Elevate Sensitivity for Intact Protein Analysis

This technical note demonstrates a sensitive method for high-throughput (HT) intact protein analysis using the Echo® MS+ system with ZenoTOF 8600 system. Overall, >5X gain in signal-to-noise (S/N) at MS1 level for intact analysis of myoglobin, Bovine Serum Albumin (BSA), and NISTmAb was observed using the Echo® MS+ system with ZenoTOF 8600 system compared to the Echo® MS+ system with ZenoTOF 7600 system (Figure 1). The development of protein-based therapeutics and vaccines requires analytical technologies that keep pace with modern research demands. HT methods are essential for performing simultaneous multicomponent analyses, enabling rapid candidate selection and formulation under tight timelines. While LC-MS remains a trusted approach, its inherently low-throughput nature makes it unsuitable for workflows that require processing thousands of samples quickly. To address this limitation, the Echo® MS+ system with ZenoTOF 8600 system presents an alternative approach for analyzing intact proteins with higher throughput and lower sample consumption. Key benefits for HT intact protein analysis using the Echo® MS+ system with ZenoTOF 8600 system • Sensitive intact protein analysis: The improved hardware provides >5X S/N gain at MS1 level, enabling confident and sensitive analysis of intact proteins with varying sizes and at sub-μM concentration, compared to the Echo® MS+ system with ZenoTOF 7600 system. • High throughput: The sample analysis speed was 1.4 sec/sample, highlighting fast sample analysis and meeting throughput needs Elevating sensitivity for high-throughput intact protein analysis using next-generation technologies in acoustic ejection and HRMS Figure 1. HT intact analysis of myoglobin (left), BSA (middle) and NISTmAb (right) using the Echo® MS+ system with ZenoTOF 7600 system and the Echo® MS+ system with ZenoTOF 8600 system. For myoglobin, mass reconstruction was done using 1μM myoglobin in water, where myoglobin with a mass of 16951.2 Da observed with S/N of 290 using ZenoTOF 7600 system (Figure 1A), compared to a S/N of 1600 using ZenoTOF 8600 system (Figure 1D). For BSA, mass reconstruction was performed using 20μM BSA in water for ZenoTOF 8600 system and 25μM BSA in water for ZenoTOF 7600 system. Figure 1B shows BSA with a mass of 66433.5 Da and a S/N of 60 for the ZenoTOF 7600 system, and Figure 1E shows BSA with a mass of 66437.6 Da and a S/N of 710 For NISTmAb, mass reconstruction was done using 33.5μM NISTmAb in histidine buffer, Figure 1C demonstrates NISTmAb G0F/G1F glycoform with mass of 148195.9 Da, with S/N of 240 using ZenoTOF 7600 system, versus Figure 1F G0F/G1F with mass of 148203.0 Da, with S/N of 2100 using ZenoTOF 8600 system. Xuejiao Yin1, ChiuCheong Aw1, Han Wang1, Chang Liu2, and Eshani Galermo3 1SCIEX, Singapore; 2SCIEX Canada; 3SCIEX, USA • Low sample consumption: Analyze as low as 3 μL per sample with nanoliter-sized sample consumption per ejection. • Streamlined data management: Acquire data in batches or individually and process data with protein reconstruction in SCIEX OS software using Analytics and Explorer modules. • Well-based monitoring: Using a target list within SCIEX OS software, different proteins can be monitored and tracked in different wells, giving greater flexibility and delivering information within a few minutes. Introduction The analysis of intact proteins and their impurities is critical to ensuring drug quality, safety, and efficacy throughout development and manufacturing. Biotherapeutics, including monoclonal antibodies and complex protein-based drugs, present unique analytical challenges due to their structural heterogeneity and the presence of low-abundant proteoforms or impurities. 1 Traditional LC-MS workflows have been widely adopted for intact mass analysis; however, the growing demand for higher throughput and improved sensitivity has necessitated innovative approaches. 