De Novo Sequencing of Antibody Light Chain Proteoforms from Patients with Multiple Myeloma

Abstract

In multiple myeloma diseases, monoclonal immunoglobulin light chains (LCs) are abundantly produced, with, as a consequence in some cases, the formation of deposits affecting various organs, such as the kidney, while in other cases remaining soluble up to concentrations of several g•L −1 in plasma. The exact factors crucial for the solubility of LCs are poorly understood, but it can be hypothesized that their amino acid sequence plays an important role. Determining the precise sequences of patient-derived LCs is therefore highly desirable. We establish here a novel de novo sequencing workflow for patient-derived LCs, based on the combination of bottom-up and top-down proteomics without database search. PEAKS is used for the de novo sequencing of peptides that are further assembled into full length LC sequences using ALPS. Top-down proteomics provides the molecular masses of proteoforms and allows the exact determination of the amino acid sequence including all posttranslational modifications. This pipeline is then used for the complete de novo sequencing of LCs extracted from the urine of 10 patients with multiple myeloma. We show that for the bottom-up part, digestions with trypsin and Nepenthes digestive fluid are sufficient to produce overlapping peptides able to generate the best sequence candidates. Top-down proteomics is absolutely required to achieve 100% final sequence coverage and characterize clinical samples containing several LCs. Our work highlights an unexpected range of modifications.

Validated Method Based on Immunocapture and Liquid Chromatography Coupled to High-Resolution Mass Spectrometry to Eliminate Isatuximab Interference with M-Protein Measurement in Serum

“…This method was then successfully validated with an LLOQ of 10.0 μg/mL alemtuzumab equivalent, allowing the detection of M-protein at very low concentrations compared to SPEP. Furthermore, the method sensitivity is higher than traditional top-down methods and is very close to that generally achieved with clonotypic peptide methods. − , …”

“…Furthermore, the method sensitivity is higher than traditional top-down methods 14 and is very close to that generally achieved with clonotypic peptide methods. [11][12][13]20 Clinical Sample Analysis. The validated method was then used to analyze 216 samples corresponding to treatment cycles from 38 patients included in the Isa-Pd arm (therapy combination of isatuximab, pomalidomide, and dexamethasone) of the ICARIA study.…”

“…The first approach, based on LC-ESI-MS/MS (tandem mass spectrometry), measures clonotypic peptides from the Ig complementarity-determining region (CDR) as surrogate markers for the M-protein . Although this MS method is considered more sensitive (limit of quantification <10 μg/mL), it requires a priori knowledge of the M-protein amino acid sequence, resulting in a more complex and costly analysis. , The patient-specific workflow is a major downside of this method, which requires prior sample analysis to identify unique peptides from the patient’s M-protein CDR. A patient-specific method then needs to be developed based upon the analysis of the unique peptide using specific multiple reaction monitoring (MRM) transitions.…”

Template-Based Assembly of Proteomic Short Reads For De Novo Antibody Sequencing and Repertoire Profiling

“…Chamot-Rooke and colleagues recently reported proteomic sequencing of multiple-myeloma light chains from patient urine using an integrated bottom-up and top-down sequencing approach. 43 Here, we used the bottom-up de novo sequencing reads from that study as a test case to reconstruct the light chains (see Supporting Figure S3A ). This includes one sample consisting of a mixture of two closely related light chains for which the variable positions are also clearly identifiable in the sequence logo from the Stitch analysis (see Supporting Figure S3B ).…”

A perspective toward mass spectrometry-based de novo sequencing of endogenous antibodies

“…Using a somewhat comparable approach, Dupré et al 103 analyzed isolated light chains from the urine of a patient affected by multiple myeloma. They assembled de novo data from peptides into a full-length sequence, using the intact mass data as a scaffold.…”