Dimethyl Labeling Coupled with Mass Spectrometry for Topographical

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Dimethyl labeling coupled with mass spectrometry for topographical characterization of primary amines on monoclonal antibodies Sin-Yi Jhan, Li-Juan Huang, Tzu-Fan Wang, Ho-Hsuan Chou, and Shu-Hui Chen Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b00320 • Publication Date (Web): 03 Mar 2017 Downloaded from http://pubs.acs.org on March 4, 2017

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Analytical Chemistry

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Dimethyl Labeling Coupled with Mass Spectrometry for Topographical Characterization of Primary Amines on Monoclonal Antibodies Sin-Yi Jhan,# Li-Juan Huang,# Tzu-Fan Wang, Ho-Hsuan Chou, Shu-Hui Chen* Department of Chemistry, National Cheng Kung University, no.1 College Road, Tainan, 701, Taiwan, Republic of China #

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these authors contributed to this work equally

*corresponding author: shchen @mail.ncku.edu.tw

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Site-specific solvent accessibility of the primary amines (mainly lysine or the

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N-termini) on proteins is of great interest in many research areas, because amines are

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an important functional group for protein conjugation. In this study, we coupled

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dimethyl labeling via reductive amination with liquid chromatography-mass

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spectrometry (LC-MS) to fully characterize the solvent accessibility of lysine residues

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and the N-termini on human immunoglobulin G (IgG). Circular dichroism (CD) and

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fluorescence spectroscopy revealed that dimethyl labeling did not alter the

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conformation of the native IgG molecule. Based on intact protein measurements, up to

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28 (light chain) and 66 (heavy chain) dimethyl tags, covering all lysine residues and

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the N-termini were sequentially incorporated into IgG molecules in1000s. All labeled

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sites were identified and quantified by a bottom-up proteomics approach. Some highly

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exposed hot-spots (for example, the N-termini of both the heavy and the light chains)

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and some buried sites (for example, K415 in the heavy chain and K39 in the light

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chain) were unambiguously revealed. This method was also used to characterize

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aggregation-induced structural changes in IgGs by increasing the temperature.

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Substantial changes in the labeling percentage of many lysine sites were observed,

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indicating a non-native aggregation triggered by thermal stress. Due to high labeling

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yields and van der Waals surface of the labeling reagents being comparable to that of

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water, dimethyl labeling is a highly promising technique for probing amines surface

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topography of proteins. It can also be used as a complementary approach to other

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methods for resolving the higher order structure of proteins by LC-MS.

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Introduction

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imaging,2 bio-inspired materials3 and protein-drug conjugates (ADCs),4-6 the latter being

Protein conjugation is widely employed in applications including biosensing,1

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currently subjected to extensive analytical characterizations or clinical trials. However,

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the efficiency of protein conjugation depends not only on the functional group, but also

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conjugation site. The resulting population of protein conjugates is generally

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pharmacokinetic parameters, drug efﬁciency, and toxicological properties.6 Thus, prior

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knowledge of the reactivity of each site of the target residue is highly valuable in

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abundance, greater chemical reactivity, and easy surface accessibility on a protein

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and NHS ester-derivatives9-10 are one of the most popular amine-reactive labeling

on the local environment, structural ﬂexibility, and solvent accessibility of the individual heterogeneous, with respect to the payload and the conjugation site on the same residue of a protein. For ADCs, this has resulted in serious problems including differences in

controlling the reaction or in characterizing conjugated products. Due to its higher molecule, lysine is one of the most popular conjugation residues. N-acetylsuccinimide7-8 reagents, which are widely used for lysine conjugation. Tandem mass tags (TMTs),11 which use EDC-NHS linking chemistry as isobaric-labeling reagents in quantitative proteomics, have been applied to characterize the properties of lysine conjugates in human IgG, by coupling the labeling method with intact protein measurements, using native MS and bottom-up proteomic techniques.12 These isotopically labeled reporter groups facilitate multiplex tandem mass spectrometry (MS/MS) experiments, thereby allowing the determination of labeling kinetics in a single experiment.13 However, more than half of the lysine residues on the TMT-labeled human IgG were neither identified nor quantified by the bottom-up proteomics technique. This was probably due to incomplete labeling or digestion, or due to low MS detectability of the labeled peptides. In order to comprehensively access the surface exposure map of all primary amine groups

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on a protein molecule, called amine surface topography, a highly efficient, fully

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sites is needed.

Stable isotope dimethyl labeling via reductive amination is a robust and inexpensive labeling method with a high reaction yield (nearly 100%) for quantitative proteomics, using liquid chromatography-mass spectrometry (LC-MS).14 Dimethyl labeling may be multiplexed,15-16 and the induced signal enhancement of the a1 ion

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can be further applied to characterize the disulfide linkages in IgG molecules.17 This labeling method uses formaldehyde as the tagging reagent, and sodium cyanoborohydride as the reducing reagent, both of which have a van der Waals

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surface similar to that of water. Thus, they are expected to act as excellent labeling reagents for probing solvent accessibility of amine groups, mainly lysine residues, on a protein. In addition, dimethyl labeling may be used as a complementary approach to other methods for resolving the higher order structure of proteins using LC-MS. For

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example, hydrogen/deuterium exchange (HDX)18-22 is able to reveal structural

accessible, and amine-specific labeling reagent with high MS detectability of the labeled


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information about the protein backbone, but it does not directly probe the structure of

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the side-chain residues. On the other hand, covalent23-25 labeling methods such as hydroxyl radical-mediated footprinting,18, 26-27 are able to react with up to ~65% of the