2 This technical note introduces an advanced workflow that combines Echo® MS+ system with ZenoTOF 8600 system, delivering significant improvements in sensitivity and throughput for intact mass analysis and determination of limit of quantitation (LOQ). Echo® MS+ system leverages acoustic droplet ejection for rapid, contact-free sample introduction, eliminating chromatographic separation and enabling ultra-fast analysis. When paired with ZenoTOF 8600 system’s enhanced ion optics and Zeno trap, this workflow achieves >5X S/N increase relative to previous platforms, enabling confident detection of low-concentration species in complex biotherapeutic samples. In this work, intact protein analyses of myoglobin, BSA, and NISTmAb were performed using the Echo® MS+ with ZenoTOF 7600 system and Echo® MS+ system with ZenoTOF 8600 system to evaluate the benefits of MS sensitivity improvements provided by the new platform. Methods Sample preparation: Myoglobin (M1882, Sigma) and BSA (A9418, Sigma) were prepared in Milli-Q water to prepare 1 mM stock solutions. Serial dilutions of myoglobin and BSA from 0.01 μM to 50 μM were prepared from the stock solution for linear dynamic range (LDR) analysis. A serial dilution of 0.6 μM to 33.5 μM NISTmAb (RM8671, NIST) was prepared using the histidine buffer. Acoustic ejection: The 70% acetonitrile (ACN) and 30% water with 0.1% formic acid (FA) as the modifier were chosen as the carrier solvent for all the experiments (Table 1). The flow rate was optimized based on the probe and electrode coupled to the mass spectrometer. Table 1. Acoustic ejection conditions. Mass spectrometry: TOFMS data were acquired using the ZenoTOF 7600 system or the ZenoTOF 8600 system. Key source and TOFMS parameters for intact analysis of myoglobin, BSA, and NISTmAb are shown in Tables 2 and 3, respectively. Table 2. TOFMS and source parameters for the myoglobin and BSA experiments. Myoglobin and BSA NISTmAb Carrier solvent 0.1% FA in 70:30 (v/v) ACN/water Fluid class AQ Ejection volume 20 nL 100 nL Rep rate 20 Hz Parameter ZenoTOF 7600 system ZenoTOF 8600 system Workflow Intact protein Polarity Positive Curtain gas 35 psi 45 psi Source temperature 300°C Spray voltage 5000 V CAD gas 7 MS Method TOFMS Start mass 700 m/z Stop mass 3000 m/z Declustering potential 80 V 70 V Collision energy 30 V 30 V ITC Fixed, 100 Accumulation time 0.1 s Time bins to sum 30 Table 3. TOFMS and source parameters for NISTmAb analysis. Data processing: Data was processed in SCIEX OS software 4.1.2.13 using the Explorer and Analytics modules. Intact analysis of myoglobin and BSA To evaluate the sensitivity improvement for Myoglobin analysis using the Echo® MS+ system with ZenoTOF 8600 system over the Echo® MS+ system with ZenoTOF 7600 system, the S/Ns for the 1 μM myoglobin sample were calculated from the reconstructed protein spectra (Figures 2A and 2B). A S/N of ~1600 was obtained using the Echo® MS+ system with ZenoTOF 8600 system (Figure 2D). This value is >5X of the S/N (~290) measured on the Echo® MS+ system with ZenoTOF 7600 system. To assess the quantitative performance for high-throughput analysis, calibration curves were generated for myoglobin, using the Echo® MS+ system coupled with ZenoTOF 8600 system. For myoglobin, good linearity was achieved across 0.1–10 μM with an excellent correlation coefficient (r²), 0.9928, without the use of an internal standard for normalization. And the LOQ was determined to be 0.1 μM (Figure 3A). Figure 2. Myoglobin analyzed using ZenoTOF 7600 system and ZenoTOF 8600 system. For 1 μM myoglobin, reconstruction with mass of 16951.2 Da with S/N around 290 using ZenoTOF 7600 system, compared to a S/N of 1600 using ZenoTOF 8600 system, indicating a 5-fold increase in MS1 sensitivity using the ZenoTOF 7600 system compared to ZenoTOF 8600 system. Figure 3. Myoglobin calibration curve and the analysis of 0.1 μM samples using Echo® MS+ system with ZenoTOF 8600 system. Good linearity was achieved across 0.1-10 μM with r2 of 0.9928 (A). Examples of reconstructed myoglobin at LOQ of 0.1 μM are shown in (B), where the S/N at 0.1 μM is around 8. The LOQ can be further improved by increasing the sample ejection volume. Parameter ZenoTOF 7600 system ZenoTOF 8600 system Workflow Intact protein Large protein (>70 kDa) Checked