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INTRODUCTION

Characterization of post-translational modifications, degradation products and

sequence variants in biotherapeutics is essential to guarantee purity and efficacy.

Top-down strategies are appealing for assessment of the abundance of

modifications that may exist independently or in tandem.

Collisional activation is the most widely used fragmentation technique and is

available on virtually all MS platforms; however, the obtainable sequence coverage

by CID in top-down applications is known to suffer for a number of reasons,

including limited proton mobility in intact proteins.

Hydrophilic interaction chromatography (HILIC) has emerged as a powerful

separation strategy, and has been demonstrated to separate protein glycoforms and

deamidation in peptides.1,2

Data independent fragmentation strategies afford a higher duty cycle than data

dependent approaches and can provide fragmentation data for all species in a single

acquisition. This strategy is particularly appealing for low abundance components for

which targeted and data dependent strategies are challenging.

DATA INDEPENDENT TOP-DOWN CHARACTERIZATION OF PROTEINS FOR BIOTHERAPEUTIC APPLICATIONS

Lindsay J. Morrison, Brad J. Williams, and Barbara J. Sullivan Waters Corporation, Beverly, MA 01915

METHODS CONTINUED

MSE, SONAR, and targeted MS/MS experiments were performed on a Waters Xevo

G2-XS Q-TOF platform. Proteins were analyzed by MSE

and in SONAR by using a fixed collision voltage of 22 eV. The 14+ charge state was selected for the targeted experiments in order to reduce the charge state of the observed fragment ions and improve fragment detection as collisional activation of lower charge states of myoglobin has been previously shown to improve sequence coverage.

3 HDMS

E

experiments were performed on a Waters SYNAPT G2-Si using a wideband enhancement (WBE) acquisition mode.

References

MSE

CONCLUSIONS

HDMSE CONTINUED

HILIC CHROMATOGRAPHY

HDMSE

Figure 4. Experimental design for top down IM-DIA strategy utilizing a charge-state based IMS wideband enhancement (WBE). (A) The entire charge state envelope of the protein is collisional-ly activated in the Trap region prior to ion mobility separation. The resulting fragment ions are then separated by IMS resulting in regions of the (B) Drift Time vs. m/z plot that separate into singly and multiply charged fragment ion series (denoted with white lines). The wideband en-hancement occurs through synchronization of the IMS drift elutions with the TOF pusher (A, bottom panel). (C) Two wideband enhancement calibrations (white lines in Panel B) were ap-plied to the top down IM-DIA analysis of myoglobin sequence variants. An example of a multiply charged wideband enhancement is shown for a MYG_HORSE fragment ion y28

4+ (772.1550 m/z) with a 5.3 fold signal improvement.

Figure 2. (A) RPLC (C4) separation of neat MYG_HORSE, MYG_PIG, and the binary mixture, indi-

cating co-elution. (B) HILIC separation of the same sample set which shows clear separation of

HORSE and PIG myoglobin. (C) Deconvoluted mass spectra of MYG_HORSE / MYG_PIG (Peaks i/ii

labeled in Panel B).

Table 1. Sequence and molecular weights of horse, pig, human, and canine myoglobin. Sequence

variant sites are highlighted in colored font.

METHODS

Pig, human, and canine myoglobin were obtained from Life Diagnostics, buffer

exchanged using a Millipore P6000 spin filter, and diluted to approximately 1 mg/mL in

H2O prior to use. Horse myoglobin was obtained from Sigma-Aldrich and prepared at 1

mg/mL. Low volumes (0.5-1.0 μL) were injected onto a 2.1 mm x 15 cm Waters 300 Å,

1.7 µm BEH amide column and HILIC chromatography was performed by ramping

solvent A (H2O with 0.1% formic acid (FA) and 0.02% trifluoroacetic acid (TFA)) from

22% A to 32% A (78% B to 68% B, ACN with 0.1% FA and 0.02% TFA) over 18 min.

Figure 1. (A) Comparison of HILIC and RPLC (C4) separation of myoglobin sequence variant mix-

ture using the same mobile phases and equivalent gradients. Note that MYG_HORSE (Peak i) and

MYG_PIG (Peak ii) co-elute in the RPLC separation. (B) Extracted corresponding mass spectrum

for each myoglobin sequence variant. (C) Expanded view of the +18 charge state (blue highlight-

ed region of Panel B).

