Rapid-Turnaround Top-Down Sequence Verification and Characterization of Modified Proteins and Oligonucleotides Using a Benchtop MALDI-TOF/TOF Instrument

Significance of the topic

The accurate and rapid verification of intact biomolecules such as proteins and oligonucleotides is critical across biopharma development, quality control and structural biology. Top-down sequencing approaches that read sequence information directly from intact molecules reduce ambiguity introduced by proteolysis or complex deconvolution algorithms and accelerate decision-making in development workflows. MALDI-based top-down sequencing (MALDI-TDS) using in-source decay (ISD) offers singly charged fragment spectra that simplify interpretation and enable LC-free, high-throughput analysis of diverse modalities including modified RNAs, labeled DNAs and PEGylated/modified proteins.

Objectives and study overview

This application-focused study demonstrates the capability of a benchtop MALDI-TOF/TOF (Bruker neofleX) to perform rapid top-down verification and characterization of modified biomolecules without LC separation. Three representative samples were analyzed to illustrate method performance and scope:

• A 29.7 kDa recombinant cutinase (Thermobifida alba estl) expression product with a disulfide bond and a minor N-terminally truncated proteoform.
• A heavily modified 39-mer RNA (2´-O-methyl, 2´-fluoro, phosphorothioate) (~12.5 kDa).
• A 5´-DBCO labeled DNA 21-mer (~7 kDa).

Key goals were intact-mass confirmation, sequence readout by MALDI-ISD, detection and localization of modifications and proteoforms, and demonstration of workflow speed and flexibility.

Methods

Samples and preparation:

• Recombinant protein: 29.7 kDa, concentration 40 pmol/µL; matrix: 2,5-DHAP for intact mass and SDHB for MALDI-ISD top-down sequencing.
• Oligonucleotides: RNA 39-mer and DNA 21-mer, each 10 pmol/µL; matrices used included 3-HPA and 2,4,6-THAP.

Acquisition and instrument settings:

• Instrument: Bruker neofleX benchtop MALDI-TOF/TOF.
• Intact mass analysis: positive linear mode.
• MALDI-ISD top-down sequencing: positive reflector mode to generate singly charged ISD fragments.
• Default application methods were used for data collection.

Data analysis:

• Spectra were interpreted using Bruker OmniScape and BioTools software.
• Fragment ion types used for sequence assignment included N-terminal c-type ions for proteins and d-/w-type ions for oligonucleotides, plus y- and z+2-type ions for C-terminal protein assignments.

Instrumentation used

• Bruker neofleX benchtop MALDI-TOF/TOF mass spectrometer.
• Matrices: 2,5-dihydroxyacetophenone (2,5-DHAP), super-DHB (SDHB), 3-hydroxypicolinic acid (3-HPA) and 2,4,6-trihydroxyacetophenone (2,4,6-THAP).
• Software: Bruker OmniScape and BioTools for MALDI-TDS data processing and sequence mapping.

Main results and discussion

Recombinant protein:

• Intact-mass MALDI-TOF confirmed the expected principal molecular weight but revealed a minor proteoform ~203 Da lower, suggesting an N-terminal dipeptide loss.
• MALDI-ISD produced extensive sequence information: N- and C-terminal readout reached ~80 amino acids from each terminus, yielding greater than 50% MS/MS sequence confirmation from a single spectrum.
• The minor proteoform was identified as an N-terminally truncated variant lacking the dipeptide MA, supported by a low-abundance series of N-terminal c-type ions matching the truncated sequence.
• A gap in the C-terminal fragment ion series (y, z+2) identified a disulfide bond between cysteines at positions 242 and 260.

Modified RNA 39-mer:

• MALDI-ISD sequencing achieved approximately 67% MS/MS coverage using d- and w-type fragment ions, sufficient to confirm the primary sequence and the complex modification pattern (2´-fluoro, 2´-O-methyl and phosphorothioate linkages).
• Singly charged ISD fragments facilitated straightforward interpretation despite extensive chemical modifications.

5´-DBCO labeled DNA 21-mer:

• Sequence verification reached roughly 85% MS/MS coverage with d-, y- and w-type fragments, and the DBCO label was localized to the 5´ end of the oligonucleotide.
• High coverage enabled confident assignment of both sequence and label position from a single MALDI-ISD dataset.

Discussion points:

• MALDI-ISD fragment spectra are dominated by singly charged ions, removing the need for deconvolution and simplifying sequence assignment compared with multiply charged ESI-MS/MS spectra.
• The method is inherently LC-free, allowing rapid switching between protein and oligonucleotide analyses without reconfiguration of LC solvents or gradients.
• Limitations include gaps in fragment series due to disulfide bridges or inaccessible bond cleavages; complementary approaches or reduction/derivatization may be required to fully map such features.

Benefits and practical applications

• Rapid turnaround: single-spectrum sequence confirmation and intact-mass verification accelerate development decisions in biopharma and support high-throughput screening of expression products and modified oligonucleotides.
• Simplicity: singly charged ISD fragments simplify data interpretation and reduce computational overhead compared with deconvolution-heavy workflows.
• Versatility: the same benchtop MALDI-TOF/TOF platform can analyze proteins, modified RNAs and labeled DNAs without LC reconfiguration, improving lab efficiency.
• Structural insight: detection of proteoforms, N-terminal truncations and disulfide linkages provides actionable information for construct evaluation, expression optimization and analytical quality control.

Future trends and potential uses

• Integration into QC pipelines: MALDI-TDS could become a routine rapid-release or in-process control tool for defined modalities where intact verification suffices.
• Expanded modification coverage: method optimization and improved fragment assignment algorithms can extend reliable readout to increasingly complex chemical modifications.
• Automation and throughput: plate-based MALDI workflows are amenable to robotic sample preparation and batch processing for higher throughput demands.
• Hybrid approaches: combining MALDI-TDS with targeted LC-MS/MS, reduction/alkylation or enzymatic cleavage may resolve remaining coverage gaps and disulfide topology questions.
• Software advances: improved spectral annotation, automated proteoform detection and confidence scoring will increase the method’s robustness for regulated environments.

Conclusion

MALDI-TDS performed on a benchtop MALDI-TOF/TOF (neofleX) is a fast, flexible and LC-free approach for top-down verification and characterization of intact proteins and modified oligonucleotides. The method yields high-confidence sequence readouts (50–85% MS/MS coverage in the presented cases), enables detection and localization of proteoforms, modifications and labels, and simplifies interpretation through singly charged ISD fragment spectra. As a rapid screening and characterization tool, MALDI-TDS can accelerate biopharma development and provide complementary structural information to conventional LC–MS workflows.

References

1. Bruker Application Note MT-142.