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Objective:To establish a matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) method for the direct detection of serum M protein without antibody enrichment, and to assess its detection performance.Methods:Method establishment. A total of 712 waste serum samples were collected from patients who applied for the M protein identification test in Beijing Chaoyang Hospital affiliated to Capital Medical University. The immunoglobulin light chain was obtained by reduction of IgG and IgA by TCEP, and the detection method was preliminarily determined. The waste serum samples from 20 healthy people were collected to determine the range of mass-to-charge ratios of κ and λ light chain ions. 8 parallel tubes and 8 batches were set up for intra-and inter-batch reproducibility evaluation. 10-fold, 100-fold and 200-fold diluted M protein from 23 positive samples were detected by established MALDI-TOF MS method, and its sensitivity was evaluated. 3 methods of IFE, SPE and MALDI-TOF MS were used to detect M protein simultaneously, and the coincidence rate between MALDI-TOF MS and IFE and SPE was calculated.Results:The repeatability within and between batches was 100%, respectively. The original, 10-, 100-and 200-fold dilutions of 23 M protein-positive samples were determined, and the detection limit of MALDI-TOF MS for M protein was 0.06-0.18 g/L. IFE as the gold standard, the overall coincidence rates of SPE and MALDI-TOF MS were 85.9% and 92.3%, respectively, and the positive coincidence rates of SPE and MALDI-TOF MS were 72.8% and 99.7%, respectively, of the 712 samples. Among the different types of M-proteins, MALDI-TOF-MS agreed 100% with IFE M-protein results for IgA, IgD, IgM, free light chain type and biclonal group, while the agreements of SPE for IgM, IgA and free light chain samples were only 66.7%, 58% and 19.5%, respectively. One positive sample in the IgG group was not detected by MALDI-TOF MS. 23 M-proteins positive samples were diluted by original, 10, 100 and 200 times to access the sensitivity of MALDI-TOF MS method. The coincidence rate of MALDI-TOF MS was 100% and IFE was 96% at 10-fold dilution. The coincidence rate of IFE was 28% and 23% of MALDI-TOF MS at 100-fold and 200-fold dilution, respectively.Conclusions:A MALDI-TOF MS method for the detection of serum M-proteins was successfully established. This method has the advantages of high detection throughput, fast speed, good sensitivity, specificity and coincidence rate.
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ObjectiveTo evaluate clinical significance of serum free light chains (sFLC) in diagnosis and response to the therapies of patients with multiple myeloma (MM).MethodssFLC (κ,λ and κ / λ ratio)were examed by immumoassay from 62 patients with MM at different stage. The results were analyzed associated with clinical data,and 35 cases of chronic renal failure(CRF)patients and 62 cases of healthy donors were taken as controls.ResultsMedium sFLC of normal κ value was (13.25±6.46) mg/L,λ value was (18.39±11.42) mg/L; and κ / λ ratio was (0.97±0.64) mg/L (range 0.33-1.61).sFLC κ and λ of CRF patients were (200.01±299.87) mg/L,(191.02±245.98) mg/L,significantly higher than that of the normal control group (t =-17.804,-16.894,both P ＜ 0.001),but the κ/λ ratio was at normal range (1.11±0.29).κ value range was at 16.20- 35 250 mg/L in newly diagnosed intact immunoglobulin MM patients with IgGκ,IgAκ and IgDκ type.The range of λ values was 15.70-4885 mg/L in IgGλ,IgAλ,IgDλ type,and κ/λ ratio was abnormal in 96.5 ％ (55/57) patients (＜0.5 or ＞1.5).The κ,λ value and κ/λ ratio were close to that of the normal after remmision.ConclusionsFLC ( κ,λ,and κ / λ ratio) are very good monitoring markers for MM.
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Objective To analyze the differentially expressed proteins in bone marrow supernatants of multiple myeloma patients by using 2-DE and MALDI-TOF-MS,and search for the special protein markers for studying the mechanism of the development and diagnosis or differential diagnosis of multiple myeloma.Methods The bone marrow supernatant samples of fourteen multiple myeloma patients,five other hematologic malignancies and five normal controls were collected.After removing albumin and IgG,the proteins in the supernatants were separated by 2-DE.Three groups images were analyzed and compared by Imagemnster 2D platinum 5.0 analysis software.Differentially expressed proteins were selected if the protein spots intensity showed more than 3 fold increase or decrease among different groups.The identities of the differentially expressed proteins with good repeatibility were determined by PMF based on by MALDI-TOF-MS or MALDI-TOF-MS/MS and NCBInr database search.Results 2-DE maps of bone marrow supernatants of the three groups could be analyzed and compared by image analysis of software.Forty-seven and fifty-eight differentially expressed protein spots were detected in multiple myeloma samples compared with normal controls and other hematologic malignancies samples,respectively.Forty-one reproducible spots were analyzed and identified by mass spectrum.Compared with other hematologic malignancies and normal controls,five up-expressed proteins and three down-expressed proteins were identified in multiple myeloma samples.They includes immunoglobulin J chain κ and λ light chain,provirus ancestral Gag polyprotein,mature oxy-cope catalytic antibody with hapten for up-expressed proteins,and hemoglobin,haptoglobin Hp2,zinc-alPha-2-glycoprotein for down-expressed proteins.These differentially expressed proteins reflect the features of muhiple myeloma,and relate to the development,progression and therapy of multiple myeloma.Conclusions Eight differentially expressed proteins in bone marrow supernatants of multiple myeloma are identified by using 2-DE and MALDI-TOF-MS.These differentially expressed proteins could be useful in studying the mechanism of the development and progression of multiple myeloma,and in developing diagnosis and differential diagnosis of multiple myeloma.