The De Novo Sequencing of Full-Length Amyloidogenic Light Chain By Peripheral Blood Mass Spectrometry

Accurate determination of full-length sequences and post-translational modifications of amyloidogenic light chains (AL-LC) is crucial for understanding the pathogenic mechanisms underlying AL amyloidosis. Traditional methods, such as indirect cDNA sequencing, often fall short in achieving full-length sequence determination. In contrast, mass spectrometry offers a more precise approach. However, previous studies have faced challenges due to limited clinical specimens and inadequate database search strategies, hindering full-length sequence acquisition. The introduction of the REmAb sequencing platform (Rapid Novor, Kitchener, ON) combined with LC-MS technology offers a promising new method for determining full-length AL-LC sequences.

In our study, we aimed to develop and validate a de novo sequencing methodology for AL-LC using LC-MS. We enrolled 17 patients with AL amyloidosis and 11 patients with other plasma cell disorders, each with a serum free light chain (dFLC) ≥50 mg/dL. Seven patients with AL amyloidosis also provided biopsy tissue specimens. We performed serum-free light chain (sFLC) enrichment, PNGF enzyme pretreatment, and LC-MS analysis on baseline peripheral blood samples. De novo sequencing was conducted using the REmAb platform, and N-glycosylation modifications were identified with pGlyco3 (pFind Lab, Beijing, China). Additionally, biopsy tissue was analyzed with LMD/MS at other institutions.

Electrophoresis results confirmed that sFLC-enriched monomers were concentrated at 17 kDa and dimers at 34 kDa, consistent with existing literature. Sequencing results from peripheral blood samples showed a high consistency of 98.85% (range 70.4-100.0%) compared to mass spectrometry data from biopsy tissues, indicating the accuracy of our methodology.

Our method also enabled the precise identification of post-translational modifications, including N-glycosylation, which was specific to AL-LC and not observed in non-AL-LC samples. Homologous modeling of protease cleavage sites revealed that these sites are primarily located at the N-terminus or external structures, with LC fragments being rare in peripheral blood, suggesting protein degradation predominantly occurs after sedimentation.

Preliminary analysis of mutation profiles indicated that non-conservative mutations in AL-LC are concentrated in the V region and differ from those in the control group. However, no specific mutation sites were identified within AL-LC of the same germline type, suggesting that multiple mutations may contribute to pathogenicity. Further validation with a larger sample size is needed.

In conclusion, we successfully established and validated a de novo full-length sequencing methodology for peripheral blood AL-LC using LC-MS. This method offers accurate sequencing results and the ability to simultaneously acquire post-translational modification information, facilitating research into AL-LC mechanisms and providing potential for improved risk prediction and clinical monitoring of AL amyloidosis patients.