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residue on a protein, but it is not suitable for probing the solvent-accessible surface area (SASA) of a specific side chain residue, due to incomplete or nonspecific labeling with different amino acids. With high specificity, reactivity, and surface

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accessibility, dimethyl labeling appears to be an excellent method to provide

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supporting information which is difficult to probe by other methods. In this study, we aimed to explore dimethyl labeling as a novel and highly

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specific covalent-labeling technique for characterizing amine surface topography or as a supplementary method for elucidating the higher order structure of proteins by

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LC-MS. Since dimethyl labeling is commonly used to label digested peptides rather than proteins in quantitative proteomics; here, we developed this method for intact proteins with a high labeling yield. We confirmed that the 3D structure of proteins remains unchanged by this labeling technique, and hence it is suitable for probing protein structure. This method was then used to generate a complete surface exposure map of lysine residues or the N-termini on a human IgG molecule, based on a bottom-up proteomics technique. In addition, we used the reported method to characterize the changes in protein structure, upon protein aggregation induced by thermal stress.

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Experimental section Formaldehyde-d2, sodium cyanoborohydride (NaBH3CN), sodium acetate, ammonium hydroxide, L-dithiothreitol (DTT), iodoacetamide (IAM), ammonium bicarbonate (NH4HCO3), guanidine hydrochloride (Gd-HCl), sequence

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grade chymotrypsin and endoproteinase Glu-C were purchased from Sigma-Aldrich (St. Louis, MO, USA). HPLC grade acetonitrile (ACN) was purchased from J. T Baker (Phillipsburg, NJ, USA). Tris(hydroxymethyl)aminomethane (Tris), sodium chloride (NaCl), and ethanol were from J.T.Baker (Phillipsburg, NJ, USA). Acetonitrile (ACN) was from Merck (Darmstadt, Germany). Formic acid (FA) was

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purchased from Thermo Fisher Scientific Inc. (Waltham, MA, USA). Water (ddH2O) was deionized to 18 MΩ by a Milli-Q system. Amicon centrifugal filters (10 kDa MWCO) were obtained from Millipore (Billerica, MA, USA). Human IgG (Avastin) ,which sequence was shown in Figure 1A, was produced by Roche Inc.

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(East Dublin, GA, USA) and is commercially available. Denatured IgG standard was generated by the reaction with Gd-HCl (6 M, pH 8) at 37℃ for 1 h and then buffer

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exchanged to sodium acetate buffer (100 mM, pH 5-6).

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Materials.

Aggregation induced by thermal stress. The IgG solution was buffer



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exchanged to citrate buffer (20 mM containing 100 mM NaCl, pH 6) using a 10-kDa

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cutoff filter to yield a final concentration of 1 µg/µL. The solution was heated to 65

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°C for 30 min and 5 h, respectively, and then cooled to room temperature.

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Dimethyl labeling on intact IgGs. Native or denatured human IgG (65 µg) was dissolved in 100 µL sodium acetate buffer (100 mM, pH 5-6) and then added with 20 µL of formaldehyde-d2 (4% in water). After vortexing for 5 min, the protein solution was added with 20µL of NaBH3CN dissolved in ddH2O to yield a final

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concentration of 1.4, 14, 28, 42, or 85 mM, respectively. The solution was incubated

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at room temperature for 30 s or 2 h with occasional mixings and then quenched by addition of 20 µL of ammonium hydroxide (7 % in ddH2O) to consume excess

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formaldehyde-d2. The thermal-stressed solutions (100 µL) were buffer exchanged to sodium acetate buffer (100 mM, pH 5-6) and then labeled with formaldehyde via the reduction of NaBH3CN (14 mM) for 30 s, following the same procedure described above.

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Intact protein measurement. The labeled protein solution was buffer exchanged to ammonia bicarbonate (50 mM NH4HCO3, pH 8) using a 10-kDa cutoff

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filter to yield a final concentration of 1 µg/µL and then reduced by DTT (10 mM) at

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37 °C for 1 hr. The solution was cooled to room temperature, added with PNGase F (enzyme:protein ratio of 1: 100), and incubated at 37 °C for 18 h. The resulting solution containing IgG heavy and light chain was diluted with 0.2% FA to a final

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concentration of 0.1 µg/µL.

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A volume of 9.8 µL of the diluted solution was injected onto Waters ACQUITY UPLC system equipped with a size exclusion column (ACQUITY UPLC BEH200,SEC 1.7 um,2.1 x150 mm Waters Corporation, Milford, MA, USA) which was online-coupled to a Xevo™ G2S QTof instrument (Waters Corporation, Milford,

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MA, USA). The IgG heavy and light chain were eluted by the mobile phase containing 10 %ACN in 0.1% FA at a flowrate of 0.3 mL/min. The column temperature was maintained at 30°C; the desolvation gas and the source temperature were set to 450 °C and 150 °C, respectively. The capillary and the cone voltage was

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set at 3kV and 40 V, respectively, and the data (m/z 800-2500) were collected by MassLynx 4.1 software. The acquired multiple charge profiles were deconvoluted using MaxEnt 1 algorithm. The number of labeled methyl group was determined based on the band maxima (heavy chain) or the weighted average of the payloads

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(light chain): Σi (relative peak area (%)i × number of methyl group).

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Bottom-up proteomics method. The labeled and reduced protein solution described above was alkylated by iodoacetamide (10 mM) in the dark for 30 min with

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occasional mixings, followed by addition of PNGase-F (enzyme:protein ratio of 1:


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100) to remove N-glycans at Fc-IgG. Then, the solution was divided into two for the

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digestion of chymotrypsin (enzyme:protein ratio of 1:25) or Glu-C (enzyme:protein

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ratio of 1:40), respectively, at 37 °C for 16 h. After digestion, the solution was dried by a freeze dryer, and then re-dissolved in ddH2O containing 0.2 %FA.

