digest IA kits have all the advantages previously outlined for fast, easy and reproducible protein digestion for quantitation and characterization applications with the added advantage of combining an immunocapture and the digestion process into a single well. This has significant benefits for quantitation studies where immunoaffinity capture is typically employed to increase sensitivity by purifying low level proteins from complex biological matrices. This step is then followed by protein digestion. The MART Digest IA kits achieve this due to their unique design where the immunoaffinity reagents (either streptavidin, protein A or protein G) and heat activated thermally stable trypsin are co-immobilized onto a single bead. Following the binding of a capture reagent to the bead, and enrichment of the target, the enzyme is activated at elevated temperatures for accelerated digestion under protein denaturing conditions. The resulting workflow is as easy as enrich, wash and digest. Magnetic and non-magnetic versions of the beads are available. Immobilized treptavidin Immobilized, heat-stable trypsin (inactive at room temperature) N N Bead Biomarker N N N N Biotinylated capture antibody Anti-biomarker Ab Bead Immobilized Protein A or G 9

10 ow does the MART Digest IA Kit work? Conventional protein enrichment and digestion Expose biotinylated capture antibody to biological sample Incubate with affinity capture resin N N epharose bead N N Conventional tryptic digest Reduction, TCEP: 60 C, min Alkylation, IAA: RT, 30 min Quenching, ACC: RT, 15 min Digestion: 37 C, h N N epharose bead N N Total time hours 10

11 Protein enrichment and digestion using the MART Digest IA kit N N Use MART Digest IA resin for affinity capture. Incubate at room temperature. N N Bead N N Digestion with MART Digest IA resin 70 C, 1 h N N Bead N N Total time 3 4 hours 11

12 Quantitation with the MART Digest IA Kit compared to a conventional approach The following is an example of using the MART Digest IA treptavidin kit for the quantitation of a biomarker (human interrferon a14) in human plasma, compared to a traditional immunocapture and digestion method. MART Digest IA Kit process The MART Digest IA protocol used involved an immunocapture step, taking 2 hours, followed by a 1 hour, high-temperature digestion with immobilized trypsin. This is compared to immunocapture with a high capacity streptavidin gel followed by overnight tryptic digestion of the biomarker protein. A IL peptide was spiked into the samples to act as an internal standard. 6.0e4 5.0e4 250 ng Low QC IL std. 6.0e4 5.0e ng igh QC IL std. Intensity, cps 4.0e4 3.0e4 2.0e4 Intensity, cps 4.0e4 3.0e4 2.0e4 1.4 e4 1.0e4 0.4 e Time, min 1.0e Time, min 0.7 tandard Curve (n = 1) Quality Controls (n = 4) Area Ratio Concentration Ratio (ng/ml) 5000 Actual Conc (ng/ml) Accuracy (%) Calc Value (ng/ml) Actual Conc (ng/ml) CV (%) Accuracy (%) Recovery with MART Digest IA kit 500 ng/ml spike 7330 (cps) Recovery 64% 12

13 Conventional streptavidin agarose process 3.0e4 250 ng Low QC IL std. 2.0e ng igh QC IL std. Intensity, cps 2.0e4 1.0e4 Intensity, cps 1.0e4 0.6 e4 0.2 e Time, min Time, min Area Ratio Concentration Ratio (ng/ml) 5000 tandard Curve (n = 1) Quality Controls (n = 4) Actual Conc (ng/ml) Accuracy (%) Calc Value (ng/ml) Actual Conc (ng/ml) CV (%) Accuracy (%) Note: Data provided courtesy of PharmaCadence Analytical ervices, atfield, PA, UA. Carmen Fernández-Metzler, Bonnie Baker, Robyn Buerger. Recovery with conventional approach 500 ng/ml spike 2778 (cps) Recovery 35% A tep Change in Protein Affinity Capture and Digestion MART Digest and MART Digest IA kits provide a significant change in protein sample preparation, by delivering workflows that are: ignificantly faster Easier to use ighly reproducible ensitive Compatible with automation 13

