MASS-FIX for the detection of monoclonal proteins and light chain N-glycosylation in routine clinical practice: a cross-sectional study of 6315 patients

Abstract

Immunoenrichment-based matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS), termed MASS-FIX, offers several advantages over immunofixation for the detection and isotyping of serum monoclonal protein, including superior sensitivity and specificity, the ability to differentiate therapeutic monoclonal antibodies, and the rapid identification of light chain (LC) N-glycosylation. We identified 6315 patients with MASS-FIX performed at our institution since 2018. Of these, 4118 patients (65%) with a wide array of plasma cell disorders (PCD), including rare monoclonal gammopathies of clinical significance, had a positive MASS-FIX. Two-hundred twenty-one (5%) of the MASS-FIX positive patients had evidence of LC N-glycosylation, which was more commonly identified in IgM heavy chain isotype, kappa LC isotype, and in diagnoses of immunoglobulin light chain (AL) amyloidosis and cold agglutinin disease (CAD) compared to other PCD. This cross-sectional study describes the largest cohort of patients to undergo MASS-FIX in routine clinical practice. Our findings demonstrate the widespread utility of this assay, and confirm that LC N-glycosylation should prompt suspicion for AL amyloidosis and CAD in the appropriate clinical context.

Dissecting the Molecular Features of Systemic Light Chain (AL) Amyloidosis: Contributions from Proteomics

“…Nowadays, an unprecedented wealth of data on immunoglobulin LCs sequences can be obtained from the direct proteomic analysis of these proteins from amyloid deposits or biological fluids. In terms of throughput, speed and sensitivity, proteomics also favorably compares with traditional biochemical methods such as Edman degradation [ 10 , 18 , 20 , 21 , 56 , 57 , 58 ] for sequencing LCs extracted from amyloid fibrils.…”

“…Recently, new LC-MS (Liquid Chromatography-Mass Spectrometry) and MALDI-TOF (Matrix Assisted Laser Desorption Ionization-Time Of Flight) MS-based approaches specifically dedicated to clinical analysis of serum LCs and free LCs have been described [ 66 , 67 , 68 , 69 , 70 , 71 , 72 ]. A major such example is the method implemented by Mayo Clinic investigators, known as MASS-FIX (based on immunoenrichment of immunoglobulins and LC subclasses, followed by MALDI-TOF MS), for the detection and evaluation of monoclonal components in plasma cell dyscrasias, including AL amyloidosis [ 9 , 10 , 73 , 74 , 75 , 76 ]. The favorable features of this approach in terms of diagnostic sensitivity and specificity supported its rapid translation into the clinical routine.…”

“…An important aspect in studying amyloid LC precursors by MS concerns the chance to characterize the PTMs that occur on these proteins in vivo, a crucial aspect to elucidate the possible factors behind stability loss and fibril deposition. Diverse chemical modifications affecting amyloidogenic LCs have been described over the years through multiple biochemical approaches, such as oxidation [ 8 , 63 , 77 ], cysteinylation [ 12 , 59 ], deamidation [ 78 ] and glycosylation [ 9 , 10 , 73 , 74 , 75 , 79 ]. Glycosylation is especially interesting in relation to AL amyloidosis, because recent reports, based on MASS-FIX-based population-based analysis of serum samples from individuals with various monoclonal gammopathies, support the concept that this PTM may be related to a LC’s ability to form amyloid deposits [ 49 ].…”

“…Glycosylation is especially interesting in relation to AL amyloidosis, because recent reports, based on MASS-FIX-based population-based analysis of serum samples from individuals with various monoclonal gammopathies, support the concept that this PTM may be related to a LC’s ability to form amyloid deposits [ 49 ]. In fact, MASS-FIX showed that peaks in the MALDI-TOF mass spectrum, corresponding to glycosylated LCs, are present in AL patients with a statistically higher frequency compared to MGUS or MM [ 9 , 10 , 73 , 74 , 75 ]. In fact, up to 33% of AL-κ and 10.2% of AL-λ patients show a pattern consistent with glycosylation, compared with 3.7% and 4.9% of patients with κ and λ non-AL plasma cell disorders [ 9 , 79 ].…”

“…The proteomic methodologies and technology used in this field encompass a wide spectrum in terms of instrumentation and applications, ranging from more traditional gel-based methods, to LC-MS/MS-based analyses, up to the most recent MALDI imaging mass spectrometry studies. Importantly, various types of ex vivo relevant specimens have been the object of analysis by proteomics in AL amyloidosis, including serum [ 8 , 9 , 10 , 11 ], urines [ 12 , 13 ], formalin-fixed paraffin-embedded (FFPE) [ 14 , 15 , 16 , 17 , 18 , 19 ] tissues, or fresh/frozen specimens such as subcutaneous abdominal fat [ 15 , 20 , 21 , 22 ]. This review will provide a critical overview of how proteomics has expanded our molecular knowledge and opened new clinical perspectives in AL amyloidosis, with a brief description of the techniques and methodologies used for each analytical task, presentation of the most recent acquisitions and a critical discussion of open issues and ongoing trends.…”