Detection of M-Protein in Acetonitrile Precipitates of Serum Using MALDI-TOF Mass Spectrometry: A Novel Methodology

Background: Multiple myeloma (MM) and plasmacytoma(s) belong to a group of clonal plasma cell dyscrasias. In 97-98% of all cases, they are characterised by the detection of a monoclonal protein (M-protein) in the blood, and sometimes in the urine. MALDI-TOF-mass spectrometry (MS) has demonstrated excellent analytical sensitivity for the screening and detection of M-protein. We present the results of a novel methodology for M-protein analysis by MALDI-TOF MS. Patients and Methods: Blood samples from patients and controls were collected after obtaining Institutional Ethics Committee approval. The work was carried out in accordance with the Declaration of Helsinki after obtaining written informed consent. Patients with confirmed multiple myeloma or plasmacytoma and M-protein detected by serum protein electrophoresis (SPEP), serum immunofixation electrophoresis (IFE), and serum free light chains (FLC) were included for MALDI-TOF MS analysis. IFE and FLC analysis were sent to independent laboratories for external validation of the MALDI-TOF MS results. Reagent-based extraction The serum fraction was separated from whole blood by centrifugation at 5000 rpm for 15 minutes and stored at -80oC until further analysis. Twenty-five μL of the serum sample was mixed with 50% acetonitrile (ACN) to form a precipitate. After precipitation and incubation, the mixture was centrifuged. The protein precipitate was washed with 20% ACN. After centrifugation, the supernatant was discarded, and the precipitate was reconstituted in a buffer comprising 10% formic acid (FA) and 50 mmol/L tris(2-carboxyethyl)phosphine hydrochloride (TCEP). The MALDI-TOF MS results were validated using immunoenrichment by anti-kappa (κ) and anti-lambda (λ) biotin-labelled antibodies immobilised on streptavidin magnetic beads. MALDI-TOF MS measurements were obtained for intact proteins using alpha-cyano-4-hydroxycinnamic acid as a matrix. The images obtained were overlaid on apparently healthy serum samples to confirm the presence of M-protein. The samples were then analysed using UltraflexTM LT, Bruker MALDI/TOF-TOF mass spectrometer. The mass spectra for each sample was exported to FlexAnalysis 3.3 (Bruker Daltonics) and background subtracted. A sample was considered positive for M-protein if there was a sharp or broad peak within the κ or λ mass/charge (m/z) range- κ m/z- [M+2H]2+: 11550-12300 Da; [M+H]+: 23100-24600 Da), and λ m/z- [M+2H]2+: 11100-11500 Da; [M+H]+: 22200-23100 Da. All the images wer