Polarity Positive Curtain gas 35 psi 45 psi Source temperature 300°C Spray voltage 5000 V CAD gas 7 MS Method TOFMS Start mass 2800 m/z Stop mass 5000 m/z Declustering potential 260 V 180 V Collision energy 70 V 70 V ITC Fixed, 100 Accumulation time 0.1 s Time bins to sum 80 120 Figure 4. BSA analyzed using ZenoTOF 7600 system and ZenoTOF 8600 system. For 20 μM BSA, reconstruction with a mass of 66430.3 Da with S/N of 60 using the ZenoTOF 7600 system, compared to an S/N around 710 using the ZenoTOF 8600 system, indicating a 12 - fold increase in S/N at the MS1 level using the ZenoTOF 7600 system compared to the ZenoTOF 8600 system . Figure 5. BSA calibration curve and the analysis of 0.2 μM samples using Echo® MS+ system with ZenoTOF 8600 system. BSA (in water) demonstrated good linearity across 0.2-10 μM with r2 of 0.9932 (A). Examples of reconstructed BSA at the LOQ of 0.2 μM are shown in (B), where the S/N at 0.2 μM is approximately 3. The same analysis was also performed on BSA. Nearly a 12- fold improvement based on S/N (~60 vs. ~710) was achieved for 20 μM BSA as displayed in Figure 4 . The results of both Myoglobin and BSA demonstrate the enhanced intact mass analysis using the Echo® MS+ system with ZenoTOF 8600 system . Therefore, the S/N gain at the MS1 level offered by the Echo® MS+ system with the ZenoTOF 8600 system can enable improved detection of low- abundance proteins, particularly when low sample consumption is required. Similarly, BSA exhibited high linearity (r² = 0.9932) across concentrations ranging from 0.2 to 10 μM (Figure 5A). The LOQ for BSA was 0.2 μM with the ejection volume of 20 nL. Performance can be further improved by increasing the ejection volume, as indicated in Figures 3B and 5B. For the reproducibility assessment, a concentration of 1 μM was evaluated for both myoglobin and BSA. Utilizing a predefined target list, a full 384 - well plate containing myoglobin and BSA samples was analyzed within 14 minutes. The target list was configured such that: • Well 1 monitored myoglobin • Well 2 monitored BSA • Well 3 monitored both myoglobin and BSA Where the same pattern was repeated across all wells. As summarized in Tables 4 and 5, the reproducibility for 1 μM myoglobin across 3 replicate runs was <8% coefficient of variation (CV), with mass accuracy <8 ppm. Similarly, BSA demonstrated reproducibility of <6% CV across 3 replicate runs, with mass accuracy <5 ppm. Table 4. Reproducibility and mass accuracy for 1 μM myoglobin on Echo ® MS+ system with ZenoTOF 8600 system . %CV Processed in individual wells Processed in the same wells Run 1 6.29% 5.89% Run 2 6.48% 6.06% Run 3 7.64% 6.53% Mass accuracy (ppm) Processed in individual wells Processed in the same wells Run 1 5.05 4.86 Run 2 7.20 7.11 Run 3 6.76 6.48 Table 5. Reproducibility and mass accuracy for 1 μM BSA on the Echo® MS+ system with ZenoTOF 8600 system. Intact analysis of NISTmAb In addition to proteins smaller than 70 kDa, such as myoglobin and BSA, the Echo® MS+ system can analyze intact proteins exceeding 70 kDa, including intact monoclonal antibodies (mAbs). Figure 6 shows data for 33.5 μM NISTmAb acquired using the Echo® MS+ system with ZenoTOF 7600 system and Echo® MS+ system with ZenoTOF 8600 system. Similar charge-state distributions of NISTmAb were observed in both systems (Figures 6A and 6E). However, a close inspection of the 36+ charge state show that the Echo® MS+ system with ZenoTOF 8600 system provided a lower “valley-to-peak” ratio (i.e., better peak resolution) of ~17% between the G0F/G0F and G0F/G1F glycoforms (Figure 6B), as compared to ~22% for Echo® MS+ system with ZenoTOF 7600 system (Figure 6F), indicating better desolvation using the Echo® MS+ system with ZenoTOF 8600 system. To evaluate the sensitivity improvement on the Echo® MS+ system with ZenoTOF 8600 system, we calculated the S/N of the G0F/G1F glycoform. A 9-fold improvement in S/N (~2100) was obtained compared to that measured on the Echo® MS+ system with ZenoTOF 7600 system (~240) (Figure 6D and 6H). A concentration range of NISTmAb was introduced to the Echo® MS+ system with ZenoTOF 8600 