SONAR

Figure 5. Singly charged wideband enhancement IM-DIA analysis of HILIC separated horse and porcine myoglobin (Figure 2). An expanded view of m/z 980-1010 with (+WBE) and without (-WBE) the singly charged wideband enhancement applied for (A) MYG_HORSE and (B) MYG_PIG. Note the b9 (m/z 1001.4237) from horse myoglobin which was previously not ob-served in top panel A. (B) Expanded view of the porcine myoglobin b9 (m/z 986.4613) fragment ion which confirms the identity of this sequence variant. (C) Extracted ion chromatograms for the b9 fragment ion for horse and porcine myoglobin with retention times at 14.79 and 16.02 min, respectively.

Figure 6. Infusion-based SONAR experiment using horse heart myoglobin. Panels A-C represent the scanning quadrupole mass range 750 – 920 m/z vs. Peak Intensity plots using quadrupole win-dows of: (A) 5 Da, (B) 10 Da, and (C) 20 Da. Species of Δ98 Da are partially resolved in (A), demon-strating the utility of SONAR for species that have similar m/z and that may not be chromato-graphically separable. Panels (D) and (E) show representative SONAR MS/MS spectra for the +21 and +20 charge state of myoglobin showing the effect of precursor charge state on the resulting fragmentation patterns. The respective insets highlight distinct differences in the fragmentation patterns, each generated using fixed collision energy of 22 eV.

OBJECTIVE

Evaluate the performance of three top-down DIA strategies (MSE, HDMS

E,

and SONAR) in combination with HILIC chromatography. Myoglobin, which has a high degree of sequence variation between species, is used as a model system to examine the extent to which sequence variants can be characterized by top-down methods.

SONAR is a novel DIA approach in which a scanning quadrupole is used convert MS data to a time domain in manner analogous to ion mobility, providing temporal separa-tion of species having different mass-to-charge ratios. In top-down experiments, this provides the opportunity to individually fragment different charge states to take ad-vantage of different fragmentation patterns. This approach trades sensitivity for selectiv-ity owing to the reduced duty cycle relative to MS

E.

Variant Sequence MW

(Mono)

HORSE GLSDG EWQQ1V LNV2WG KVEA3D I4A5GHG QEVLI RLFT6G7 HPETL E8KFDK FKHLK T9EA10EM KA11SED LKKHG T12V13VLT ALGGI LKKKG HHEAE LK14PLA QSHAT KHKIP I15KYLE FISD16A17 IIH18VL H19SKHP20 GDFG21A DA22Q23G24A MT25KAL ELFRN26 DI27AA28K29 YKELG FQG

16,941.0

PIG GLSDG EWQL1V LNV2WG KVEA3D V4A5GHG QEVLI RLFK6G7 HPETL E8KFDK FKHLK S9ED10EM KA11SED LKKHG N12T13VLT ALGGI LKKKG HHEAE LT14PLA QSHAT KHKIP V15KYLE FISE16A17 IIQ18VL Q19SKHP20 GDFG21A DA22Q23G24A MS25KAL ELFRN26 DM27AA28K29 YKELG FQG

16,942.9

HUMAN GLSDG EWQL1V LNV2WG KVEA3D I4P5GHG QEVLI RLFK6G7 HPETL E8KFDK FKHLK S9ED10EM KA11SED LKKHG A12T13VLT ALGGI LKKKG HHEAE IK14PLA QSHAT KHKIP V15KYLE FISE16C17 IIQ18VL Q19SKHP20 GDFG21A DA22Q23G24A MN25KAL ELFRK26 DM27AS28N29 YKELG FQG

17,041.9

CANINE GLSDG EWQI1V LNI2WG KVET3D L4A5GHG QEVLI RLFK6N7 HPETL D8KFDK FKHLK T9ED10EM KG11SED LKKHG N12T13VLT ALGGI LKKKG HHEAE LK14PLA QSHAT KHKIP V15KYLE FISD16A17 IIQ18VL Q19SKHS20 GDFH21A DT22E23A24A MK25KAL ELFRN26 DI27AA28K29 YKELG FQG

