Subset of Kappa and Lambda Germline Sequences Result in Light Chains with a Higher Molecular Mass Phenotype

In
our previous work, we showed that electrospray ionization of
intact polyclonal kappa and lambda light chains isolated from normal
serum generates two distinct, Gaussian-shaped, molecular mass distributions
representing the light-chain repertoire. During the analysis of a
large (>100) patient sample set, we noticed a low-intensity molecular
mass distribution with a mean of approximately 24 250 Da, roughly
800 Da higher than the mean of the typical kappa molecular-mass distribution
mean of 23 450 Da. We also observed distinct clones in this
region that did not appear to contain any typical post-translational
modifications that would account for such a large mass shift. To determine
the origin of the high molecular mass clones, we performed de novo
bottom-up mass spectrometry on a purified IgM monoclonal light chain
that had a calculated molecular mass of 24 275.03 Da. The entire
sequence of the monoclonal light chain was determined using multienzyme
digestion and de novo sequence-alignment software and was found to
belong to the germline allele IGKV2-30. The alignment of kappa germline
sequences revealed ten IGKV2 and one IGKV4 sequences that contained
additional amino acids in their CDR1 region, creating the high-molecular-mass
phenotype. We also performed an alignment of lambda germline sequences,
which showed additional amino acids in the CDR2 region, and the FR3
region of functional germline sequences that result in a high-molecular-mass
phenotype. The work presented here illustrates the ability of mass
spectrometry to provide information on the diversity of light-chain
molecular mass phenotypes in circulation, which reflects the germline
sequences selected by the immunoglobulin-secreting B-cell population.

在我们此前的研究中，已证实从正常血清中分离得到的完整多克隆κ链与λ链经电喷雾电离（electrospray ionization）处理后，可呈现两种截然不同的高斯型分子质量分布，以此表征轻链库（light-chain repertoire）的组成特征。在对超过100例患者的大样本队列进行分析时，我们观察到一组低强度的分子质量分布峰，其均值约为24250 Da，较典型κ链分子质量分布的均值（23450 Da）高出约800 Da。同时我们在该质量区间内发现了独特的克隆型，这类克隆似乎不具备任何可解释该显著质量偏移的典型翻译后修饰（post-translational modification）。为明确该高分子质量克隆型的来源，我们对一株经纯化的IgM单克隆轻链开展了自下而上（bottom-up）从头测序（de novo）质谱分析法（mass spectrometry）检测，该轻链的理论计算分子质量为24275.03 Da。通过多酶消化法与从头序列比对软件，我们确定了该单克隆轻链的全序列，经比对发现其归属生殖系等位基因IGKV2-30。对κ链生殖系序列的比对分析显示，共有10条IGKV2家族与1条IGKV4家族的序列在其CDR1（Complementarity-determining region 1，互补决定区1）区域存在额外氨基酸插入，由此产生了高分子质量表型。我们同时对λ链生殖系序列开展了比对分析，结果显示功能性生殖系序列的CDR2（Complementarity-determining region 2，互补决定区2）区域与FR3（Framework region 3，骨架区3）区域存在额外氨基酸插入，进而导致高分子质量表型。本研究阐明了质谱分析法可用于获取循环中轻链分子质量表型的多样性信息，而该多样性恰恰反映了免疫球蛋白分泌型B细胞群体所选择的生殖系序列特征。