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A volume of 1µL of the resulting solution was injected onto Waters nanoACQUITY UPLC system equipped with a pre-column (Waters, 0.180 mm ×20 mm, 5 µm C18) followed by a nanocolumn (Waters, 75 µm × 25 cm, 1.7 µm C18) in

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series coupled online to an LTQ-Orbitrap XL mass spectrometer (Thermo Fisher

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Scientific, San Jose, CA, USA). Mobile phase A was 0.1 % FA in water, while mobile phase B was 0.1 % FA in ACN. The elution program consisted of an isocratic elution

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at 5 % B for 5min, a linear gradient from 5 % to 40% B over 35 min followed by another linear gradient from 40 % to 90 % B over 5 min, and finally an isocratic elution at 90 % B for 5 min. The flow rate was maintained at 0.3 µL/min. The LTQ-Orbitrap XL mass spectrometer was operated as the followings: survey full-scan MS spectra (m/z 300-2000) were acquired in the Orbitrap with a mass resolution of 60,000 at m/z 400 (with an ion target value of 5 × 105 ions). The five most intense

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peaks with charge state ≥ 2 were selected for sequencing and fragmented in the ion trap with normalized collision energy of 35%, activation q = 0.25, activation time of 30 ms, and one microscan. The target value was 1× 104. The ion selection threshold

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was 5000 counts, and the maximum allowed ion accumulation times were 500 ms for full scans and 30 ms for CID. For the identification of peptide sequence and the labeling sites, the acquired raw data were searched by PEAKS Studio 7.5 (Bioinformatics Solutions Inc., Waterloo, Ontario, Canada) using de-novo search function as well as the database search function against the theoretical sequence of Avastin. The following parameters were

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used: a mass tolerance of 5 ppm for the precursor ion and 0.8 Da for the product ion; Dimethyl:2H(4) (K), Dimethyl:2H(4) (N-term) as the variable modification; carbamidomethyl-cysteine as the fixed modification; and one allowable miscleavage. Only sequences identified with a probability score > 20 (P 20 for the labeling site were counted.

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Moreover, each identified sequences were further confirmed by manual extractions. The labeling percentage of a specific site was calculated by dividing the peak area of extracted ion chromatograms (XICs) derived from all labeled peptides containing a specific labeled site divided by the sum of the peak area of the corresponding un-labeled and labeled peptides.

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Circular dichroism (CD) Spectroscopy. Far-UV CD spectra were recorded at 25 °C on a JASCO J-815 spectropolarimeter (JASCO Inc., Japan) equipped with a temperature-controlling liquid system. The spectra ranging from 200–260 nm were



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collected using cuvettes with a 1-mm path length, 0.1-nm resolution, 1.0-s response

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time, and 100 nm/min scanning speed. All the measurements were performed under a

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nitrogen flow. Each scan was repeated ten times to obtain an averaged value. The results were expressed as mean residue ellipticities [θ] in units of degrees cm2 dmol-1,

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which is defined as [θ] = 10θobs (lc)-1, where θobs is the observed ellipticity in degrees, c is the concentration in moles per liter, and l is the length of the light path in

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centimeters.

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Trp fluorescence measurements. Both the un-labeled and labeled IgG solutions were prepared in Tris buffer (20 mM, pH 7.5) with a final concentration of 0.6 µΜ.

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Their fluorescence spectra ranging from 305 to 500 nm were acquired by a fluorescence spectrophotometer JASCO FP-6500 (JASCO Corporation, Tokyo, Japan) with an excitation wavelength of 295 nm.

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Dynamic light scattering (DLS). The native or heated IgG solutions were diluted with ddH2O to suitable concentrations for DLS measurement. A volume of 1.5

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mL of the native or stressed IgG solution (0.25-1 µg/µL) was placed into the sample compartment of a Zetasizer Nano‐ZS90 system (Malvern Instruments, Worcestershire,

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U.K.). The hydrodynamic radii of IgG molecules was obtained based on the average of at least 4 repeated measurements.

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Molecular modeling. Molecular models of IgG (Avastin) were constructed using the SWISS MODEL protein modeling server, based on an X-ray crystal structure of human IgG (PDB code:1IGT). Energy minimization was carried out in Discovery Studio 2.5 (Accelrys, Burlington, MA, USA) using the CHARMM force field in LibDock. The SASA values were calculated and displayed using PyMol software (http://www.pymol.org/).

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Results and discussions

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reaction course of dimethyl labeling of intact human IgG was monitored using excess labeling reagents (156 mM formaldehyde and 85 mM NaBH3CN) on quadrupole-time of flight (Q-TOF) LC/MS. The reaction was quenched by adding ammonium

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hydroxide (NH4OH) at intervals of 20, 40, 60, 100, 300 and1000 s, after the reaction started. Multiple-charge precursor ion profiles of both the heavy and light chains of the IgG molecule were detected. As shown in Figure 1B, the deconvoluted mass spectra

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revealed a broad parabolic mass profile (23.6–25 kDa) for the light chain, containing

Labeling yield investigated using intact protein measurement. To investigate whether dimethyl labeling via reductive amination can achieve a high labeling yield for intact proteins, similar to that for peptides used in quantitative proteomics,28 the


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multiple bands (different payloads) with a 16-Da (methyl group tagged by one

