Hitching timsTOF to the proteogenomics wagon takes autoantibody clonal analysis to the next level of sensitivity.

Pulmonary alveolar proteinosis (PAP) is a disorder causing accumulation of surfactant material in the lung. The autoimmune variant (aPAP) predominates, thought to be caused by elevated levels of granulocyte-macrophage colony-stimulating factor (GM-CSF) autoantibodies (GMAbs). Polyclonal, and with strong neutralizing capacity, these interfere with alveolar macrophage function and maturation to impair surfactant catabolism. Circulating GM-CSF autoreactive B cells (GMARBs) are potential precursors for GMAb-producing plasma and B cells, however the mechanism involved remains unclear.

Proteogenomics identifies peptides from enzymatically digested proteins, comparing their LC-MS/MS spectra against a cDNA database derived using Next Gen Sequencing (NGS), and has been used to characterise autoantibody sequence diversity in other diseases.  Proteomic analysis of polyclonal antibodies nevertheless remains difficult: complementarity-determining regions (CDRs), part of the antibodies’ variable chain, are so highly variable the number of peptides per clone is insufficient for detection by std LC-MS/MS. In contrast, non-CDRs feature high clonal sequence homology, making clone-specific sequence discrimination difficult.

Hashimoto et al here adopts a recent LC-MS/MS advancement – timsTOF, together with IonOpticks Aurora Series UHPLC columns, to identify GMAb-derived peptides compared against a cDNA database derived from GMARBs, in the process evaluating analytical accuracy of the new technology.

timsTOF incorporates both trapped ion mobility spectrometry (TIMS) and quadrupole time-of-flight (TOF), to combine accurate determination of the mass:charge ratio with separation based on size:charge. Generating additional separation power and increased peak capacity over conventional LC-MS/MS, it uses the parallel accumulation serial fragmentation (PASEF) method, enabling  high sequencing speeds without loss of sensitivity. Together with an outstanding chromatography delivered by IonOpticks Aurora Series, GMAb peptides were matched to almost all cDNA clones of GMARBs generated by NGS, suggesting most GMARBs differentiate into plasma cells to generate GMAbs in aPAP cases. Additionally, this  approach was able to identify many sequences in the hypervariable CDR3 region, signifying the high sensitivity of this LC-MS-MS system.

Read the full paper
Proteogenomic analysis of granulocyte macrophage colony- stimulating factor autoantibodies in the blood of a patient with autoimmune pulmonary alveolar proteinosis
Sci Rep
. 10, 18 March 2020. doi: https://doi.org/10.1038/s41598-020-61934-y

Hashimoto A, Takeuchi S, Kajita R, Yamagata A, Kaku Ri, Tanaka T & Nakata K.

Commentary by Andreia Almeida, PhD.

About the author
Andreia is an application scientist at IonOpticks playing a key role on the utilisation and development of next generation HPLC columns. Andreia has completed her PhD at Institute for Glycomics – Griffith University in Gold Coast with a strong focus on Glycomics & Glycoproteomics.