Automated Immunoprecipitation, Sample Preparation,\nand Individual Ion Mass Spectrometry Platform for Proteoforms

Charge\ndetection mass spectrometry (CDMS) is a well-established\ntechnique that provides direct mass spectral outputs regardless of\nanalyte heterogeneity or molecular weight. Over the past few years,\nit has been demonstrated that CDMS can be multiplexed on Orbitrap\nanalyzers utilizing an integrated approach termed individual ion mass\nspectrometry (I<sup>2</sup>MS). To further increase adaptability,\nrobustness, and throughput of this technique, here, we present a method\nthat utilizes numerous integrated equipment components including a\nKingfisher system, SampleStream platform, and Q Exactive mass spectrometer\nto provide a fully automated workflow for immunoprecipitation, sample\npreparation, injection, and subsequent I<sup>2</sup>MS acquisition.\nThis automated workflow has been applied to a cohort of 58 test subjects\nto determine individualized patient antibody responses to SARS-CoV-2\nantigens. Results from a range of serum donors include 37 subject\nI<sup>2</sup>MS spectra that contained a positive COVID-19 antibody\nresponse and 21 I<sup>2</sup>MS spectra that contained a negative\nCOVID-19 antibody response. This high-throughput automated I<sup>2</sup>MS workflow can currently process over 100 samples per week and is\ngeneral for making immunoprecipitation-MS workflows achieve proteoform\nresolution.