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formaldehyde molecule) interval in between. This indicated that either some sites were not labeled or were only labeled with one formaldehyde molecule (methylation),

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resulting in a heterogeneous distribution of IgG labeled with different payloads during the early phase of the reaction. However, the mass spectral peak width decreased with time. After 1000 s of the reaction, a relatively sharp peak with a mass shift

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corresponding to practically complete (14 sites for the light chain) dimethyl labeling

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was detected, indicating a decrease in heterogeneity as the reaction proceeded towards completeness. The same reaction kinetics was observed for the heavy chain

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(corresponding to 33 sites). As shown in Figure 1B (right), a broad band was detected at 20, 40, 60, 100, 300 and 1000 s, with increasing mass shift and decreasing peak

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width. Resolution of the mass spectrometer was not sufficient to resolve different masses under the broad band of the heavy chain. Like the light chain, however, a highly heterogeneous payload profile associated with a broad peak was observed during the early phase (20 s) of the reaction, which became less heterogeneous, displaying relatively narrower peaks, as the reaction approached completeness. A relatively sharp peak with mass shift corresponding to 33 sites of dimethyl labeling was detected, indicating complete dimethyl labeling at 1000 s. These results confirm that complete dimethyl labeling can be achieved for intact proteins, as long as the reaction time is sufficient. As seen in Figure 1C, the number of methyl groups incorporated increased exponentially with the reaction time and reached a plateau after 1000 s (or 17 min) of

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the reaction, which was much longer than that for peptides 300 s (or 5 min)).14 Control experiments with the denatured IgG standard, which is lack of 3D structure, was also conducted. In principle, the amine group was expected to become more

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exposed and its solvent accessibility becomes in-distinguishable among different sites as an IgG molecule is denatured. As shown, the denatured IgG standard was completely labeled at about 300 s (or 5 min) after the reaction which is comparable to that for a peptide14 but much quicker than 1000 s (or 17 min) for a native IgG molecule. Moreover, as shown in Figure 1D (see results below for the bottom-up

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approach), the labeling percentage among different lysine sites became in-distinguishable for the denatured IgG standard as compared to that for the native one. Once all the lysine sites become equally accessible by denaturing, the labeled percentages are in-distinguishable, indicating the labeling is controlled by the

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accessibility of individual sites rather than reactivity of the labeling. These results indicate that dimethyl labeling is able to dictate the protein conformation or the surface exposure of primary amines via monitoring the labeled IgGs and the distribution of the labeled sites.



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Conformational changes induced by labeling. The effect of dimethyl labeling on

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the conformation and structure of a protein was examined by UV-circular dichroism (CD) and Trp fluorescence spectroscopy.29 Because the maximum wavelength of tryptophan was about 280-300 nm for excitation and 300-350 nm for emission

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depending on the polarity of the local environment, we have chosen an excitation wavelength of 295 nm and emission ranging from 305 to 500 nm for this investigation. As shown in Figure 2A, UV-CD of the completely dimethylated IgG molecule

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showed comparable ellipticity with that of the unlabeled IgG, ranging from 200 to 260 nm, which indicates that the secondary structure of IgG is conserved even after

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labeling. Moreover, as shown in Figure 2B, the intrinsic fluorescence spectrum of the completely dimethylated IgG molecule showed an emission profile (peak maxima at

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334 nm) comparable with that of the unlabeled IgG (peak maxima at 333 nm), ranging from 310 to 460 nm, which indicates the exposure of tryptophan residues. This indicates that the higher order structure of IgG molecules was also conserved after labeling. Labeled lysine sites revealed by bottom-up proteomics approach. Since our aim was to reveal different solvent accessibilities of different sites of lysine residues or N-termini, the reaction was carried out for 30 s. Based on the results shown in Figure 1B, about 80% labeling was achieved after 30 s by using excess labeling reagents. In order to access the early phase of the reaction, the concentration of NaBH3CN reducing agent was adjusted, ranging between 1.4 to 85 mM, while keeping the concentration of formaldehyde in excess. In addition, a solution treated with 85 mM NaBH3CN for 2 h, which was shown to yield complete labeling (Figure 1B), was used

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as control for side-by-side comparison. All labeled samples were digested with chymotrypsin or GluC and subjected to further analysis by the bottom-up technique.

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As shown in Figure 3, the XICs (left) as well as the corresponding MS spectra (right) of the unlabeled, methylated, and dimethylated peptides of the same backbone were identified based on MS/MS sequencing (Supplementary Figure S1). The unlabeled peptide eluted slightly earlier than the labeled ones. Although the methylated and dimethylated peptides co-eluted, they were differentiated based on their MS or

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MS/MS spectra (Figure S1). We noticed that a majority of the labeled peptides was dimethylated, and only a few methylated peptides were identified from the samples treated with low concentrations of the NaBH3CN reagent. As shown in Figure 3, the

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signal intensity of the labeled peptide increased at the expense of the unlabeled peptide, as the NaBH3CN concentration increased. At a concentration of 85 mM, almost complete dimethyl labeling was observed after 30 s, and complete labeling was observed after 2 h, which are consistent with those observed via intact protein

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measurements (Figure 1).


