ABSTRACT
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.
ABSTRACT
One of significant characteristics of matrix-assisted laser desorption/ionization tandem time of flight mass spectrometry ( MALD-TOF/TOF ) high-energy collision induced dissociation ( CID ) is to produce abundant immonium ( IM ) ions that can offer a wealth of information for peptide composition. However, MALDI-TOF/TOF is generally used for routine protein identification based on database search or de novo sequencing combined with chemical derivation. Consequently, the characteristics of IM ions may not be fully explored and utilized. Here, a total of 239 MS/MS spectra are used to explore the fragmentation features of IM ions with MALDI TOF/TOF spectrometry and their application for peptide identification. IM ion signals can be observed for 14 kinds of amino acids including histidine etc with a positive rate of more than 50%. We have found that the chemical nature of the amino acids and position effects are the two main factors that affect the intensity of fragment ions. In addition, false positive IM ions are mainly derived from Arg, Lys, Leu and Ile residues or mixture peptides. Besides the compositional information, partial sequence information can also be obtained by a comparison of the relative intensity of IM ions. These findings are helpful when performing manual interpretations and could be useful for improving current peptide search algorithms.