system to determine the LOQ, where as low as 0.6 μM was quantified when ejecting 100 nL (Figure 7). Previous testing of NISTmAb on the Echo® MS+ system with ZenoTOF 7600 system established an LOQ of 33.5 μM.3 Therefore, about 56-fold improvement in LOQ was achieved. Table 6. Reproducibility and mass accuracy for 3.3 μM NISTmAb on Echo® MS+ system with ZenoTOF 8600 system. %CV G0F/ G0F G0F/ G1F G1F/ GF G1F/ G2F G2F/ G2F Run 1 4.74% 4.43% 4.17% 4.44% 7.38% Run 2 4.43% 4.08% 4.27% 5.10% 5.80% Run 3 3.61% 3.52% 3.44% 3.95% 4.99% Mass accuracy (Da) G0F/ G0F G0F/ G1F G1F/ GF G1F/ G2F G2F/ G2F Run 1 9.26 10.59 8.94 6.11 5.28 Run 2 9.25 10.58 9.09 6.13 5.18 Run 3 9.27 10.64 9.23 6.00 4.91 %CV Processed in individual wells Processed in the same wells Run 1 5.43% 4.99% Run 2 5.02% 4.61% Run 3 5.34% 4.40% Mass accuracy (ppm) Processed in individual wells Processed in the same wells Run 1 4.64 4.19 Run 2 2.31 2.40 Run 3 2.20 2.14 Figure 7. NISTmAb (0.6 μM) was analyzed using Echo® MS+ system with ZenoTOF 8600 system. All the major glycoforms, i.e. G0F/G0F, G0F/G1F, G1F/G1F, G1F/G2F, and G2F/G2F were successfully integrated in the reconstruction spectrum using 0.6 μM NISTmAb on the ZenoTOF 8600 system. Figure 6. NISTmAb (33.5 μM) was analyzed using the ZenoTOF 7600 system and ZenoTOF 8600 system. Figures 6A and 6E illustrate the charge envelope of the NISTmAb in both ZenoTOF 7600 and 8600 systems. Figure 6B and 6F zoomed into the charge state with the highest intensity to show the resolution using valley-to-peak ratio in ZenoTOF 7600 system and ZenoTOF 8600 system. All the major glycoforms were detected and confidently assigned to the data from both the ZenoTOF 7600 and ZenoTOF 8600 system. Intensity around 40-60 cps was observed for G0F/G0F, G0F/G1F, and G1F/G1F (Figure 6C, ZenoTOF 7600 system) while the intensity was 500-700 cps on ZenoTOF 8600 system (Figure 6G). For the G0F/G1F glycoform, the S/N is around 240 on the ZenoTOF 7600 system (Figure 6D), compared to S/N of 2100 on the ZenoTOF 8600 system (Figure 6H). This indicates a 10-fold increase in S/N at the MS1 level. The MS sensitivity gain offered by ZenoTOF 8600 system enabled the detection of lower concentration protein samples. Conclusions • More than 5-fold improvement in S/N at MS1 level was achieved using Echo® MS+ system with ZenoTOF 8600 system compared to the previous platform, enabling enhanced intact mass measurements of proteins. • Detection of lower-concentration, low-abundance species (0.1-0.6 μM) with reduced sample load was demonstrated on the Echo® MS+ system with ZenoTOF 8600 system. • When combined with the Echo® MS+ system, the ZenoTOF 8600 system demonstrates great linearity with r2 greater than 0.99 across concentrations from 0.1 μM to 10 μM without using an internal standard for normalization. • High analytical throughput (1.4 sec per sample), high signal reproducibility (CV <10%), high mass accuracy (<10 ppm for myoglobin and BSA and <11 Da for NISTmAb), and low sample volume requirements (as low as 3 μL per sample) were easily achieved on the Echo® MS+ system with ZenoTOF 8600 system, demonstrating a powerful platform for the HT analysis at the intact protein level. • All core workflows, including batch calibration, data acquisition, protein reconstruction (via BioToolKit and Intact Quant), and data processing (via Target list and Analytics)—are supported and can be executed within a single integrated environment using SCIEX OS software. 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MKT-37238-A Headquarters 250 Forest Street | Marlborough, MA 01752 USA Phone 508 -383-7700 sciex.com International Sales For our office locations please call the division headquarters or refer to our website at sciex.com/offices References 1. Liu, C. High- throughput MS for intact protein analysis. Bioanalysis. 2023 Aug;15(16):1017 - 1019. 2. Enhanced sensitivity of intact LC - MS approaches to expand the capability of biotherapeutics analysis . SCIEX technical note, MKT- 35364 -A. 3. Accelerating intact biotherapeutic analysis using acoustic ejection mass spectrometry (AEMS). SCIEX Poster, MKT - 36402 -A.