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We used a peptide score of 20 as a filter for the sequence identification and A

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score of 20 as the filter to locate labeling sites on the identified peptide sequence. As

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shown in Supplementary Table S1, a nearly full coverage of the IgG sequence was achieved for the sample with high NaBH3CN concentration using chymotrypsin digestion alone, except K453 of the heavy chain, which was identified by GluC

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digestion. As shown, each lysine residue was covered by one or more identified peptides. The labeling (methylated or dimethylated) percentage was calculated based

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on the average of three repeated measurements. A map of the reaction kinetics, which represents the primary amine (mainly Lys residue) surface topography, is shown in Table 1. The labeling percentage of a majority of Lys residues under different reaction

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times was successfully determined, except some lysine sites such as K211–K224 and K326–340, which were not identified in the solution with low NaBH3CN

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concentrations. This is likely due to heterogeneous labeling of multiple Lys sites on a single peptide, since it is hard to resolve multiple masses associated with the same peptide backbone without sufficient fragment ions in the MS/MS spectra. For example, five Lys sites (K211-K224) were present in the peptide backbone of

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ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL, and six Lys sites (K326-340) were present in the peptide backbone of KCKVSNKALPAPIEKTISKAKGQPREPQVY. Only their fully labeled peptide ions were unambiguously identified from the samples treated with high NaBH3CN concentration (85 mM). As shown in Table 1, some highly exposed sites such as the N-termini of both

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the heavy and the light chains were almost completely labeled under low NaBH3CN concentrations, indicating that these sites have a high solvent accessibility and faster reaction kinetics. According to molecular modeling analysis shown in Figure 4, the

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N-termini had the greatest SASA for the heavy chain (98 Å2, 93 %), as well as for the light chain (105 Å2, 100 %), which is consistent with our results. Likewise, the data also revealed some highly buried sites including K415, which had the lowest labeling percentage among the heavy chain residues and K39, which had the lowest labeling percentage among the light chain residues (Table 1). Consistent with our experimental

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findings, the lowest SASA value was for K415 (2.7 Å2, 2.5%) for the heavy chain, and K39 (34 Å2, 32%) for the light chain, according to the modeling studies (Figure 4). In earlier studies,12 the most exposed N-termini of both the heavy and the light chain,

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as well as the least exposed K415 residue of the heavy chain, were not identified by TMT labeling. Nevertheless, consistent with the results of dimethyl labeling, TMT labeling also identified K39 of the light chain to have the lowest solvent accessibility.12 There is, however, an exception between the experimental and the

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modeling results. As indicated in Table 1, the K98 residue of the heavy chain was



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identified to have a high solvent accessibility by both the dimethyl labeling (Table 1)

2 3 4

and the TMT labeling.12 In contrast, molecular modeling indicated a small SASA for the K98 site on the heavy chain (10 Å2, 10%) (Figure 4). Based on the bottom-up analysis shown in Figure 1D, the labeling percentage of Lys 98 is in-distinguishable to

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those of other sites of the denatured standard in contrast to that of the native one, indicating the high labeling percentage of Lys98 was due to its surface exposure rather than selective reactivity. Therefore, such in-consistency between experimental result

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and the modeling is likely due to discrepancies of the modeling rather than reactivity of the labeling. For example, the solvent molecules involved in salt bridge formation

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may not be fully considered in the modeling and needs to be investigated further.

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Effect of thermal stress-induced aggregation on solvent accessibility. Aggregation of monoclonal antibodies (mAbs) by thermal or other types of stress is a

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common yet poorly understood phenomenon in the field of therapeutic development.30-32 Aggregation of proteins is a key hindrance to formulation and stability assessments, with a recent interest in the 0.1–1 µm aggregate size range, identified as an area of concern for immunogenicity, and hence, also for product safety. We applied dimethyl labeling to monitor the changes in Lys surface topography of IgG during aggregation. For thermally induced aggregation study, incubation temperature (65 °C) of a few degrees below the unfolding point of the least stable CH2 domain (70.1 °C) was used to retain a major proportion of the structure in native state during antibody aggregation. The growth of the IgG aggregate was first monitored by the naked eye, followed by DLS. As shown in Supplementary Figure S2,

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IgG aggregation induced by thermal stress (65 °C) was not visible at the 30-min time point, but the protein size was observed to increase from 10 to 30 nm by DLS, indicating the formation of protein oligomers in response to thermal stress. As the heating time increased to 5 h, protein aggregates were clearly visible with the naked eye near the bottom of the vial, and the size of the aggregates was measured to be greater than 200 nm by DLS.

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Dimethyl labeling was also applied to IgG samples heated to 65 °C for 30 min and 5 h. The labeling percentage of each Lys site on the IgG molecule was calculated

31 32 33

based on three repeated measurements and compared with those obtained without applying thermal stress. As shown in Figure 5, substantial changes in the labeling percentage were observed by applying thermal stress, indicating that elevated

34 35 36 37

temperatures triggered non-native aggregation. This is consistent with previous reports based on HDX patterns.20 As shown in Figure 5, the labeling percentage of most lysine sites increased upon heating for 30 min, decreased as the heating time increased to 5 h, suggesting the formation of soluble oligomers of unfolded IgG

38

formed by thermal stress at 30 min, and then accumulated at an intermediate time


Page 10 of 23

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1

interval before converting to large insoluble aggregates at 5 h. Such thermal

2 3

stress-induced unfolding is consistent with previous observations based on Trp fluorescence and ANS binding measurements20 in which, the thermal-stressed IgG

4 5 6

sample formed by incubation at 70 °C for 10 min exhibited a more unfolded structure than that of the fully unfolded control containing 6 M guanidine HCl. These results suggest that enhanced protein unfolding was triggered by the thermal stress, which

7

resulted in increased labeling or residue exposure. This unfolding was then followed

8 9 10 11

by protein aggregation possibly via the unfolded surface, resulting in decreased labeling. Our data also showed that dimethyl labeling is complementary to the HDX approach in resolving structural changes induced by thermal stress. In previous

