Development of an LC-MS/MS peptide mapping protocol for the NISTmAb

Peptide mapping is a component of the analytical toolbox used within the biopharmaceutical industry to aid in the identity confirmation of a protein therapeutic and to monitor degradative events such as oxidation or deamidation. These methods offer the advantage of providing site-specific information regarding post-translational and chemical modifications that may arise during production, processing or storage. A number of such variations may also be induced by the sample preparation methods themselves which may confound the ability to accurately evaluate the true modification levels. One important focus when developing a peptide mapping method should therefore be the use of sample preparation conditions that will minimize the degree of artificial modifications induced. Unfortunately, the conditions that are amenable to effective reduction, alkylation and digestion are often the same conditions that promote unwanted modifications. Here we describe the optimization of a tryptic digestion protocol used for peptide mapping of the NISTmAb IgG1κ which addresses the challenge of balancing maximum digestion efficiency with minimum artificial modifications. The parameters on which we focused include buffer concentration, digestion time and temperature, as well as the source and type of trypsin (recombinant vs. pancreatic; bovine vs porcine) used. Using the optimized protocol we generated a peptide map of the NISTmAb which allowed us to confirm its identity at the level of primary structure. Graphical abstract Peptide map of the NISTmAb RM 8671 monoclonal antibody. Tryptic digestion was performed using an optimized protocol and followed by LC-UV-MS analysis. The trace represents the total ion chromatogram. Each peak was mapped to peptides identified using mass spectrometry data. Electronic supplementary material The online version of this article (10.1007/s00216-018-0848-6) contains supplementary material, which is available to authorized users.


Document S1
Detailed protocol for optimized tryptic digestion of the NISTmAb Buffer Preparation. Mass measurements for solution preparation were performed using a Mettler-Toledo AL54 analytical balance (calibrated annually). PH measurements were read using the Orion Star™ A211 pH Meter (Thermo Scientific) fitted with the ROSS Sure Flow Combination pH Electrode (Thermo Scientific). Protein concentration was measured using the Thermo Scientific™ NanoDrop 2000 spectrophotometer. Details regarding the reagents used can be found in the Materials and Methods section of the main manuscript.
The 0.1 mol/L Tris, pH 7.8 solution was prepared as follows: 1) 2.127 g Tris-HCl and 0.7877 g Tris base were added to a 200 mL volumetric flask; 2) the flask was filled to the 200.0 mL mark with LC-MS grade water; 3) while recording pH with a calibrated pH meter, the solution was adjusted to pH 7.8 ± 0.02 by drop-wise addition of 1 mol/L hydrochloric acid; 4) the solution was sterile filtered through a 0.22 µm cellulose acetate membrane; and 5) stored at 2 °C to 8 °C until use.
The 10 mmol/L EDTA in 0.1 mol/L Tris, pH 7.8 solution was prepared as follows: 1) 29.2 mg ethylenediaminetetraacetic acid (EDTA) were added to a 10 mL volumetric flask; 2) the flask was filled to the 10 mL mark with 0.1 mol/L Tris, pH 7.8; 3) the solution was sterile filtered through a 0.22 µm cellulose acetate membrane; and 5) stored at 2 °C to 8 °C until use.
Denaturing Buffer (6 mol/L guanidine HCl, 1 mmol/L EDTA in 0.1 mol/L Tris, pH 7.8) was prepared as follows: 1) 5.732 g guanidine HCl and 1 mL of 10 mmol/L EDTA were added to a 10 mL volumetric flask; 2) the flask was filled to 10 mL with 0.1 mol/L Tris, pH 7.8; 3) the solution was sterile filtered through a 0.22 µm cellulose acetate membrane; and 5) stored at 2 °C to 8 °C until use.
Formulation buffer (12.5 mmol/L L-histidine/12.5 mmol/L L-histidine HCl, pH 6.0) was prepared as follows: 1) 1.3129 g histidine monohydrochloride monohydrate and 0.9704 g Lhistidine were placed in a beaker and diluted with ~450 mL LC-MS grade water; 2) while recording pH with a calibrated pH meter, the solution was adjusted to pH 6.00 ± 0.02 by dropwise addition of 1 mol/L hydrochloric acid; 3) the solution was transferred to a 500 mL volumetric flask, the beaker rinsed with LC-MS grade water and the flask volume was adjusted to 500.0 mL using the rinse water; 4) the solution was sterile filtered through a 0.22 µm cellulose acetate membrane into a sterile plastic bottle; and 5) stored at 2 °C to 8 °C. 500 mmol/L DTT was prepared as follows: 1) the tube containing 7.7 mg of dithiothreitol (DTT) (pre-weighed by manufacturer; Pierce No-Weigh Format DTT Cat #20291) was briefly centrifuged to bring all particles to the bottom; 2) 100 µL of LC-MS grade water were added to the tube; 3) the contents of the tube were mixed by gentle pipetting; and 3) the solution was stored in small volume aliquots at -80 °C. 500 mmol/L IAM was prepared as follows: 1) the tube containing 56 mg of iodoacetamide (IAM) (pre-weighed by manufacturer; Sigma pre-weighed vials, Cat #A3221) was briefly centrifuged to bring all particles to the bottom; 2) 600 µL of LC-MS grade water were added to the tube; 3) the contents of the tube were mixed by gentle pipetting; and 4) the solution was stored in small volume aliquots at -80 °C.
Digestion Buffer (1 mol/L urea in 0.1 mol/L Tris, pH 7.8) was prepared at the time of use as follows: 1) 300.3 mg urea were added to a 5 mL volumetric flask; 2) the flask was filled to 5 mL with 0.1 mol/L Tris, pH 7.8; and 3) the solution was sterile filtered through a 0.22 µm cellulose acetate membrane. 0.05 mol/L Acetic Acid was prepared as follows: 1) ≈ 25 mL of LC-MS grade water were added to a 25 mL volumetric flask; 2) 0.072 mL acetic acid (99.9% purity) were added to the flask while under a fume hood, using a glass syringe with stainless steel needle and polytetrafluoroethylene (PTFE) plunger; 3) the flask was filled to the 25 mL mark with LC-MS grade water; 4) the solution was sterile filtered through a 0.22 µm PTFE membrane into an amber glass reagent bottle with PTFE-lined cap; and 5) stored at 2 °C to 8 °C.
1 µg/µL Trypsin was prepared as follows: 1) 25 µL of 0.05 mol/L acetic acid were added to a 25 µg vial of trypsin (Recombinant, proteomics grade, expressed in Pichia pastoris; Sigma Cat # 03708985001); 2) the solution was mixed by gentle pipetting; and 3) stored in small volume aliquots at -80 °C. 0.1 % formic acid in LC-MS grade water was purchased as a pre-formulated solution.
Optimized Tryptic Digestion Protocol. The optimized tryptic digestion protocol is summarized below. The protocol begins with denaturing, reduction and alkylation of 500 µg of NISTmAb, and proceeds with the digestion of 50 µg of the reduced and alkylated NISTmAb. The buffer exchange and digestion (Step 4 through Step 6) may be scaled up to use a larger amount of reduced and alkylated NISTmAb if desired.

