Because of low throughput and limited robustness, nano-scale liquid chromatography has been a bottleneck for advancing proteomics in biomedical research. Here, we developed and evaluated two new LC concepts—“pre-formed gradients” and “offset gradients for peptide re-focusing”—that are both implemented in the Evosep One instrument. We evaluated robustness with more than 2000 HeLa runs, demonstrated absence of cross-contamination with crude plasma samples, high proteome coverage by fractionated HeLa and routinely measuring more than 5000 proteins/sample in just 21 minutes.

Neurodegenerative diseases are a growing burden, and there is an urgent need for better biomarkers for diagnosis, prognosis, and treatment efficacy. Structural and functional brain alterations are reflected in the protein composition of cerebrospinal fluid (CSF). Alzheimer's disease (AD) patients have higher CSF levels of tau, but we lack knowledge of systems-wide changes of CSF protein levels that accompany AD. Here, we present a highly reproducible mass spectrometry (MS)based proteomics workflow for the in-depth analysis of CSF from minimal sample amounts. From three independent studies (197 individuals), we characterize differences in proteins by AD status (> 1,000 proteins, CV < 20%). Proteins with previous links to neurodegeneration such as tau, SOD1, and PARK7 differed most strongly by AD status, providing strong positive controls for our approach. CSF proteome changes in Alzheimer's disease prove to be widespread and often correlated with tau concentrations. Our unbiased screen also reveals a consistent glycolytic signature across our cohorts and a recent study. Machine learning suggests clinical utility of this proteomic signature.