12 13 14 15 16 17 18 19 20 21 22 23

studies,20 the Fab fragment was identified to be involved in the intermolecular interactions critical for protein aggregation. Consistent with this observation, in our study, the labeling percentage of most lysine sites in the Fab region changed substantially by thermal stress (Figure 5). In contrast, the HDX pattern of the Fc portion did not change substantially by thermal stress, except the C-terminal region (430–452). However, substantial changes in the labeling percentage of lysine residues in the Fc region, including the C-terminal region (430–452), were detected by dimethyl labeling (Figure 5). We propose that such discrepancies observed for the Fc region could be due to differences in solvent accessibilities between the backbone (probed by HDX pattern) and the side chain residues (probed by dimethyl labeling). On the other hand, the lack of detection of many reporter peptides in the CH2 domain of the Fc region, such as 284–312, 324–254, and 405–410, by the HDX method20

24 25

could have posed uncovered information. In contrast, there are complementarity-determining regions (CDRs) observed to involve in thermal

26 27 28 29 30

stress-induced aggregation by HDX approach but such phenomena could not be probed by dimethyl labeling due to the lack of lysine residues in the CDRs. This shows that the higher order structure of proteins can be better revealed by combining both HDX and dimethyl labeling approach with LC-MS. It is worth mentioning that dimethyl labeling is superior to HDX approach in the

31 32 33 34

experimental side because dimethyl labeling is covalent and irreversible with high labeling yield (near 100%). Thus, the reaction kinetics can be well controlled and studied by monitoring the products during the reaction course (either by changing the reaction time or concentration of the reactants). In contrast, HDX using non-covalent

35 36 37

bonding suffers from back exchange and scrambling during CID. Thus, dimethyl labeling can be easily identified by MS2 using CID. Potential artifacts are anticipated for HDX approach in the identification of the labeled protein backbone.

38



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Conclusion

1 2 3 4

Because dimethyl labeling is mainly governed by solvent accessibility rather than reactivity, the labeling method yielded a comprehensive topographical map of the primary amines on human IgG, including highly exposed and highly buried sites,

5 6

without altering the protein conformation. In addition, dimethyl labeling was also shown to supplement the HDX approach in revealing structural changes caused by

7

protein aggregation induced by thermal stress. In view of the robustness, simplicity,

8 9

specificity as well as an almost complete labeling yield for intact proteins, dimethyl labeling coupled with LC-MS is a highly promising method for probing the primary

10 11 12

amine or the lysine surface topography of a protein. In the same context, dimethyl labeling is also an excellent complementary approach to other methods for resolving the higher order structure of proteins by LC-MS. We believe this method is applicable

13 14 15

for therapeutic proteins and its applications for ADCs remain to be examined.

16 17 18 19

Acknowledgement This work was supported by Ministry of Science and Technology, Taiwan, Republic of China (NSC 102-2113-M-006-005-MY3).

20 21 22

Supporting Information Available: Supplement information containing Figure S1. LC-MS/MS spectra; Figure S2. Dynamic light scattering for native IgG (top) or thermal stressed-IgG; and Table S1. Peptides of human IgG (Avastin) identified by the

23 24

digestion of chymotrypsin or GluC is available free of charge via the Internet at http://pubs.acs.org

25

.

26 27 28 29

References (1) Niemeyer, C. M. Angew Chem Int Ed Engl 2010, 49, 1200-1216. (2) Medintz, I. L.; Uyeda, H. T.; Goldman, E. R.; Mattoussi, H. Nat Mater 2005, 4, 435-446.

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(3) Zhao, Y.; Sakai, F.; Su, L.; Liu, Y.; Wei, K.; Chen, G.; Jiang, M. Adv Mater 2013, 25, 5215-5256.

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(4) Chudasama, V.; Maruani, A.; Caddick, S. Nat Chem 2016, 8, 113-118. (5) Gordon, M. R.; Canakci, M.; Li, L. Y.; Zhuang, J. M.; Osborne, B.;

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Thayumanavan, S. Bioconjug Chem 2015, 26, 2198-2215. (6) Strop, P.; Liu, S. H.; Dorywalska, M.; Delaria, K.; Dushin, R. G.; Tran, T. T.; Ho, W. H.; Farias, S.; Casas, M. G.; Abdiche, Y.; Zhou, D.; Chandrasekaran, R.; Samain, C.; Loo, C.; Rossi, A.; Rickert, M.; Krimm, S.; Wong, T.; Chin, S. M.; Yu, J.; Dilley, J.;

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Shelton, D. L.; Rajpal, A. Chem Biol 2013, 20, 161-167.

2 3

(7) Behrens, C. R.; Liu, B. Mabs-Austin 2014, 6, 46-53. (8) Scholten, A.; Visser, N. F. C.; van den Heuvel, R. H. H.; Heck, A. J. R. J Am Chem

4 5 6

Soc 2006, 17, 983-994. (9) Sochaj, A. M.; Swiderska, K. W.; Otlewski, J. Biotechnol Adv 2015, 33, 775-784. (10) Kvaratskhelia, M.; Miller, J. T.; Budihas, S. R.; Pannell, L. K.; Le Grice, S. F. J.

7 8

P Natl Acad Sci USA 2002, 99, 15988-15993. (11) Hung, C. W.; Tholey, A. Anal Chem 2012, 84, 161-170.

9 10

(12) Gautier, V.; Boumeester, A. J.; Lossl, P.; Heck, A. J. Proteomics 2015, 15, 2756-2765.

11

(13) Zhou, Y.; Vachet, R. W. Anal Chem 2013, 85, 9664-9670.

