Next-generation protein sequencing and individual ion mass spectrometry enable complementary analysis of interleukin-6.

Abstract

The vast complexity of the proteome currently overwhelms any single analytical technology in capturing the full spectrum of proteoform diversity. In this study, we evaluated the complementarity of two cutting-edge proteomic technologies-single-molecule protein sequencing and individual ion mass spectrometry-for analyzing recombinant human IL-6 (rhIL-6) at the amino acid, peptide, and intact proteoform levels. For single-molecule protein sequencing, we employed the recently released Platinum instrument. Next-generation protein sequencing (NGPS) on Platinum utilizes cycles of N-terminal amino acid recognizer binding and aminopeptidase cleavage to enable parallelized sequencing of single peptide molecules. We found that NGPS produces single amino acid coverage of multiple key regions of IL-6, including two peptides within helices A and C, which harbor residues that reportedly impact IL-6 function. For top-down proteoform evaluation, we used individual ion mass spectrometry (I²MS), a highly parallelized Orbitrap-based charge detection MS platform. Single ion detection of gas-phase fragmentation products (I²MS²) gives significant sequence coverage in key regions of IL-6, including two regions within helices B and D that are involved in IL-6 signaling. Together, these complementary technologies delivered a combined 52% sequence coverage, offering a more complete view of IL-6 structural and functional diversity than either technology alone. This study highlights the complementarity of these protein detection methods to cover protein segments relevant to biological interactions more comprehensively.