TECHNICAL MANUAL AccuMAP™ Low pH Protein Digestion Kits Instructions for Use of Products VA1040 and VA1050

AccuMAP™ Low pH Protein Digestion Kits All technical literature is available at: www.promega.com/protocols/ Visit the web site to verify that you are using the most current version of this Technical Manual. E-mail Promega Technical Services if you have questions on use of this system: [email protected] Promega Corpora on · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · 608-274-4330 · Fax 608-277-2516 www.promega.com TM504 · 5/

1. Description The AccuMap™ Low pH Protein Digestion Kit is designed for accurate, reproducible characterization of biotherapeutic proteins by peptide mapping using LC/MS and/or UV HPLC. The entire sample preparation procedure is performed at low (mildly acidic) pH to suppress artificial deamidation and disulfide bond scrambling. The kit also contains an optional agent for suppression of protein oxidation during sample preparation. Features • Suppression of sample preparation-induced artificial deamidation, disulfide bond scrambling and oxidation over the course of sample preparation. Pre-existing...

2. Product Components and Storage Conditions PRODUCT SIZE CAT.# AccuMAP™ Low pH Protein Digestion Mini Kit 1 each VA1040 Contains sufficient reagents to prepare and digest 500pg of protein. Includes: • 1ml AccuMAP™ Denaturing Solution (contains GuHCl) Storage Conditions: Store all kit contents at -65°C or below until ready to use. Before using either kit, see Section 8.B for information on preparation of reagents and solutions. Promega Corporation ■ 2800 Woods Hollow Road ■ Madison, WI 53711-5399 USA ■ Toll Free in USA 800-356-9526 ■ 608-274-4330 ■ Fax 608-277-2516 3

Protocols for Sample Preparation Under Reducing Conditions Materials to Be Supplied by the User • NANOpure® (or equivalent grade) water • • trifluoroacetic acid (TFA) urea (for use in Sections 3.B and 4.B) The protocols in this technical manual do not include a ‘capping’ step with excess reducing agent following alkylation. A capping step is commonly used to inactivate excess alkylating agent. In contrast, we allow alkylation to continue over the course of the digestion reaction effectively extending the incubation of a reduced protein with IAM to 4.5 hours. This extended alkylation period is required...

Add 10µl of AccuMAP™ 10X Low pH Reaction Buffer. Add 2µl of AccuMAP™ 100X Oxidation Suppressant Solution if needed. If suppressant is not needed, add 2µl of distilled water. Note: We recommend that you add the suppressant if a protein solution contains polysorbates or other compounds that decompose into oxidizing agents or have inherent protein oxidizing activity. You must clean up the sample prior to UV HPLC or LC/MS analysis if suppressant is used as this reagent can damage reverse phase columns. Complete the digestion using additional AccuMAP™ Low pH Resistant rLys-C or AccuMAP™ Modified Trypsin...

3.B. Sample Preparation with Urea (continued) Reduce and Alkylate 1. Add 5µl of protein solution (50µg protein quantity) to 42µl of 9M urea/1.4X AccuMAP™ Low pH Reaction Buffer mix. Add 1µl of 100mM TCEP, mix and incubate for 30 minutes at 37°C. Add 2µl of 300mM IAM, mix and incubate protected from light for 30 minutes at 37°C. Cover the heating block with aluminum foil to protect from light if needed. Add 10µl of AccuMAP™ Low pH Resistant rLys-C, mix and incubate protected from light for 1 hour at 37°C. Add 10µl of AccuMAP™ 10X Low pH Reaction Buffer. Add 2µl of AccuMAP™ 100X Oxidation Suppressant...

3.C. Sample Preparation with Desalting by Size Exclusion Chromatography Some sample preparation protocols make use of a desalting step following denaturing, reduction and alkylation in order to remove the denaturants and other components that inhibit proteolytic activity. While these desalting procedures can be effective in that regard, they also have significant drawbacks that stem from loss of protein and protein refolding. If possible, use the protocol described in Section 3.A, which is optimized for high reproducibility and digestion efficiency. This protocol is optimized for sample preparation...

Add TFA to a final concentration of 2% (for example, add 11µl of 20% TFA). Check a small amount of sample with pH paper to ensure the reaction is properly acidified. Store digests at –20°C or below until ready for analysis. We recommend that you clean up digested protein with solid phase extraction or a trapping column prior to UV HPLC or LC/MS analysis to remove salts and impurities. 3.D. Sample Preparation of Proteolytically Resistant Proteins This protocol is optimized for the digestion of 50μg of protein at a concentration of 10mg/ml. For less concentrated protein samples, please refer to Table...

Complete the digestion using AccuMAP™ Low pH Resistant rLys-C or AccuMAP™ Modified Trypsin and AccuMAP™ Low pH Resistant rLys-C as follows: Digest with AccuMAP™ Low pH Resistant rLys-C: Add 218µl of NANOpure® water and 10µl of AccuMAP™ Low pH Resistant rLys-C. Mix and incubate protected from light overnight at 37°C. Digest with AccuMAP™ Modified Trypsin and AccuMAP™ Low pH Resistant rLys-C: Add 198µl of NANOpure® water and 10µl of AccuMAP™ Low pH Resistant rLys-C. Mix and incubate protected from light overnight at 37°C. In the morning, add 20µl of AccuMAP™ Modified Trypsin. Mix and incubate for...

4.A. Sample Preparation with Guanidine HCl (continued) Digest 4. Add 10µl of AccuMAP™ 10X Low pH Reaction Buffer. Complete the digestion using AccuMAP™ Low pH Resistant rLys-C or AccuMAP™ Modified Trypsin and AccuMAP™ Low pH Resistant rLys-C as follows: Digest with AccuMAP™ Low pH Resistant rLys-C: Add 80µl of NANOpure® water and 10µl of AccuMAP™ Low pH Resistant rLys-C. Mix and incubate for 3 hours at 37°C (the reaction can be continued overnight if desired). Digest with AccuMAP™ Modified Trypsin and AccuMAP™ Low pH Resistant rLys-C: Add 60µl of NANOpure® water, 10µl of AccuMAP™ Low pH Resistant...