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(14) Hsu, J. L.; Huang, S. Y.; Chow, N. H.; Chen, S. H. Anal Chem 2003, 75, 6843-6852. (15) Boersema, P. J.; Raijmakers, R.; Lemeer, S.; Mohammed, S.; Heck, A. J. R. Nat

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Protoc 2009, 4, 484-494. (16) Zhou, Y. P.; Vachet, R. W. Anal Chem 2013, 85, 9664-9670. (17) Huang, S. Y.; Chen, S. F.; Chen, C. H.; Huang, H. W.; Wu, W. G.; Sung, W. C.

18 19

Anal Chem 2014, 86, 8742-8750. (18) Xu, G. H.; Chance, M. R. Chem Rev 2007, 107, 3514-3543.

20 21 22

(19) Rand, K. D.; Zehl, M.; Jorgensen, T. J. D. Accounts Chem Res 2014, 47, 3018-3027. (20) Zhang, A.; Singh, S. K.; Shirts, M. R.; Kumar, S.; Fernandez, E. J. Pharm

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Res-Dordr 2012, 29, 236-250.

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(21) Majumdar, R.; Middaugh, C. R.; Weis, D. D.; Volkin, D. B. J Pharm Sci-Us 2015, 104, 327-345.

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(22) Zhang, A.; Hu, P.; MacGregor, P.; Xue, Y.; Fan, H.; Suchecki, P.; Olszewski, L.;

27 28

Liu, A. Anal Chem 2014, 86, 3468-3475. (23) Borotto, N. B.; Zhou, Y.; Hollingsworth, S. R.; Hale, J. E.; Graban, E. M.;

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Vaughan, R. C.; Vachet, R. W. Anal Chem 2015, 87, 10627-10634.

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(24) Mendoza, V. L.; Vachet, R. W. Anal Chem 2008, 80, 2895-2904. (25) Mendoza, V. L.; Vachet, R. W. Mass Spectrom Rev 2009, 28, 785-815. (26) Deperalta, G.; Alvarez, M.; Bechtel, C.; Dong, K.; McDonald, R.; Ling, V.

33 34 35 36

Mabs-Austin 2013, 5, 86-101. (27) Maleknia, S. D.; Downard, K. M. Chem Soc Rev 2014, 43, 3244-3258. (28) Shen, P. T.; Hsu, J. L.; Chen, S. H. Anal Chem 2007, 79, 9520-9530. (29) Chattopadhyay, A.; Rawat, S. S.; Kelkar, D. A.; Ray, S.; Chakrabarti, A. Protein

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1

Chromatogr B Analyt Technol Biomed Life Sci 2016, 1032, 189-197.

2

(31) Wang, W.; Nema, S.; Teagarden, D. Int J Pharm

3 4

(32) Sahin, E.; Grillo, A. O.; Perkins, M. D.; Roberts, C. J. J Pharm Sci 2010, 99, 4830-4848.

2010, 390, 89-99.

5 6


Page 15 of 23

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1 2

Figure legends

3

Theoretical sequence of the human IgG (Avastin) (B) Deconvoluted intact mass

4

spectra of the native IgG light chain (left) and heavy chain (right) measured at 0, 20,

5

40, 60, 100, 300 and 1000 s (top to bottom) after performing the reaction using excess

6

labeling reagents (156 mM formaldehyde and 85 mM NaBH3CN ) (C) Plot of labeling

7

percentage versus reaction time for the native (diamond) and denatured (square)

8

heavy and light chain of IgGs based on the average of three repeated measurements

9

and the error bar indicates the standard deviation (n=3). The dashed lines indicate the

10

theoretical numbers based on the sequence. (D) The labeling percentage of different

11

lysine sites on the native (diamond) and denatured (square) heavy and light chain of

12

IgGs.

Figure 1. Dimethyl labeling of intact native and the denatured IgG standards. (A)

13

14



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1 2 3


Page 16 of 23

Page 17 of 23

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1

2 3 4



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1 2

Figure 2. Structural changes in IgG molecules before (solid line) and after (dashed

3

line) dimethyl labeling, probed by (A) CD spectroscopy and (B) Trp fluorescence

4

(spectra recorded at 25 °C with an excitation wavelength of 295 nm and emission

5

ranging from 305 to 500 nm.)

6

7 8 9 10


Page 18 of 23

Page 19 of 23

1

Figure 3. Extracted ion chromatograms of the unlabeled and labeled-peptide ions of

2

the same backbone (LNNFYPREAKVQW) measured for the solutions treated with

3

156 mM formaldehyde followed by NaBH3CN concentration of 1.4, 14, 28, 42, 85

4

mM

5

spectra of the unlabeled, methylated, and dimethylated ions are shown in the right.

for 30 s and 85 mM NaBH3CN for 2 h (bottom to top). The corresponding mass

labeled (dimethylated or methylated) peptides 848.9598 z=2

100

35.10

+32.0572 Da

90

35.08

80

+16.0286 Da

70

34.9135.03

Relative Abundance

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


34.91

34.89

60 840.9461 z=2

50 40 832.9319 z=2

30

34.91

20

free peptide 10

33.5 34.0 34.5 35.0 35.5 36.0 36.5 Time (min)

0 830

835

840

845

850

855

m/z

6 7 8 9 10

Figure 4. Molecular model and the solvent-accessible surface area (SASA) values of

11

IgG molecule (Avastin). The highly exposed sites (N-termini of the heavy and the

12

light chain, K98 of the heavy chain) are indicated in red. The most buried sites (K415

13

and K39 of the heavy and light chain, respectively) are indicated in blue.

