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.

Non‐alcoholic fatty liver disease ( NAFLD ) affects 25% of the population and can progress to cirrhosis with limited treatment options. As the liver secretes most of the blood plasma proteins, liver disease may affect the plasma proteome. Plasma proteome profiling of 48 patients with and without cirrhosis or NAFLD revealed six statistically significantly changing proteins ( ALDOB , APOM , LGALS 3 BP , PIGR , VTN , and AFM ), two of which are already linked to liver disease. Polymeric immunoglobulin receptor ( PIGR ) was significantly elevated in both cohorts by 170% in NAFLD and 298% in cirrhosis and was further validated in mouse models. Furthermore, a global correlation map of clinical and proteomic data strongly associated DPP 4, ANPEP , TGFBI , PIGR , and APOE with NAFLD and cirrhosis. The prominent diabetic drug target DPP 4 is an aminopeptidase like ANPEP , ENPEP , and LAP 3, all of which are up‐regulated in the human or mouse data. Furthermore, ANPEP and TGFBI have potential roles in extracellular matrix remodeling in fibrosis. Thus, plasma proteome profiling can identify potential biomarkers and drug targets in liver disease.

Plasma and serum are rich sources of information regarding an individual's health state, and protein tests inform medical decision making. Despite major investments, few new biomarkers have reached the clinic. Mass spectrometry (MS)‐based proteomics now allows highly specific and quantitative readout of the plasma proteome. Here, we employ Plasma Proteome Profiling to define quality marker panels to assess plasma samples and the likelihood that suggested biomarkers are instead artifacts related to sample handling and processing. We acquire deep reference proteomes of erythrocytes, platelets, plasma, and whole blood of 20 individuals (> 6,000 proteins), and compare serum and plasma proteomes. Based on spike‐in experiments, we determine sample quality‐associated proteins, many of which have been reported as biomarker candidates as revealed by a comprehensive literature survey. We provide sample preparation guidelines and an online resource ( http://www.plasmaproteomeprofiling.org) to assess overall sample‐related bias in clinical studies and to prevent costly miss‐assignment of biomarker candidates.

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Highlights d Plasma proteome profiling of independent Roux-en-Y gastric bypass cohorts d Global correlation maps of the plasma proteome reveal functional networks d Systemic inflammation and lipid transport are the major remodeled processes d Gastric bypass has specific and common effects to other weight loss interventions