Serum samples from multiple myeloma (MM) patients are collected regularly for conventional tests to quantify the M-protein longitudinally. However, the limited sensitivity of conventional tests makes quantification of minimal residual disease (MRD) difficult. MRD can be quantified with highly sensitive molecular techniques on bone marrow, but bone marrow procedures are invasive and cannot be performed regularly. In this study, we introduce highly sensitive targeted mass spectrometry (MS-MRD) to quantify M-protein from patient serum. Therefore, we can follow each patient’s course of disease and dynamically measure MRD.

A total of 929 serum samples were prepared and assessed with MS-MRD from 41 MM patients that participated in the IFM 2009 clinical trial study (ClinicalTrials.gov identifier NCT01191060). Previously, 1 or 2 clonotypic peptides from the M-protein sequence of each patient were selected to assess with MS-MRD. Every selected clonotypic peptide had a synthesized stable isotope-labelled (SIL) peptide used for M-protein quantification. In a 96-well plate (Axygen, Corning, NY, USA), 2 µL of patient serum samples and 2 µL of SIL-peptides were diluted in a 500 µL buffer solution with 50 mM ammonium bicarbonate and 27% acetonitrile. Then, 30 µL of serum dilutions were treated with 30 µL of 0.2 % Rapigest. Samples were reduced with 6 µL 10 mmol/L dithiotreitol for 30 min at 60 °C and alkylated with 3 µL 14 mmol/L iodoacetamide for 30 min at room temperature. Samples were digested overnight at 37°C with 4 µL 200 ng/µL trypsin (Promega, Madison, WI, USA). The next day, the samples were acidified with 1.5 µL 25% trifluoroacetic acid and incubated for 30 min at 37°C. Finally, the samples were filtered on a pre-wetted 0.45 µm Multiscreen-HA filter plate (Millipore, Merck, Darmstadt, Germany) and 2 µL prepared sample was loaded in the mass spectrometer. Peptides were separated on an EasySpray C18 column (0.15 mm x 100 mm) during a 30 minute gradient from 2 to 31 % acetonitrile in 0.1 % aqueous formic acid. Clonotypic peptides and SIL-peptides were measured with targeted MS (Orbitrap Exploris 120, Thermo Fisher Scientific) with the following MS parameters: 60000 resolution, 2 m/z isolation window, RF lens of 70%, an internal mass calibration with a lock mass of 445.12003 m/z, and an MS1 scan was included after every 20 spectra. The collision energy of each clonotypic peptide was between 18 to 36 normalized collision energy. All serum samples of 3 patients were experimentally prepared and measured in 3 independent experiments over a period of 6 months. The results of targeted MS were analyzed in Skyline. The software tool Peakfit was applied to automatically select peptide peak boundaries, additionally the results were manually reviewed. The dot product between the clonotypic peptide and SIL-peptide in Skyline was used to accept or reject peptides as valid signals (threshold 0.89). Based on the dot product, one of the two quantified clonotypic peptides of each patient’s M-protein was selected to qualify for the M-protein. For patients with a free light chain M-protein (n=3) and patients with a light chain escape M-protein (n=3) the light chain clonotypic peptide was selected. Available data of conventional methods (serum protein electrophoresis; SPEP) for all patients who were M-protein positive at diagnosis were used to adjust the M-protein concentration quantified with MS-MRD.

Between 13 to 31 serum samples per MM patient (n=41) collected from the IFM 2009 clinical trial study.