# Development of Highly Multiplex Targeted Proteomics Assays in Biofluids Using a Nominal Mass Ion Trap Mass Spectrometer

## TL;DR

This paper introduces a new method using a mass spectrometer to measure over 2000 proteins at once, enabling large-scale clinical studies of diseases like Alzheimer's.

## Contribution

The novel use of a hybrid nominal mass instrument and gas-phase fractionation libraries allows highly multiplex targeted proteomics assays.

## Key findings

- The Stellar mass spectrometer can target over 2000 peptides in a single injection.
- Targeted assays successfully detect Alzheimer's- and Parkinson's-associated proteins in cerebrospinal fluid.
- Survey-style PRM assays capture disease-related protein differences without prior hypotheses.

## Abstract

The development of targeted assays that monitor biomedically relevant proteins is an important step in bridging discovery experiments to large scale clinical studies. Targeted assays are currently unable to scale to hundreds or thousands of targets. We demonstrate the generation of large-scale assays using a novel hybrid nominal mass instrument. The scale of these assays is achievable with the Stellar mass spectrometer through the accommodation of shifting retention times by real-time alignment, while being sensitive and fast enough to handle many concurrent targets. Assays were constructed using precursor information from gas-phase fractionation data-independent acquisition (DIA). We demonstrate the ability to schedule methods from orbitrap and linear ion trap acquired gas-phase fractionation DIA library, and compare the quantification of a matrix-matched calibration curve from orbitrap DIA and linear ion trap parallel reaction monitoring (PRM). Two applications of these proposed workflows are shown with a cerebrospinal fluid neurodegenerative disease protein PRM assay and with a Mag-Net enriched plasma extracellular vesicle protein survey PRM assay. In cerebrospinal fluid, our assay targets proteins discovered previously to be associated with Alzheimer’s disease in a small independent sample set. For the Mag-Net enriched plasma survey assay, we observe that proteins selected based on their measurement robustness are still able to capture differences in abundance across disease groups in a small sample set. These highlight the application of highly multiplex, targeted protein assays in clinical research.

•Gas phase fractionation libraries enable rapid development of targeted proteomics assays.•The Stellar MS can be used to target >2000 peptides in a single injection.•A nominal mass ion trap mass spectrometer offers excellent quantitative performance.•Targeted assays detect known Alzheimer's- and Parkinson's-associated proteins.•Survey-style PRM assays capture disease biology without prior hypotheses.

Gas phase fractionation libraries enable rapid development of targeted proteomics assays.

The Stellar MS can be used to target >2000 peptides in a single injection.

A nominal mass ion trap mass spectrometer offers excellent quantitative performance.

Targeted assays detect known Alzheimer's- and Parkinson's-associated proteins.

Survey-style PRM assays capture disease biology without prior hypotheses.

Targeted proteomics assays are essential for translating biomarker discoveries into clinical applications, but current methods struggle to scale beyond a few hundred targets. This study demonstrates that the Stellar mass spectrometer, combined with gas-phase fractionated DIA libraries and real-time retention time alignment, enables highly multiplex PRM assays measuring over 2000 peptides in a single injection. Applied to cerebrospinal fluid and plasma extracellular vesicles, these assays successfully quantify neurodegenerative disease-associated proteins, bridging discovery proteomics and large-scale clinical studies.