Sample and Blank Preparation
a. 50 µL of 10 mg/mL NISTmAb were added to 445 µL Denaturing Buffer in a polypropylene tube b. in a separate tube, a blank was prepared by adding 50 µL of Formulation Buffer to 445 µL Denaturing Buffer 2. Reduction a. 5 µL of 500 mmol/L DTT were added to each tube b. the contents of each tube were mixed gently c. the tubes were incubated at 4°C for 60 min, in the dark 3. Alkylation a. 10.2 µL of 500 mmol/L IAM were added to each tube b. the contents of each tube were mixed gently c. the tubes were incubated at 4°C for 60 min, in the dark 4. Buffer Exchange (using Zeba™ Spin 7K MWCO size-exclusion desalting columns (P/N 89882, Thermo Scientific, Waltham, MA)) a. fresh Digestion Buffer was made and used to prepare two size exclusion spin columns for buffer exchange according to the manufacturer's specifications b. after spin columns were exchanged to Digestion Buffer and the alkylation incubation period was complete the columns were placed in clean, labeled 1.5 mL polypropylene tubes c. 100 µL of the reduced and alkylated NISTmAb were added to the center of one column bed without disturbing the bed with the pipet tip d. 100 µL of the blank were added to the center of the remaining column bed e. the columns were centrifuged according to the manufacturer's specifications to collect the sample and blank which were then in Digestion Buffer 5. Protein Concentration a. the contents of the buffer-exchanged blank tube were used to "blank" the spectrophotometer b. the protein concentration of the buffer-exchanged NISTmAb was measured by UV-VIS at 280 nm absorbance using the manufacturer default settings for IgG 6. Digestion a. the measured protein concentration was used to determine the volume needed for 50 µg of buffer-exchanged NISTmAb and this volume was transferred to a new polypropylene tube: b. 2.78 µL of 1 µg/µL trypsin were added to the 50 µg aliquot of NISTmAb (a 1:18 (w/w) enzyme:NISTmAb ratio) c. the volume of Digestion Buffer needed to dilute the sample to 0.5 µg/µL was determined:  Table S1 Representative deconvoluted masses of PS 8670 subunit peaks The species comprising each UV peak shown in Fig. 1, Fig. 2, Fig. 3, ESM Fig. S1 and ESM Fig. S2 were identified by deconvolution of their corresponding MS spectra. Observed and calculated average masses representative of each species are shown. LC = light chain; HC = heavy chain.  Figure 7 and ESM Figure S3 were correlated with the peptide identifications. Lower case letters in the peptide sequence reflect the location of the observed modification. nd = not detected. Peptide location is described as "H" for Heavy Chain or "L" for Light Chain, followed by the first and last residue numbers relative to the N-terminal residue of that chain.