Department of Laboratory Medicine and Pathology and ‡Biomedical Statistics and Informatics, Mayo Clinic , Rochester, Minnesota 55905, United States.
J Proteome Res. 2014 Mar 7;13(3):1419-27. doi: 10.1021/pr400985k. Epub 2014 Feb 11.
A monoclonal gammopathy is defined by the detection a monoclonal immunoglobulin (M-protein). In clinical practice, the M-protein is detected by protein gel electrophoresis (PEL) and immunofixation electrophoresis (IFE). We theorized that molecular mass could be used instead of electrophoretic patterns to identify and quantify the M-protein because each light and heavy chain has a unique amino acid sequence and thus a unique molecular mass whose increased concentration could be distinguished from the normal polyclonal background. In addition, we surmised that top-down MS could be used to isotype the M-protein because each immunoglobulin has a constant region with an amino acid sequence unique to each isotype. Our method first enriches serum for immunoglobulins followed by reduction using DTT to separate light chains from heavy chains and then by microflow LC-ESI-Q-TOF MS. The multiply charged light and heavy chain ions are converted to their molecular masses, and reconstructed peak area calculations for light chains are used for quantification. Using this method, we demonstrate how the light chain portion of an M-protein can be monitored by molecular mass, and we also show that in sequential samples from a patient with multiple myeloma the light chain portion of the M-protein was detected in all samples, even those negative by PEL, IFE, and quantitative FLC. We also present top-down MS isotyping of M-protein light chains using a unique isotype-specific fragmentation pattern allowing for quantification and isotype identification in the same run. Our results show that microLC-ESI-Q-TOF MS provides superior sensitivity and specificity compared to conventional methods and shows promise as a viable method of detecting and isotyping an M-protein.
单克隆丙种球蛋白血症定义为由单克隆免疫球蛋白（M 蛋白）检测。在临床实践中，M 蛋白通过蛋白凝胶电泳（PEL）和免疫固定电泳（IFE）进行检测。我们推测，可以使用分子量代替电泳模式来识别和定量 M 蛋白，因为每条轻链和重链都具有独特的氨基酸序列，因此具有独特的分子量，其浓度的增加可以与正常的多克隆背景区分开来。此外，我们推测自上而下的 MS 可用于对 M 蛋白进行免疫分型，因为每种免疫球蛋白都有恒定区，其氨基酸序列对每种免疫型都是独特的。我们的方法首先富集血清中的免疫球蛋白，然后使用 DTT 还原将轻链与重链分离，然后通过微流 LC-ESI-Q-TOF MS。将多重电荷的轻链和重链离子转化为它们的分子量，并对轻链进行重建峰面积计算以进行定量。使用这种方法，我们证明了可以通过分子量监测 M 蛋白的轻链部分，并且还表明在多发性骨髓瘤患者的连续样本中，M 蛋白的轻链部分在所有样本中均被检测到，甚至在 PEL、IFE 和定量 FLC 均为阴性的样本中也是如此。我们还使用独特的同种型特异性片段模式展示了对 M 蛋白轻链进行自上而下的 MS 免疫分型，允许在同一运行中进行定量和同种型鉴定。我们的结果表明，与传统方法相比，微 LC-ESI-Q-TOF MS 具有更高的灵敏度和特异性，并有望成为一种可行的检测和免疫分型 M 蛋白的方法。