14 rdering Information Part Number Description MART Digest Kits MART Digest Kit Trypsin and collection plate B MB MART Digest Kit Trypsin, bulk resin option MART Digest Kit Trypsin, magnetic bulk resin option MART Digest Kit Trypsin, filter/collection plate B MB MART Digest Kit Trypsin, bulk resin option with filter/collection plate MART Digest Kit Trypsin, magnetic bulk resin option with filter/collection plate MART Digest Kit Trypsin, LAµ/collection plate B MB MART Digest Kit Trypsin, bulk resin option with LAµ/collection plate MART Digest Kit Trypsin, magnetic bulk resin option with LAµ/collection plate mart Digest Kit oluble Trypsin and collection plate MART Digest Kit Chymotrypsin and collection plate B MB MART Digest Kit Chymotrypsin, bulk resin option MART Digest Kit Chymotrypsin, magnetic bulk resin option MART Digest Kit Chymotrypsin, filter/collection plate B MB MART Digest Kit Chymotrypsin, bulk resin option with filter/collection plate MART Digest Kit Chymotrypsin, magnetic bulk resin option with filter/collection plate MART Digest Kit Chymotrypsin, LAµ/collection plate B MB MART Digest Kit Chymotrypsin, bulk resin option with LAµ/collection plate MART Digest Kit Chymotrypsin, magnetic bulk resin option with LAµ/collection plate MART Digest Kit Proteinase K and collection plate B MB MART Digest Kit Proteinase K, bulk resin option MART Digest Kit Proteinase K, magnetic bulk resin option MART Digest Kit Proteinase K, filter/collection plate B MB MART Digest Kit Proteinase K, bulk resin option with filter/collection plate MART Digest Kit Proteinase K, magnetic bulk resin option with filter/collection plate MART Digest Kit Proteinase K, LAµ/collection plate B MB MART Digest Kit Proteinase K, bulk resin option with LAµ/collection plate MART Digest Kit Proteinase K, magnetic bulk resin option with LAµ/collection plate 14

15 Part Number Description MART Digest IA Kits MART Digest IA Kit, treptavidin (Av) non-magnetic MART Digest IA Kit, Av non-magnetic with LAµ PE and collection plate MART Digest IA Kit, Av magnetic with LAµ PE and collection plate MART Digest IA Kit, Av magnetic MART Digest IA Kit, Protein A non-magnetic MART Digest IA Kit, Protein A non-magnetic with LAµ PE and collection plate MART Digest IA Kit, Protein A magnetic with LAµ PE and collection plate MART Digest IA Kit, Protein A magnetic MART Digest IA Kit, Protein G non-magnetic MART Digest IA Kit, Protein G non-magnetic with LAµ PE and collection plate MART Digest IA Kit, Protein G magnetic with LAµ PE and collection plate MART Digest IA Kit, Protein G magnetic Complementary Products Part Number Description Thermo cientific 96 well Vacuum Manifold Vacuum Pump (NA) Vacuum Pump (EU) LAμ RP PE Plate F MART Digest Filter Plate KingFisher Duo Prime Purification ystem KingFisher Flex Purification ystem 15

16 Characterize the full diversity of biomolecules Thermo cientific solutions for biomolecule separations LAµ PE Plates MIA D.A.R.T.s Versette Automated Liquid andler Webeal KingFisher Flex Purification ystem ample Preparation ample andling eparation and Detection Data Management BioLC/UPLC Columns Watson LIM and Data Manager BioPharma Finder Mass Informatics Platform Q Exactive BioPharma Platform Vanquish UPLC ystem Find out more at thermofisher.com/martdigest For Research Use nly. Not for use in diagnostic procedures Thermo Fisher cientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher cientific and its subsidiaries unless otherwise specified. TG21544-EN 0816