Analysis of the N-terminal Amino Acid Sequence of IgG Antibodies Using a Protein Sequencer (PPSQ ) Isocratic System

Significance of the topic

The reliable determination of N-terminal amino acid sequences is a cornerstone in structural characterization and identity verification of biopharmaceutical proteins such as monoclonal antibodies. Small changes in primary structure arising from manufacturing, expression host, or post‑translational events can alter functionality, safety, or regulatory classification. Edman degradation performed on dedicated protein sequencers remains a robust method for direct N‑terminal sequencing, complementing mass spectrometry for applications where stepwise release and unambiguous residue identification are required.

Objectives and overview of the study

This application note demonstrates the use of the Shimadzu PPSQ-51A/53A isocratic protein sequencer (SPD-40 detector) to obtain the N‑terminal amino acid sequences of humanized IgG1 produced in Chinese hamster ovary (CHO) cells. The goal was to verify the N‑terminal residues of separated heavy and light chains after SDS‑PAGE and electroblotting, and to illustrate analytical performance, practical sample preparation, and interpretive outcomes for antibody identity confirmation.

Methodology

• Sample: Humanized IgG1κ expressed in CHO cells (source: National Institute of Advanced Industrial Science and Technology).
• Pretreatment: Denaturation and reduction followed by SDS‑PAGE (Novex TBE 4–20% gel). Samples were heat‑treated at 95 °C for 25 min prior to loading. Electrophoresis at 100 V for 1 h.
• Protein transfer: Electroblotting onto PVDF membrane (FluoroTrans) with transfer at 25 V for 2 h.
• Membrane staining and band excision: Brief Coomassie R‑250 staining (1 min) and destaining (50% methanol, 1 min) to visualize heavy and light chain bands, which were excised for sequencing.
• Edman degradation and detection: Sequential Edman cleavage on the PPSQ‑51A/53A; released phenylthiohydantoin (PTH) amino acids analyzed by HPLC.

Used instrumentation

• Protein sequencer: PPSQ‑51A/53A (Shimadzu) isocratic system equipped with SPD‑40 UV detector (detection at 269 nm, STD cell).
• Analytical column: Wakopak Wakosil PTH‑II, 250 mm L × 4.6 mm I.D.
• Chromatographic conditions: PTH‑amino acids mobile phase (manufacturer formulation), flow rate 1.0 mL/min, column temperature 40 °C.
• Gel and blotting supplies: Novex TBE gels, NuPAGE buffers, FluoroTrans PVDF membranes; Coomassie Brilliant Blue R‑250 for staining.

Main results and discussion

• Chromatographic separation: A 25 pmol PTH‑amino acid standard mixture provided clear baseline separation for the canonical residues except cysteine, which is not detected by this PTH‑HPLC approach.
• Light chain (L‑chain): Sequential Edman cycles produced identifiable PTH peaks enabling assignment of residues up to the 21st position in the variable region. The determined N‑terminal sequence was Asp‑Ile‑Gln‑Met‑Thr‑Gln‑Ser‑Pro‑Ser‑Ser‑Leu‑Ser‑Ala‑X‑X‑Gly‑Asp‑Arg‑X‑Thr (X indicating ambiguous or undetermined positions). Cycles 14, 15 and 19 showed ambiguous or missing signal, consistent with lower signal intensity, overlapping peaks, or modifications that block or obscure residue identification.
• Heavy chain (H‑chain): The first five cycles yielded Glu‑Val‑Gln‑Leu‑Val‑Glu‑Ser‑Gly‑Gly‑Gly‑Leu‑Val‑Gln‑Pro‑Gly‑Gly‑Ser‑Leu‑Arg‑Leu from the N‑terminus. Database comparison identified this region as corresponding to the human immunoglobulin heavy chain variable region gene IGHV3‑72.
• Interpretation notes: The approach reliably confirmed N‑terminal identity and origin (variable regions) for both chains. However, occasional cycles produced multiple co‑eluting PTH peaks or no clear increases, which limits continuous read length and requires cautious interpretation. Modifications, blocked N‑termini, or residue-specific detectability (e.g., cysteine) account for some limitations.

Key analytical considerations and limitations

• Edman sequencing requires a free, unblocked N‑terminus; N‑terminal modifications or certain PTMs can prevent residue release and detection.
• Cysteine is not detected using the described PTH‑HPLC conditions unless chemically modified beforehand; hence cysteine positions may appear as gaps or ambiguous cycles.
• Sequence read length is constrained by cumulative losses in yield and signal across cycles; typical reliable readout is tens of residues depending on sample quality.
• High purity and clean separation of chains (SDS‑PAGE and PVDF transfer/excision) are essential to avoid mixed signals from co‑migrating species.

Benefits and practical applications

• Direct and specific N‑terminal residue identification aids identity testing, comparability studies, and verification of expression constructs for therapeutic antibodies.
• The PPSQ isocratic setup with simple software reduces operational complexity and running costs relative to gradient systems, enabling routine application in QC and research labs.
• Edman sequencing complements mass spectrometry: it provides unambiguous N‑terminal residue order and is particularly useful when database entries are absent or when confirming signal peptide cleavage sites and engineered N‑terminal sequences.

Future trends and potential uses

• Integration with orthogonal techniques: Coupling Edman sequencing workflows with LC‑MS/MS and intact mass analysis will provide richer information on N‑terminal modifications and sequence confirmation across platforms.
• Improved chemistry and detectors: Enhanced reagents for cysteine detection, higher‑sensitivity detectors, and improved HPLC media could extend read length and residue coverage.
• Automation and throughput: Streamlined sample handling (automated gel excision, robotic blot processing) and software-driven data interpretation will support higher throughput for biopharma QC and comparability testing.
• Bioinformatics integration: Direct database matching and automated annotation of variable regions and germline gene assignment will accelerate identity confirmation for monoclonal antibodies and antibody discovery programs.

Conclusion

The PPSQ‑51A/53A protein sequencer using Edman degradation and PTH‑HPLC provides a robust and user‑friendly method for N‑terminal sequencing of antibody chains following SDS‑PAGE and PVDF transfer. The method successfully identified N‑terminal variable region residues of both heavy and light chains from a humanized IgG1, demonstrating utility for identity verification in biopharmaceutical development. Limitations such as inability to directly detect cysteine, potential blocked termini, and reduced signal in later cycles must be considered in experimental design and data interpretation.

References

• Shimadzu Corporation. PPSQ‑51A/53A Protein Sequencer application note (First Edition: Jun. 2026).
• National Institute of Advanced Industrial Science and Technology (AIST) — source of CHO‑derived humanized IgG1κ sample (as cited in the application note).