17,195.1

Figure 7. (A) Sequence coverage of the four myoglobin variants and (B) number of variant sites identified using HILIC LC-MS strategies. The criteria used for variant site identification was two adjacent fragment ions of the same b/y series flanking the site. Note that HDMSE data was col-lected on a SYNAPT G2-Si on a different day with slightly different sample preparation; hence the data is not directly comparable. SONAR data was collected over the 20+ and 21+ charge states and the spectra from both averaged prior to deconvolution. Sequence coverage maps for horse myoglobin (LC-MS) are shown in (C). The lower protein sequence coverage of the LC-MS SONAR experiment was postulated to be a result of the inherently lower duty cycle; consequently, tar-geted, MSE, and SONAR were performed on horse myoglobin using direct infusion to decouple the effect of signal. Sequence coverage and the number of variant sites identified by these meth-ods are shown in (D) and (E), respectively. Independent deconvolution of each charge state sam-pled by SONAR and subsequent combination of the peak lists was found to provide the best per-formance, hence the “SONAR combined” column represents this treatment. In (F), sequence cov-erage maps are shown for SONAR of horse myoglobin from LC-MS and the infusion experiment. To facilitate comparison, the spectra from 20+ and 21+ charge states from the infusion experi-ment were averaged and deconvolved to simulate the treatment of the data in (A) and (B).

Figure 3. Targeted (A) and MSE (B) MS/MS spectra and the respective coverage maps from pig myoglobin. Zoomed views from m/z 672 to 762 are shown in (C) and (D), highlighting improved fragment ion signal resulting from fragmentation of the entire charge state envelope. (E) Se-quence coverage of the four myoglobin variants from targeted and MSE strategies using a 10 ppm fragment ion threshold. (F) Proteoform characterization score (PCA) for the four myoglobin variants, reported from Prosight Lite. Dotted yellow and green lines indicate thresholds for pro-tein identification and proteoform characterization, respectively.4

1. Badgett, M. J.; Boyes, B.; Orlando, R., The Separation and Quantitation of Peptides with and without Oxidation of Methionine and De-amidation of Asparagine Using Hydrophilic Interaction Liquid Chromatography with Mass Spectrometry (HILIC-MS). Journal of The American Society for Mass Spectrometry 2017, 28 (5), 818-826.

2. Lauber, L. A; McCall, S. A.; Alden, B. A.; Ireneta, P. C.; Koza, S. M, Developing High Resolution HILIC Separations of Intact Glycosylated Proteins Using a Wide-Pore Amide-Bonded Stationary Phase. http://www.waters.com/webassets/cms/library/docs/720005380en.pdf

3. Shaw, J. B.; Li, W.; Holden, D. D.; Zhang, Y.; Griep-Raming, J.; Fellers, R. T.; Early, B. P.; Thomas, P. M.; Kelleher, N. L.; Brodbelt, J. S., Complete Protein Characterization Using Top-Down Mass Spectrometry and Ultraviolet Photodissociation. Journal of the American Chemical Society 2013, 135 (34), 12646-12651.

4. LeDuc, R. D.; Fellers, R. T.; Early, B. P.; Greer, J. B.; Thomas, P. M.; Kelleher, N. L., The C-Score: A Bayesian Framework to Sharply Im-prove Proteoform Scoring in High-Throughput Top Down Proteomics. Journal of Proteome Research 2014, 13 (7), 3231-3240.

Traditional ion mobility-assisted data independent acquisition (HDMSE, IM-DIA) utilizes

ion mobility to separate precursors followed by post-IMS fragmentation, providing an additional dimension of separation to UPLC-MS

E experiments. The geometry of the

SYNAPT G2-Si allows ions to be collisionally activated before (Trap) or after (Transfer) IMS separation. In the case of top-down IM-DIA described here, IM separation is used after collisional activation to provide additional separation of the fragment ions.

HILIC chromatography provides excellent separation of sequence variants, as illustrated by myoglobin.

HILIC LC-MS in combination with MSE provides improved sequence coverage

and variant site identification for low abundance sequence variants compared to targeted MS/MS.

HDMSE with wideband enhancement provides improved fragment ion signal

and clarity of fragment ions, particularly for low abundance species. SONAR offers an alternate DIA approach with enhanced selectivity at the cost

of some sensitivity. Infusion-based experiments show high potential for the ap-proach in cases where the user is not sample limited.