New Interface for Faster Proteoform Analysis: Immunoprecipitation Coupled with SampleStream-Mass Spectrometry

In collaboration with:

 

 

Description: Different proteoform products of the same gene can exhibit differing associations with health and disease, and their patterns of modifications may offer more precise markers of phenotypic differences between individuals. However, currently employed protein-biomarker discovery and quantification tools, such as bottom-up proteomics and ELISAs, are mostly proteoform-unaware. Moreover, the current throughput for proteoform-level analyses by liquid chromatography-mass spectrometry (LC-MS) for quantitative top-down proteomics is incompatible with population-level biomarker surveys requiring robust, faster proteoform analysis. To this end, we developed immunoprecipitation coupled to SampleStream mass spectrometry (IP-SampleStream-MS) as a high-throughput, automated technique for the targeted quantification of ApoAI and ApoCIII proteoforms.

General study design comparing two approaches for profiling of apolipoprotein proteoforms. Serum samples of 25 well-phenotyped CARDIA participants were submitted to either reverse-phase LC-MS (top) or IP-SampleStream-MS. The IP-SampleStream-MS approach to proteoform profiling starts from a targeted immunoprecipitation (left). Immunoprecipitation is parallelized and automated using magnetic beads and an automated sample-handling platform (Thermo KingFisher). Then, samples are transferred to SampleStream for buffer exchange, concentration, and course filtering based on molecular weight. Each sample is then automatically injected into the MS, allowing for targeted observation of the proteoform profile of different samples in quick succession. Herein, we compared the efficacy and throughput of these two approaches toward characterizing associations between proteoform abundance and phenotype.

 

Project status: Finalized

Publications:

– New Interface for Faster Proteoform Analysis: Immunoprecipitation Coupled with SampleStream-Mass Spectrometry. Henrique dos Santos Seckler, Hae-Min Park, Cameron M. Lloyd-Jones, Rafael D. Melani, Jeannie M Camarillo, John T. Wilkins, Philip D. Compton, and Neil L. Kelleher. Journal of the American Society for Mass Spectrometry 2021 32 (7), 1659-1670. DOI: 10.1021/jasms.1c00026

Congress Presentations:

– ASMS 2021