Shotgun Characterization of the Circulating IgM Antigenome of an Infectious Pathogen by Immunocapture-LC–MS/MS from Dried Serum Spots

One of the main challenges in compiling the complete
collection
of protein antigens from pathogens for the selection of vaccine candidates
or intervention targets is to acquire a broad enough representation
of them to be recognized by the highly diversified immunoglobulin
repertoire in human populations. Dried serum spot sampling (DSS) retains
a large repertoire of circulating immunoglobulins from each individual
that can be representative of a population, according to the sample
size. In this work, shotgun proteomics of an infectious pathogen based
on DSS sampling coupled with IgM immunoprecipitation, liquid chromatography–mass
spectrometry (LC–MS/MS), and bioinformatic analyses was combined
to characterize the circulating IgM antigenome. Serum samples from
a malaria endemic region at different clinical statuses were studied
to optimize IgM binding efficiency and antibody leaching by varying
serum/immunomagnetic bead ratios and elution conditions. The method
was validated using Plasmodium falciparum extracts identifying 110 of its IgM-reactive antigens while minimizing
the presence of human proteins and antibodies. Furthermore, the IgM
antigen recognition profile differentiated between malaria-infected
and noninfected individuals at the time of sampling. We conclude that
a shotgun proteomics approach offers advantages in providing a high-throughput,
reliable, and clean way to identify IgM-recognized antigens from trace
amounts of serum. The mass spectrometry raw data and metadata have
been deposited with ProteomeXchange via MassIVE with the PXD identifier
PXD043800.

在汇编病原体蛋白质抗原的完整集合，以选择疫苗候选物或干预靶点时，面临的主要挑战之一是获取足够广泛的代表性，以便它们能够被人类群体中高度多样化的免疫球蛋白谱所识别。根据样本量，干燥血清斑点采样（DSS）能够保留每个个体中大量循环免疫球蛋白，这些免疫球蛋白可以代表整个群体。在本研究中，基于DSS采样结合IgM免疫沉淀、液相色谱-质谱联用（LC-MS/MS）以及生物信息学分析的散弹蛋白质组学被联合运用，以表征循环IgM抗原组。研究了对疟疾流行地区的不同临床状态的血清样本，以优化IgM结合效率和抗体洗脱，通过调整血清/免疫磁性珠比例和洗脱条件。该方法通过鉴定恶性疟原虫提取物中的110种与其IgM反应的抗原并进行验证，同时最大限度地减少人蛋白和抗体的存在。此外，IgM抗原识别谱在采样时区分了疟疾感染者和非感染者。我们得出结论，散弹蛋白质组学方法在提供一种高通量、可靠且纯净的方式来识别血清中的IgM识别抗原方面具有优势。质谱原始数据和元数据已通过MassIVE存入ProteomeXchange，使用PXD标识符PXD043800。