Next-generation MALDI top-down sequencing of protein biotherapeutics – expanding the scope of timsTOF technology

Significance of the topic

MALDI-based top-down sequencing has become an essential tool for precise characterization of protein biotherapeutics by enabling direct analysis of intact molecular ions and their fragmentation products. Incorporating Trapped Ion Mobility Spectrometry (TIMS) adds a further gas-phase separation dimension, boosting sequence coverage, mass accuracy and spectral clarity. This capability is particularly valuable for confirming terminal modifications, mapping glycosylation sites and validating complex biopharmaceuticals in research and quality control settings.

Objectives and overview of the study

• Demonstrate next-generation MALDI top-down sequencing (MALDI-TDS) performance on the timsTOF fleX platform with ultrahigh resolution (RP ~60,000) and mass accuracy (<2 ppm).
• Evaluate method robustness and sequence coverage using a model protein (bovine carbonic anhydrase II) and relevant biotherapeutic targets (adalimumab subunits, recombinant SARS-CoV-2 S-glycoprotein RBD).
• Highlight the benefits of integrating TIMS for enhanced separation of N- and C-terminal fragments and cleaner MS/MS (T3-Sequencing) spectra.

Methodology and used instrumentation

Protein samples (CA II, adalimumab fragments, SARS-CoV-2 RBD) were prepared via reduction, deglycosylation where needed, and spotted onto Anchorchip targets with sDHB matrix. Red phosphorous clusters provided broadband m/z calibration across 1,000–15,000 Da.
Data acquisition was performed on a Bruker timsTOF fleX equipped with dual ESI/MALDI source, smartbeam 3D laser (1 kHz) and TIMS cell. MALDI-TDS spectra covered m/z 1,000–15,000 and were recorded at 60,000 RP. TIMS ramps (300–800 ms) enabled separation of singly and doubly charged ISD fragments. Selected fragments underwent CID-MS/MS (T3-Sequencing) at 50–120 eV. Data processing used Savitzky–Golay smoothing, SNAP peak picking and Bruker BioPharma Compass® and BioTools software.

Main results and discussion

Bovine carbonic anhydrase II delivered an intact molecular ion envelope and extensive ISD fragmentation, yielding a sequence validation percentage (SVP) of 86.9% without charge deconvolution. High resolution in the low m/z region enabled reliable readout of near-terminal residues.
TIMS separation distinguished N- and C-terminal ISD fragments in mobility space, simplifying spectrum interpretation and achieving combined SVP of 72% when including doubly charged fragments. TIMS also purified T3-Sequencing spectra by removing isobaric interferences, providing unambiguous confirmation of N-terminal c18 fragments.
For adalimumab subunits, single-spectrum MALDI-TDS analyses confirmed intact masses within ≤0.5 ppm and C-/N-terminal sequences (SVP 68–83%), including detection of C-terminal lysine loss on the Fc/2 fragment and glycoform assignments.
Recombinant SARS-CoV-2 RBD analysis identified core 1 and core 2 O-glycoforms by a 365 Da mass difference in the [M+2H]2+ envelope. ISD fragmentation confirmed an N-terminal pyroglutamine and localized O-glycosylation exclusively to Thr-6. TIMS-enhanced T3 spectra validated glycan losses stepwise, providing definitive glycosylation site assignment.

Benefits and practical applications

• Simultaneous high-accuracy intact mass measurement and direct terminal sequencing in a single MALDI-TDS run.
• No requirement for charge state deconvolution due to isotopic resolution of multiply charged ions.
• TIMS adds orthogonal separation for clearer spectra, aiding analysis of unknown sequences or unexpected modifications.
• Rapid, LC-free MALDI workflow complements ESI-based methods on the same instrument, streamlining biopharma analytics.

Future trends and potential applications

As MALDI-TDS with TIMS matures, it may be extended to even larger proteins, highly heterogeneous glycoproteins and full antibody sequencing. Advances in automated data interpretation and AI-driven spectral annotation will further accelerate workflows. Emerging TIMS modes and refined collision methods promise deeper coverage of post-translational modifications and discovery of low-abundance variants.

Conclusion

Next-generation MALDI-TDS on the timsTOF fleX platform offers unparalleled resolution (<2 ppm, RP 60,000), integrated TIMS separation and dual ESI/MALDI capabilities. This approach delivers rapid, high-confidence terminal sequencing, intact mass verification and PTM mapping in a single, streamlined analysis, making it a powerful addition to biotherapeutic characterization toolkits.

References

• Srzentić K et al. J. Am. Soc. Mass Spectrom. 2020, 31(9):1783–1802
• Gstöttner C et al. Anal. Chem. 2021, 93(17):6839–6847
• Gstöttner C et al. Recombinant SARS-CoV-2 Receptor Binding Domain: Comprehensive Top-Down Sequence Confirmation and O-Glycosylation Site Determination, Bruker Application Note MT-132
• Yoo C et al. J. Am. Soc. Mass Spectrom. 2009, 20(2):326–333
• Ayoub D et al. MAbs 2013, 5(5):699–710
• Resemann A et al. MAbs 2016, 8(2):318–330
• Welsink T, Wolfenstetter S. InVivo Application Note 1885914
• Suckau D, Resemann A. Anal. Chem. 2003, 75(21):5817–5824