H_Lys98 10 A2

L_Lys39 34 A2

90o

L_Asp1 105 A2 H_Glu1 2 98 A

H_Lys415 2.7 A2

14 15 16



1

Figure 5. Effect of thermal stress on labeling percentage of the N-termini and lysine

2

residues on the light chain (top) and the heavy chain (bottom) of native IgG ( ),

3 4

thermal-stressed IgG by heating at 65 °C for 30 min ( ) or 5 h ( ). The data were the average of the labeling percentage calculated based on three repeated measurements

5

of the reaction using 156 mM formaldehyde and 14 mM NaBH3CN. The error bar was

6

the standard deviation. Residues covered by Fab or Fc domain were circled. Light Chain

Fab-VL

120%

Modification

100% 80% 60% 40% 20%

K207

K190

K188

K183

K169

K149

K145

K126

K107

K103

K45

K42

N-term

K39

0%

Residue

Heavy Chain Fc-CH2

Fab-VL/CH1

Fc-CH3

120% 100%

Modification

80% 60% 40% 20%

Residue

7 8 9 10


K445

K420

K415

K398

K376

K366

K346

K344

K340

K332

K328

K326

K323

K296

K294

K280

K254

K252

K153

K139

K127

K98

K76

K43

0% -20%

N-term

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 23

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1

Table 1. The labeling percentage of each amine sites on the light or the heavy chain of

2

human IgG under the reduction of different concentrations (1.4 - 85mM) of

3

NaBH3CN for 30 s or 85 mM for 2 h. N/D means peptides covering these sites were

4

not identified based on the thresholds for database search. The data were also plotted

5

in Figure 5 for native IgG with standard deviations (n=3) indicated as error bar.

6

Site

1.4mM

14mM

28mM

42mM

85mM

85mM_2h

Light chain N-term

52.60%

99.80%

100.00%

100.00%

100.00%

100.00%

K39

13.40%

28.40%

20.40%

40.70%

99.00%

100.00%

K42

13.40%

35.40%

39.50%

41.40%

99.00%

100.00%

K45

40.60%

78.40%

56.70%

56.80%

99.00%

100.00%

K103

28.00%

24.10%

50.00%

52.00%

100.00%

100.00%

K107

17.00%

25.90%

44.80%

54.90%

100.00%

100.00%

K126

37.70%

69.50%

60.30%

67.30%

100.00%

100.00%

K145

36.10%

55.60%

64.60%

72.90%

100.00%

100.00%

K149

70.60%

95.80%

95.00%

75.90%

100.00%

100.00%

K169

45.40%

57.40%

68.20%

62.60%

96.70%

100.00%

K183

16.90%

33.30%

30.80%

39.30%

100.00%

100.00%

K188

38.50%

74.60%

60.90%

77.10%

100.00%

100.00%

K190

55.30%

98.90%

87.00%

93.10%

100.00%

100.00%

K207

75.90%

87.40%

83.30%

74.80%

100.00%

100.00%

Heavy Chain N-term

91.70%

100.00%

100.00%

100.00%

100.00%

100.00%

K43

27.80%

45.40%

49.50%

57.00%

99.80%

100.00%

K65

70.30%

90.70%

91.10%

92.80%

100.00%

100.00%

K76

43.60%

61.10%

62.50%

72.10%

99.60%

100.00%

K98

98.10%

100.00%

100.00%

100.00%

99.60%

100.00%

K127

20.20%

40.60%

55.20%

77.20%

100.00%

100.00%

K139

39.30%

58.00%

77.00%

71.90%

97.20%

100.00%

K153

53.80%

88.80%

88.00%

84.60%

99.80%

100.00%

100.00%

100.00%

100.00%

100.00%

100.00%

100.00%

K211 K216 K219

N/D



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1 2

Page 22 of 23

K220

100.00%

100.00%

K224

100.00%

100.00%

K228

2.60%

40.20%

70.50%

32.30%

100.00%

100.00%

K252

19.90%

68.10%

62.20%

85.00%

100.00%

100.00%

K254

19.90%

69.30%

66.70%

86.60%

100.00%

100.00%

K280

44.80%

68.80%

63.70%

77.70%

100.00%

100.00%

K294

57.20%

79.90%

81.00%

80.50%

100.00%

100.00%

K296

64.10%

83.90%

91.30%

88.80%

100.00%

100.00%

K323

18.90%

37.10%

38.10%

46.00%

99.10%

100.00%

K326

0.00%

96.20%

N/D

100.00%

100.00%

K328

0.10%

95.10%

100.00%

100.00%

K332

17.90%

43.10%

42.60%

100.00%

100.00%

K340

9.10%

72.40%

68.10%

100.00%

100.00%

K344

0.00%

6.90%

52.60%

44.70%

100.00%

100.00%

K346

0.00%

2.40%

5.40%

16.80%

100.00%

100.00%

K366

53.70%

81.20%

58.40%

79.20%

100.00%

100.00%

K376

45.60%

50.20%

51.10%

64.10%

78.20%

100.00%

K398

61.00%

63.70%

61.80%

79.00%

100.00%

100.00%

K415

0.00%

13.60%

30.80%

26.10%

91.90%

100.00%

K420

27.10%

47.40%

49.40%

60.10%

99.90%

100.00%

K445

0.00%

26.20%

40.70%

46.70%

93.90%

100.00%

K453

38.00%

83.00%

81.50%

95.90%

100.00%

100.00%

0

N/D

100%

3 4 5


Page 23 of 23

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

TOC

2 3

4 5


Dimethyl Labeling Coupled with Mass Spectrometry for Topographical

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